EA048681B1 - MODIFIED IMMUNOGLOBULINS TO TARGET AMYLOID DEPOSITS - Google Patents

Description

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 62/936,002, filed November 15, 2019, and U.S. Provisional Application No. 63/074,912, filed September 4, 2020, the contents of which are incorporated herein by reference in their entirety.

Submitting a list of sequences in an ASCII text file

The content of the ASCII text file submitted herein is incorporated by reference in its entirety: sequence listing machine-readable form (CRF) (file name: 165992000140SEQLIST.TXT, date of recording: November 12, 2020, size: 52 kb).

Field of technology

This application relates to modified immunoglobulins for targeting amyloid deposits, humanized antibodies that bind to human amyloid fibrils, and antibody-peptide fusion proteins, as well as methods of using them.

State of the art

Amyloidosis is a lethal protein folding disorder characterized by the aggregation and deposition of protein fibrils and heparan sulfate proteoglycan in vital organs and tissues (Merlini, G. et al. (2003) N. Engl. J. Med. 349, 583-596; Merlini, G. et al. (2004) J. Intern. Med. 255, 159-178; DeLorenzi, E. et al. (2004) Curr. Med. Chem. 11, 1065-1084; Merlini, G. (2004) Neth. J. Med. 62, 104-105). Continued accumulation of amyloid invariably leads to organ dysfunction and severe morbidity or death. Deposits may be cerebral, as in patients with Alzheimer's, Huntington's, or prion diseases, or peripheral, as in patients with light chain amyloidosis (AL) and type 2 diabetes. Further subdivision into localized or systemic subgroups indicates whether the precursor protein is produced locally (at the site of deposition) or circulates in the bloodstream and is deposited at distant anatomical sites, respectively (Westermark, P. et al. (2007) Amyloid. 14, 179-183). Amyloid may affect any organ or tissue, but the kidney, pancreas, liver, spleen, nervous tissue, and heart are the main sites of deposition in patients with familial or sporadic forms of peripheral amyloidosis. Alzheimer's disease currently affects more than 4 million Americans, and this figure is expected to increase to more than 16 million by 2050. It is by far the most common form of amyloidosis and has the greatest socioeconomic impact. In contrast, peripheral (or systemic) amyloidosis is an orphan disease, but more than 5,000 new patients are diagnosed each year in the United States alone.

Of these, the major peripheral amyloidosis is light-chain amyloidosis (AL), a sporadic dyscrasia of monoclonal plasma cells resulting in the deposition of fibrils composed of immunoglobulin light chain proteins. AL accounts for approximately two-thirds of all cases of peripheral amyloidosis and has an estimated incidence of ~1.4 per 100,000 persons per year in the United States, comparable to that of acute lymphocytic leukemia and chronic myeloid leukemia (Group, U. S. C. S. W. (2007) United States Cancer Statistics: Morbidity and Mortality Web Report 1999–2003, U.S. Department of Health and Human Services and Centers for Disease Control and Prevention, National Cancer Institute, Atlanta). Although AL is five times less common than its related plasma cell dyscrasia, multiple myeloma, it is arguably more devastating, with a median survival of only 13.2 months, partly due to the rapidly progressive nature of organ destruction, the lack of effective antiamyloid drugs, and the failure to effectively diagnose the disease before organ failure occurs. Less than 5% of all patients with AL survive 10 years or more from diagnosis (Comenzo, R. L. et al. (2002) Blood 99, 42764282). Furthermore, patients with cardiac AL amyloidosis have a median survival of less than 5 months.

ATTR is a form of systemic amyloidosis. Twenty-five percent of patients with ATTR amyloidosis die within 24 months of diagnosis. (Gertz and Dispenzieri JAMA 324(1)79-89 (2002).) Current treatments do not prevent organ damage. ATTR amyloidosis is caused by transthyretin (TTR)-based fibrils. Transthyretin is a protein made by the liver that helps transport thyroid hormone and vitamin A into the blood. TTR is normally a tetramer made up of four single-chain monomers. In hereditary ATTR amyloidosis, mutations in the TTR gene are thought to destabilize the protein and cause the tetramer to dissociate into monomers, which aggregate into amyloid fibrils. In wild-type ATTR amyloidosis, the normal TTR protein becomes unstable, misfolds, and forms amyloid fibrils.

These amyloid fibrils then accumulate in many organs throughout the body, such as in the wrist, in a narrow passage called the carpal tunnel. This can cause carpal tunnel syndrome, which causes numbness and tingling in the arm and shoulder. In the spinal canal, which can cause a narrowing of the spinal column (spinal stenosis). In the heart, which can cause heart failure and/or an abnormal heart rhythm called atrial fibrillation.

Another common form of peripheral amyloidosis in the United States is inflammation-associated amyloidosis (IAA), which is associated with chronic inflammatory diseases such as

- 1 048 681 such as arthritis, tuberculosis and familial Mediterranean fever. The incidence of AA is highest in some regions of Europe, and the frequency varies among ethnic groups (Buck, F. S. et al. (1989) Mod. Pathol. 2, 372-377). In areas where familial Mediterranean fever is common and untreated, the incidence of AA may reach 100%. In Europe, the incidence, based on pathological studies conducted in Denmark, is estimated at 0.86% (Lofberg, H. et al. (1987) Acta pathologica, microbiologica, et immunologica Scandinavica 95, 297-302); however, in patients with rheumatoid or psoriatic arthritis, the incidence of AA may reach 26%. Such a high prevalence may require screening programs for earlier detection of the disease. Amyloid deposition is associated with persistent elevations in plasma concentrations of serum amyloid A protein (sAA), the precursor of amyloid fibrils (Rocken, C. et al. (2002) Virchows Arch. 440, 111-122). AA differs from AL in the type of precursor protein deposited, but both share common mechanistic features associated with fibril formation and deposition (Rocken, C. et al. (2006) 7. Pathol. 210, 478-487; Rocken, C. et al. (2001) Am. J. Pathol. 158, 1029-1038).

In addition to disorders in which the etiopathology of amyloid is well established, fibrillar deposits with the structural and tinctorial properties of amyloid have been identified in other syndromes, although their relationship to the disease state remains to be established. For example, in type 2 diabetes, islet amyloid precursor protein (IAPP) is deposited as amyloid in the islets of Langerhans (Jaikaran, E. T. et al. (2001) Biochim. Biophys. Acta 1537, 179-203). Aggregation of IAPP results in the formation of oligomeric structures that are toxic to pancreatic cells (Lin, C. Y. et al. (2007) Diabetes 56, 1324-1332). Thus, it is suggested that the formation of IAPP amyloid in patients with type 1 diabetes mellitus promotes the destruction of β-cells and facilitates the transition to insulin dependence (Jaikaran, E. T. et al. (2001) Biochim. Biophys. Acta 1537, 179-203). In another example, plaques containing amyloid fibrils composed of apolipoprotein A-I have been identified in more than half of patients with carotid artery atherosclerosis (Westermark, P. et al. (1995) Am. J. Pathol. 147, 1186-1192; Mucchiano, G. I. etal. (2001) 7. Pathol. 193, 270-275). The deposition of these fibrils is more common in older patients, but apoA-I is certainly present in the early stages of plaque development (Vollmer, E. et al. (1991) Virchows Arch. A. Pathol. Anat. Histopathol. 419, 79-88). As a final example, amyloid Apo-A-I has also recently been identified in knee menisci obtained from patients undergoing knee replacement surgery, and it may contribute to physical wear and tear of the joint (Solomon, A. et al. (2006) Arthritis Rheum. 54, 35453550).

In total, more than 29 proteins have been chemically or serologically identified as constituents of fibrils in amyloid deposits. It is the nature of these proteins that allows disease differentiation, treatment, and prognosis. Although amyloid fibrils are associated with a clinically heterogeneous group of diseases and can form from structurally distinct and functionally distinct precursor proteins, the deposits themselves share a number of remarkably similar characteristics, including fibril structure, fibril epitopes, and the accumulation of similar accessory molecules, including heparan sulfate proteoglycans (SHBG). Amyloid is a heterogeneous complex that includes, in addition to fibrils, glycosaminoglycans (GAGs) and, in particular, perlecan SHBG (Ancsin, J. B. (2003) Amyloid 10, 67-79; Ailles, L. et al. (1993) Lab. Invest. 69, 443-448; Kisilevsky, R. (1994) Mo/. Neurobiol. 9, 23-24; Kisilevsky, R. (1990) Lab. Invest. 63, 589-591; Snow, A. D. et al. (1987) Lab. Invest. 56, 120123; Li, J. P. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 6473-6477). A partial list of amyloid and amyloid-associated disorders is shown in Fig. 1.

To date, the most effective therapeutic intervention to remove amyloid deposits, which may promote organ function recovery and lead to improved prognosis, is the use of amyloid-reactive antibodies as an immunotherapy agent. Several immunotherapeutic agents (antibodies) have been developed for amyloid-associated diseases, including the monoclonal antibody 11-1F4 for the treatment of AL amyloidosis, NEOD001 for patients with AL amyloidosis, GSK2398852 (an anti-SAP monoclonal antibody) for amyloidosis, solanezumab for Alzheimer's disease, intravenous IgG (IVIG) for Alzheimer's disease, and bapineuzumab for Alzheimer's disease. Each of these approaches has limitations or has not met the initial results in late-stage (phase 2/3) clinical trials.

The essence of the invention

Provided herein are modified immunoglobulins and antibody-peptide fusion proteins comprising an amyloid-reactive peptide linked to an antibody or a functional fragment thereof that bind to human amyloid fibrils. The modified immunoglobulins and antibody-peptide fusion proteins provided herein unexpectedly exhibit increased affinity for human amyloid fibrils due to the binding of the antibody and the amyloid-reactive peptide to human amyloid fibrils. In some embodiments, the peptide provides enhanced amyloid deposit clearance activity of the antibody. In particular, the Fc of the antibody recruits macrophages that are capable of phagocytosing and removing amyloid fibrils and deposits, such as by opsonization, and the amyloid-reactive

- 2 048681 peptide is able to bind various amyloid fibrils and heparan sulfate glycosaminoglycans. Surprisingly, humanized amyloid antibodies conjugated to the N-terminal amyloid-reactive peptide provide significantly better opsonization than chimeric antibodies that bind to amyloid fibers. Moreover, some humanized antibodies, such as VH9/VL4, bind to amyloid fibrils with higher affinity than murine or chimeric antibodies.

Also provided in this document are methods for detecting and treating amyloidosis or using the modified immunoglobulins provided in this document.

Additionally, provided herein are nucleic acids encoding modified immunoglobulins. In some embodiments, provided herein is a host cell comprising a nucleic acid encoding a modified immunoglobulin. In some embodiments, the host cell is a CHO cell.

In one aspect, the present invention relates to a modified immunoglobulin comprising: an amyloid-reactive peptide; and an Ig antibody or a functional fragment thereof that binds to human amyloid fibrils, wherein the Ig antibody or a functional fragment thereof comprises a heavy chain and a light chain, wherein the peptide and the Ig antibody or a functional fragment thereof are linked together at the N-terminal end of the Ig light chain and/or the N- and/or C-terminal end of the Ig heavy chain.

In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence that has at least 85% identity to any of the amino acid sequences set forth in SEQ ID NOs: 1-14.

In some embodiments, the amyloid-reactive peptide and the Ig antibody or functional fragment thereof are linked together at the N-terminal end of the Ig light chain.

In some embodiments, the modified immunoglobulin comprises a spacer sequence between the amyloid-reactive peptide and the Ig antibody or functional fragment thereof.

In some embodiments, the modified immunoglobulin comprises at least two amyloid-reactive peptides, wherein the amyloid-reactive peptides are the same peptide or different peptides.

In some embodiments, the Ig antibody or functional fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDRL1 as set forth in SEQ ID NO: 20, CDRL2 as set forth in SEQ ID NO: 21, and CDRL3 as set forth in SEQ ID NO: 22; and wherein the VH comprises CDRH1 as set forth in SEQ ID NO: 17, CDRH2 as set forth in SEQ ID NO: 18, CDRH3 as set forth in SEQ ID NO: 19.

In some embodiments, the Ig antibody or functional fragment thereof is a chimeric antibody or functional fragment thereof.

In some embodiments, the Ig antibody or functional fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein a) the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; or b) the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20; 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, and VH comprising 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: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the Ig antibody or functional fragment thereof comprises human framework sequences.

In some embodiments, the Ig antibody comprises a human Fc region.

In some embodiments, the modified immunoglobulin comprises at least two amyloid-reactive peptides, wherein the peptides are the same peptide or different peptides.

In some embodiments, the modified immunoglobulin binds to rVX6Wil, AB, Αβ(1-40), IAAP, ALk4, Άίλ1, or ATTR fibrils.

In another aspect, the present document provides an antibody-peptide fusion protein comprising an antibody that binds human amyloid fibrils fused to an amyloid-reactive peptide, wherein said antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH).

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In some embodiments, the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the antibody is a chimeric antibody.

In some embodiments, a) the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; or b) the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20; 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, and VH comprising 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: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; or c) VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, 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, and VH comprises CDRH1 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: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, VL comprises Leu at position 46 and Phe at position 87, and VH comprises Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94.

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

In some embodiments, VL comprises one or more amino acid residues selected from the group consisting of: Tyr at position 36; Leu at position 37; Leu at position 46; Leu at position 85; and Phe at position 87. In some embodiments, VH comprises one or more amino acid residues selected from the group consisting of: Val at position 37; Leu at position 48; Leu at position 67; Ser at position 68; Lys at position 71; Ser at position 76; Val at position 78; Leu at position 79; Phe at position 80; Thr at position 89; Val at position 93; and Thr at position 94, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, VL comprises Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87, and VH comprises Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93, and Thr at position 94.

In some embodiments, VL comprises Leu at position 46 and Phe at position 87, and VH comprises Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94.

In some embodiments, the VL comprises an amino acid sequence set forth in the group consisting of SEQ ID NO: 32-42

In some embodiments, the VH comprises an amino acid sequence set forth in the group consisting of SEQ ID NO: 43-63.

In some embodiments, VL comprises the amino acid sequence set forth in SEQ ID NO: 34, and VH comprises the amino acid sequence set forth in SEQ ID NO: 48.

In some embodiments, VL comprises the amino acid sequence set forth in SEQ ID NO: 35, and VH comprises the amino acid sequence set forth in SEQ ID NO: 51.

In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence set forth in SEQ ID NO: 1-14.

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In some embodiments, the amyloid-reactive peptide is fused to the N-terminus of VL or VH.

In some embodiments, the amyloid-reactive peptide is fused to the N-terminus of VL or VH using a spacer. In some embodiments, the spacer is a peptide spacer. In some embodiments, the spacer includes the amino acid sequence GGGYS.

In some embodiments, the antibody-peptide fusion protein binds to rVX6Wil, Av, Av(1-40), IAAP, ALk4, LS, or ATTR fibrils.

In another aspect, the present document provides a pharmaceutical composition comprising a modified immunoglobulin or an antibody-peptide fusion protein.

In another aspect, the present document provides nucleic acid(s) encoding a modified immunoglobulin and an antibody-peptide fusion protein. In another aspect, the present document provides a vector comprising the nucleic acid(s). In another aspect, the present document provides a host cell comprising the vector.

In another aspect, the present invention relates to a method for producing a modified immunoglobulin or an antibody-peptide fusion protein, comprising culturing a host cell, see above, under conditions suitable for expressing a vector encoding the modified immunoglobulin or the antibody-peptide fusion protein, and isolating the modified immunoglobulin or the antibody-peptide fusion protein.

In another aspect, the present invention relates to a method of treating a subject having an amyloid-associated disorder, comprising administering to the subject an effective amount of a modified immunoglobulin or an antibody-peptide fusion protein.

In some embodiments, the amyloid-associated disorder is amyloidosis.

In some embodiments, the amyloid-associated disorder is selected from the group consisting of AL-, AH-, AB2M-, ATTR-, transthyretin, AA-, AApoAI-, AApoAII-, AGel-, ALys-, ALEct2-, AFib-, ACys-, ACal-, AMed-, AIAPP-, APro-, AIns-, APrP-, or AB-amyloidosis.

In some embodiments, the subject is a human.

In another aspect, the present invention relates to a method of targeting an amyloid deposit for clearance, comprising contacting the amyloid deposits with a modified immunoglobulin or an antibody-peptide fusion protein as provided herein. In some embodiments, the amyloid deposit is removed. In some embodiments, the amyloid deposit is opsonized with the modified immunoglobulin or the antibody-peptide fusion protein.

In yet another aspect, the present document provides a method of targeting an amyloid deposit for clearance comprising contacting the amyloid deposit with a modified immunoglobulin and an antibody-peptide fusion protein.

In some embodiments, targeting an amyloid deposit to clear the amyloid deposit.

In some embodiments, clearance results from opsonization of the amyloid deposit.

In some embodiments, the half-life of the amyloid-reactive peptide in the modified immunoglobulin or antibody-peptide fusion protein is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to the amyloid-reactive peptide alone.

In another aspect, the document provides a method for producing a modified immunoglobulin, comprising: providing a first expression vector and a second expression vector, wherein the first expression vector comprises a first nucleic acid sequence encoding an Ig antibody light chain or a functional fragment thereof; wherein the second expression vector comprises a second nucleic acid sequence encoding an Ig antibody heavy chain or a functional fragment thereof; and wherein the first expression vector and/or the second expression vector comprise a third nucleic acid sequence that encodes a first peptide, wherein the third nucleic acid sequence is located adjacent to the first nucleic acid sequence and/or the second nucleic acid sequence; and inserting the first and second expression vectors into a cell, wherein expression of the first and second expression vectors in the cell results in the formation of an immunoglobulin attached to the first peptide.

In some embodiments, the first expression vector and/or the second expression vector comprise a fourth nucleic acid sequence that encodes a second peptide, wherein the fourth nucleic acid sequence is located adjacent to the first nucleic acid sequence and/or the second nucleic acid sequence.

In some embodiments, expression of the first and second expression vectors in a cell results in the formation of an immunoglobulin linked to the first peptide and the second peptide.

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In some embodiments, the spacer nucleic acid sequence is located between the third nucleic acid sequence and the first nucleic acid sequence and/or the second first nucleic acid sequence.

In another aspect, the present invention relates to a method of treating a subject that has or is suspected of having an amyloid-associated disease, comprising: i) administering to the subject a modified immunoglobulin or an antibody-peptide fusion protein, wherein the modified immunoglobulin or antibody-peptide fusion protein comprises a detectable label; and ii) determining whether a signal associated with the detectable label can be detected in the subject; and b) if a signal is detected, administering to the subject an agent for treating amyloidosis.

In some embodiments, if no signal is detected, the method further comprises monitoring the subject for subsequent occurrence of amyloid deposits.

In some embodiments, the method further comprises determining the signal intensity and comparing the signal to a threshold value, above which the subject is determined to have amyloid deposits.

In some embodiments, treating amyloidosis comprises administering to the subject a modified immunoglobulin or an antibody-peptide fusion protein.

In some embodiments, administration of a modified immunoglobulin or antibody-peptide fusion protein results in the removal of amyloid deposits in a subject.

In another aspect, the present invention provides a method for detecting amyloid deposits in a subject, comprising administering to the subject a modified immunoglobulin or an antibody-peptide fusion protein, wherein the modified immunoglobulin or antibody-peptide fusion protein comprises a detectable label, and detecting a signal from the modified immunoglobulin or antibody-peptide fusion protein.

In some embodiments, the subject is determined to lack amyloid deposits or to have monoclonal gammopathy of unknown significance (MGUS), multiple myeloma (MM), or one or more related plasma cell diseases.

In another aspect, the present invention provides a method for detecting a ligand, comprising: a detectable label of a modified immunoglobulin or antibody-peptide fusion protein according to any one of the paragraphs, wherein the modified immunoglobulin or antibody-peptide fusion protein has a binding affinity for the ligand; contacting the ligand with the modified immunoglobulin or antibody-peptide fusion protein; and detecting a signal from the detectable label, thereby detecting the ligand.

In some embodiments, the modified immunoglobulin or antibody-peptide fusion protein is conjugated to a detectable label.

In some embodiments, the modified immunoglobulin or antibody-peptide fusion protein comprises a spacer that is N-terminal to the amyloid-reactive peptide.

In some embodiments, the amyloid-reactive peptide is attached to the N-terminus of the VH antibody.

In one aspect, provided herein is a humanized antibody that binds to human amyloid fibrils, wherein the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDRL1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19, wherein the VL comprises one or more amino acid residues selected from the group consisting of: Tyr at position 36; Leu at position 37; Leu at position 46; Leu at position 85; and Phe at position 87; wherein VH comprises one or more amino acid residues selected from the group consisting of: Val at position 37; Leu at position 48; Leu at position 67; Ser at position 68; Lys at position 71; Ser at position 76; Val at position 78; Leu at position 79; Phe at position 80; Thr at position 89; Val at position 93; and Thr at position 94; wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, VL comprises Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87, and VH comprises Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93, and Thr at position 94.

In some embodiments, VL comprises Leu at position 46 and Phe at position 87, and VH comprises Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94.

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In some embodiments, the VL comprises an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42.

In some embodiments, the VH comprises an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63.

In some embodiments, VL comprises the amino acid sequence set forth in SEQ ID NO: 34, and VH comprises the amino acid sequence set forth in SEQ ID NO: 48.

In some embodiments, VL comprises the amino acid sequence set forth in SEQ ID NO: 35, and VH comprises the amino acid sequence set forth in SEQ ID NO: 51.

In another aspect, provided herein is a humanized antibody that binds to human amyloid fibrils, wherein the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein: a) the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; or b) the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20; a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21 and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22 and a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NOs: 71-81; and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; or c) VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and CDRL3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and 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: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, VL comprises one or more amino acid residues selected from the group consisting of: Tyr at position 36; Leu at position 37; Leu at position 46; Leu at position 85; and Phe at position 87; wherein VH comprises one or more amino acid residues selected from the group consisting of: Val at position 37; Leu at position 48; Leu at position 67; Ser at position 68; Lys at position 71; Ser at position 76; Val at position 78; Leu at position 79; Phe at position 80; Thr at position 89; Val at position 93; and Thr at position 94; wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the antibody is a full-length antibody, a Fab fragment, or an scFv.

In some embodiments, the antibody comprises an Fc region.

In some embodiments, the Fc region is of the IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the humanized antibody or antibody-peptide fusion protein is conjugated to a detectable label.

In some embodiments, the humanized antibody binds to rV^6Wil fibrils, Per125 wtATTR extract, KEN hATTR extract, SHI ΛΕλ liver extract, and/or TAL ALk liver extract.

In some embodiments, the humanized antibody binds to rVX6Wil, Αβ, Αβ(1-40), IAAP, ALk4, Λλ1, or ATTR fibrils.

In yet another aspect, the present document provides a pharmaceutical composition comprising a humanized antibody. In another aspect, the present invention relates to nucleic acid(s) encoding the humanized antibody. In yet another aspect, the present document provides a vector comprising the nucleic acid(s). In yet another aspect, the present document provides a host cell comprising the vector.

In another aspect, the present invention relates to a method for producing a humanized antibody, comprising culturing a host cell under conditions suitable for expressing a vector encoding the humanized antibody, and isolating the humanized antibody.

In another aspect, the present invention provides a method of treating a subject having an amyloid-associated disorder comprising administering to the subject an effective amount of a humanized antibody. In some embodiments, the amyloid-associated disorder is amyloidosis. In some embodiments, the amyloid-associated disorder is selected from the group consisting of AL-, AH-, Αβ2M-, ATTR-, transthyretin, AA-, AApoAI-, AApoAII-, AGel-, ALys-, ALEct2-, AFib-, ACys-, ACal-, AMed-, AIAPP-, APro-, AIns-, APrP-, or Αβamyloidosis. In some embodiments, the subject is a human.

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In another aspect, the present invention relates to a method of treating a subject that has or is suspected of having an amyloid-associated disease, comprising: determining whether the subject has an amyloid deposit by: detectably labeling a humanized antibody, administering the humanized antibody to the subject, determining whether a signal associated with the detectable label can be detected in the subject; and, if the signal is detected, administering to the subject an agent for treating amyloidosis.

In some embodiments, if no signal is detected, the method further comprises monitoring the subject for subsequent occurrence of amyloid deposits.

In some embodiments, the method further comprises determining the signal intensity and comparing the signal to a threshold value, above which the subject is determined to have amyloid deposits. In some embodiments, treating amyloidosis comprises administering a humanized antibody to the subject.

In another aspect, the present invention relates to a method for identifying amyloid deposits in a subject, comprising detectably labeling a humanized antibody, administering the humanized antibody to the subject, and detecting a signal from the humanized antibody.

In some embodiments, the subject is determined to be free of amyloid deposits or to have monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), or one or more related plasma cell diseases.

Brief description of graphic materials

Fig. 1 shows a partial list of amyloid and amyloid-related disorders.

Fig. 2 shows a schematic representation of an Ig-peptide hybrid. Panel A shows a schematic of the p5 peptide (SEQ ID NO: 1) fused to an Ig light chain at the N-terminus. Panel B shows an Ig-peptide hybrid showing an Ig structure with two Ig light chains fused to the p5 peptide (SEQ ID NO: 1).

Figure 3 shows a schematic representation of the proposed mechanism of action of the Ig-peptide hybrid for clearing amyloid deposits (not to scale). The Ig-peptide binds amyloid through peptide interactions with amyloid fibrils (or heparan sulfate glycosaminoglycans), which recruit macrophages, which then engulf (phagocytose) the amyloid for destruction.

Figure 4 shows an autoradiogram of ¹²⁵ I-Igp5 after SDS-PAGE. Under non-reducing conditions the protein functioned as a single Ig, which upon reduction was shown to contain both the heavy and light chains of p5, consistent with an intact Ig molecule.

Fig. 5 shows the biodistribution of ¹²⁵ I-Igp5 in AA-amyloid (AA) and healthy amyloid-free (WT) mice 20 h after injection.

Figure 6 shows SPECT-CT images of ¹²⁵ I-Igp5 in AA mice and healthy wild-type controls 20 h after Igp5 injection, demonstrating uptake in amyloid-laden liver and spleen of AA mice, as well as the long half-life in the blood pool of the reagent in wild-type animals. H, heart; L, liver; S, spleen. Red/yellow color indicates the presence of ¹²⁵ I-Igp5.

Figure 7 shows the in vitro phagocytosis of pHrodo Red-labeled rV^6Wil fibrils in the presence of human THP1 monocytes/macrophages. The increase in fluorescence intensity indicates the presence of labeled amyloid substrate in the low pH environment of the macrophage phagolysosome. MOPC 31c is a control mouse monoclonal antibody that is not reactive to the fibrils.

Figure 8 shows an autoradiogram of ¹²⁵ I-Igp5 after SDS-PAGE. Igp5 was purified from tissue culture supernatant, radiolabeled with ¹²⁵ I, and the product was characterized by SDS-PAGE using mouse 11-1F4 (IgG1K) as a control. The protein was analyzed under both reducing (R) and non-reducing (NR) conditions.

Figure 9 shows the binding of ¹²⁵ I-Igp5 to various amyloid-associated substrates. Panel A shows the binding of ¹²⁵ I-m11-1F4 and ¹²⁵ I-Igp5 to κ4-peptide-coated beads or rV^6Wil fibrils. ¹²⁵ I-m11-1F4 binds κ4-peptide-coated beads but not rV^6Wil fibrils, whereas ¹²⁵ I-Igp5 binds both substrates. Panel B shows the quantification of ¹²⁵ I-Igp5 and m11-1F4 binding to various synthetic amyloid fibrils and amyloid extracts. Panel C shows the correlation between ¹²⁵ I-Igp5 and ^|: 5[-p5 binding to substrate. Binding of ¹²⁵ I-Igp5 to various synthetic amyloid fibrils and amyloid extracts is significantly enhanced compared to m11-1F4, where reactivity correlates with that of the p5 peptide alone, indicating that binding is peptide-driven.

Figure 10 shows microautoradiography (ARG) analysis and Congo red staining of various tissues. Panel A shows ¹²⁵ I-Igp5 retention in AA amyloid from mouse liver and spleen, as well as amyloid binding in other tissues. ARG and Congo red staining demonstrate specific retention ^{of 125} I-Igp5 in AA amyloid from mouse liver and spleen, as well as amyloid binding in other tissues. Panel B shows the results of ARG analysis of tissues without amyloid deposits. No specific reactivity was observed with tissues without amyloid deposits in healthy (WT) mice, in which the blood pool was the only identified source of ¹²⁵ I-Igp5.

Fig. 11A shows a schematic diagram of mIgG-p5. From left to right, the diagram shows the C- to N-terminal regions of mIgG-p5, including the Ig light chain (IgLC, 220 amino acids) sequence, the spacer sequence, the p5 peptide (31 amino acids), the spacer sequence, and the N-terminal secretory leader sequence. Fig. 11B shows the amino acid sequence of the mIgG-p5 regions, including, from left to right, the N-terminus of the Ig light chain (starting at amino acid residues DVVMTQTP (SEQ ID NO: 82)), the spacer sequence at the C-terminus of the p5 peptide (amino acid residues VTPTV (SEQ ID NO: 24)), the p5 peptide (amino acid residues KAQKAQAKQAKQAQKAQKAQAKQAKQ (SEQ ID NO: 1)), and the spacer sequence at the N-terminus (amino acid residues AQAGQAGQAQGGGYS (SEQ ID NO: 23)). In Fig. 11B, the amino acid sequences are depicted from the C- to the N-terminus. Fig. 11C shows a putative structural model of the p5 peptide fused to the N-terminus of the Ig light chain. In Fig. 11D shows an autoradiogram of ^125I -labeled mIgG-p5, ^125I -m11-1F4, and ^125I -p5 after SDS-PAGE. Each protein is shown under non-reducing (NR) or reducing (R) conditions, and the relative positions of the full-length antibody (Ig), heavy chain (HC), light chain (LC), and p5 peptide are indicated. Fig. 11E shows the biodistribution of ^125I -labeled mIgG-p5 in wild-type mice without amyloid deposits 24, 48, or 72 hours after injection of ^125I -mIgG-p5. Fig. 11F shows the biodistribution of ^125I -labeled mIgG-p5 in mice with AA amyloidosis (predominantly in the liver and spleen) 24, 48, or 72 hours after injection of ^125I -mIgG-p5. In each of Figs. 11E and 11F, the ordinate shows the biodistribution rate as a percentage of the injected dose per gram of tissue, and the abscissa indicates the tissue type. Fig. 11G shows microautoradiographic images showing ^125I -labeled mIg-p5 in mice with AA amyloidosis 24 hours after injection. 24 hours are shown in black; 48 hours are shown in dark gray, and 72 hours are shown in light gray.

Figure 12 shows the annotated amino acid sequences of VH (top, SEQ ID NO: 15) and VL (bottom, SEQ ID NO: 16) of the parent murine antibody m11-1F4. CDRs are shown in boxes, canonical framework residues are underlined, and VH-VL interaction surface residues are shown in bold and italics.

Figure 13A shows europium-linked immunosorbent assay (EuLISA) data measuring the binding of chimeric (c) 11-1F4 and the humanized variants, VH10/VL4, VH9/VL4, VH8/VL4, VH7/VL4, or VH6/VL3, to synthetic rV^6Wil light chain amyloid-like fibrils. Figure 13B shows EuLISA data measuring the binding of VH6/VL3-p5 (6-3-p5) 70% pure, VH6/VL3-p5R (6-3-p5R) 65% pure, C11-1F4, or VH6/VL3 to rV^6Wil fibrils. In Figure 13C shows EuLISA data measuring the binding of VH9/VL/4-p5R to rVX6Wil fibrils, Per125 wtATTR extract, Ken ATTR extract, SHI AL-λ liver extract, or TAL ALk liver extract. Fig. 13D shows EuLISA data measuring the binding of VH9/VL/4-p5 to rVX6Wil fibrils, Per125 wtATTR extract, Ken ATTR extract, SHI AL-λ liver extract, or TAL ALk liver extract. Fig. 13E shows EuLISA data measuring the binding of c11-1F4, m111F4, or VH9/VL4 to rVX6Wil fibrils. Fig. 13F shows EuLISA data measuring VH6/VL3-p5 binding to Sno ATTR extract (dark gray circles) or Ken ATTR extract (light gray circles) and C11-1F4 binding to Sno ATTR extract (black squares). Fig. 13G shows EuLISA data measuring VH6/VL3-p5R binding to Per125 wtATTR (gray circles, see label), Sno ATTR extract (dark gray circles) or Ken ATTR extract (light gray circles) and C11-1F4 binding to Sno ATTR extract (black squares). The log-transformed molar concentration of the monoclonal antibody (-log(M)) is shown on the x-axis and the binding level (femtomoles europium) is shown on the y-axis.

Figure 14 shows the results of ¹²⁵ I-mIgp5 binding to rV^6Wil amyloid-like fibrils and human amyloid extracts obtained from tissues in an affinity adsorption assay. The y-axis shows the percentage of ¹²⁵ I-mIgG-p5 bound, and the percentage bound for each sample is indicated above the histogram bars. The x-axis shows the type of amyloid extract tested, including, from left to right, rV^6Wil fibrils (71% binding), SNO hereditary (h) ATTR (12% binding), KEN hATTR (15% binding), Per 125 wtATTR (31% binding), Per253 wild-type (WT) ATTR (17% binding), ALk HIG extract (10% binding), ALk TAL extract (37% binding), ALλ, SHI extract (34% binding), and ALλ, TYL extract (21% binding). Error bars represent standard deviation.

Figure 15A shows the uptake of pHrodo Red-labeled rV^6Wil fibrils by human THP-1 macrophages alone or in the presence of control human IgG (h), ch11-1F4, muIgp5 (produced in the expiHEK293 cell line), VH6/VL3-p5, or VH6/VL3-p5R, as indicated from left to right on the x-axis. The y-axis shows the uptake of rV^6Wil fibrils (measured in fluorescence units), and the error bars represent the standard deviation. Figure 15B shows the phagocytosis of pHrodo Red-labeled rV^6Wil fibrils by macrophages in the presence of THP-1 alone or with control human IgG, c11-1F4, muIgp5 (produced in the expiHEK293 cell line), VH6/VL3-p5R, and VH6/VL3-p5R.

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1F4, mIgp5, VH9/VL4-p5, or VH9/VL4-p5R, as indicated from left to right on the x-axis. The y-axis shows the phagocytosis rate (fluorescence units), and the error bars represent the standard deviation. Fig. 15C shows phagocytosis of pHrodo Red-labeled rV^6Wil fibrils by macrophages in the presence of hIgG control, 5 μg Rituxan (chimeric mAb as a negative control), 5 μg C11-1F4, 5 μg VH6/VL3, 5 μg VH9/VL4, VH6/VL3-p5R, or VH6/VL3-p5, as indicated from left to right on the x-axis. The y-axis shows the level of phagocytosis (pHrodo fluorescence) and the error bars represent the standard deviation.

Figure 16 shows the pharmacokinetic (PK) analysis of ^125I -labeled VH9/VL4 antibody administered intravenously to wild-type (WT) mice. The x-axis shows the time after administration in hours, and the y-axis shows the blood radioactivity level in counts per minute (cpm). A double exponential decay equation is used to fit the curves.

Fig. 17A shows the biodistribution of ¹²⁵ I-VH9/VL4-p5 in wild-type mice without amyloid deposits. Fig. 17B shows the biodistribution of ¹²⁵ I-VH9/VL4-p5R in wild-type mice without amyloid deposits. In both Fig. 17A and Fig. 17B, the x-axis indicates the organ sampled, including from left to right muscle, liver, pancreas, spleen, left kidney, right kidney, stomach, upper intestine, lower intestine, heart, lung, and blood; the y-axis indicates the biodistribution rate as a percentage of the administered dose per gram of tissue.

Figure 18 shows peptide capture ELISA data measuring the stability of VH6/VL3-p5 in mouse plasma. 100 nM VH6/VL3-p5 was added to either PBS at 4°C (darker gray circles), PBS at 37°C (middle dark gray circles), EDTA-anticoagulated plasma at 37°C (middle light gray circles), or heparin-anticoagulated plasma at 37°C (lightest gray circles). The x-axis indicates time after administration in days, and the y-axis indicates antibody-ligand binding in optical density units (AU).

Detailed description of the essence of the invention

Provided herein are modified immunoglobulins that bind amyloids. In some embodiments, the modified immunoglobulins are peptide-Ig fusions.

I. Definitions.

Unless otherwise indicated, technical terms are used according to their generally accepted usage. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); and Robert A Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710) and other similar sources. As used herein, the singular forms a, b, and c are intended to include the plural unless the context clearly dictates otherwise. The word for example is used herein to indicate a non-limiting example. Thus, the word for example is a synonym for the expression as an example. As used in this document, the term contains means includes.

Ranges may be stated herein as from about one specific value and/or to about another specific value. When such a range is expressed, the other aspect includes from one specific value and/or to another specific value. It will further be understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. Similarly, when values are expressed as approximate values, using the preceding word about, it will be understood that the specific value forms the other aspect. In certain exemplary embodiments, the term about is understood as within a range of normal tolerance in the art, for example, within 2 standard deviations of the mean. About or approximately can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context otherwise requires, all numerical values given herein may be modified by the term about. In addition, terms used herein such as example, illustrative, or exemplary are not intended to demonstrate a preference, but rather to explain that the aspect discussed below is simply one example of the aspect presented.

It is further understood that all base sizes or amino acid sizes and all molecular weights or molecular weights given for nucleic acids or polypeptides are approximate and are provided for description. It is further understood that all base sizes or amino acid sizes and all molecular weights or molecular weights given for nucleic acids or polypeptides are approximate and are provided for description. In case of conflict, this specification, including explanations of terms, will control. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

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To facilitate an overview of the various embodiments of the present invention, the following explanations of specific terms are provided:

Administration: Administering the composition to the subject by the selected route. For example, if the intravenous route is selected, the composition is administered by injecting the composition into the subject's vein. In some examples, peptides are administered to the subject.

The terms amyloids, amyloid deposits, amyloid fibrils, and amyloid fibers refer to insoluble fibrous protein aggregates that share common structural features. Protein aggregates have a tertiary structure, such as that formed by the aggregation of any of several different proteins, and consist 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 amyloids in organs can lead to amyloidosis. Although they are diverse in origin, all amyloids share the common morphological properties 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 ultrastructural features and common X-ray and infrared spectra.

Amyloidosis refers to a pathological condition or disease characterized by the presence of amyloids, such as the presence of amyloid deposits. Amyloid diseases or amyloidosis are diseases associated with the formation, deposition, accumulation, or retention of amyloid fibrils. Such diseases include, but are not limited to, Alzheimer's disease, Down syndrome, hereditary predisposition to cerebral hemorrhage with Dutch amyloidosis, and cerebral beta-amyloid angiopathy. Other amyloid diseases such as systemic AA amyloidosis, AL amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis of type II diabetes are also amyloid diseases.

The term amyloidogenic refers to the formation or propensity to form amyloid deposits. For example, some soluble monomeric proteins can undergo extensive conformational changes that cause them to aggregate into well-ordered, unbranched fibrils 8–10 nm wide, culminating in the formation of amyloid aggregates. For example, more than thirty proteins have been found to form amyloid deposits (or amyloids) in humans. Not all proteins in a diverse class of proteins, such as the immunoglobulin light chains, are capable of forming amyloids, i.e., some proteins are non-amyloidogenic, meaning that they do not have a tendency to form amyloids. However, other proteins in this class can form amyloid deposits and are thus amyloidogenic. In addition, within the class of light chain proteins, some may be considered more amyloidogenic than others based on their ability to readily form amyloid fibrils. Some light chain proteins are considered non-amyloidogenic or less amyloidogenic due to their inability to readily form amyloid fibrils in patients or in vitro.

Animal: Living multicellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both humans and non-human mammals. Similarly, the term subject includes both humans and veterinary subjects. In some examples, a subject is a subject, such as a subject suffering from an amyloid disease.

Clearance: The terms clear or clearance refer to a reduction or removal to a measurable degree. For example, clearance of amyloid deposits, as described herein, refers to a reduction or removal of deposits to a measurable or discernible degree. Clearance may result in 100% removal, but does not necessarily mean 100%. Rather, clearance may result in less than 100% removal, such as about 10%, 20%, 30%, 40%, 50%, 60%, or more removal.

Conjugate: As used herein, the term conjugate refers to the product of the linking or combination of two or more materials, and the resulting product has at least two distinct elements, such as at least two domains. The materials being joined may be the same or may be different. Such linking may be via one or more linking groups. For example, a protein conjugate is obtained by combining two or more amino acid sequences. A conjugate of two proteins, for example, results in a single protein that has a domain corresponding to each of the separately linked proteins.

An antibody refers to single-chain, double-chain, and multichain proteins and glycoproteins belonging to the classes of polyclonal, monoclonal, chimeric, and heteroimmunoglobulins (monoclonal antibodies are preferred); it also includes synthetic and genetically engineered versions of these immunoglobulins. An antibody fragment includes Fab, Fab', F(ab')2, and Fv fragments, as well as any portion of an antibody that has specificity for a desired target epitope or epitopes. A monoclonal antibody is an antibody produced by a single clone of B lymphocytes. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells with immune cells of the spleen.

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An epitope refers to a site on an antigen that is recognized by an antibody, which is determined by the specificity of the antibody's amino acid sequence. Epitopes are also called antigenic determinants. For example, an epitope may be part of a recombinant protein that is recognized by a particular antibody. Additionally, an epitope may be a conformational epitope and a linear epitope.

A chimeric antibody refers to an antibody that includes sequences derived from two different antibodies, usually from different species. Most often, chimeric antibodies include fragments of human and mouse antibodies, typically human constant regions and mouse variable regions.

A humanized antibody refers to an antibody derived from a non-human antibody, usually a mouse antibody, and a human antibody that retains or substantially retains the antigen-binding properties of the original antibody but is less immunogenic in humans.

The complementarity determining region or CDR refers to the amino acid sequences that together determine the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The immunoglobulin light and heavy chains each have three CDRs, designated L-CdRI, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. By definition, the light chain CDRs are linked by residues at positions 24 and 34 (L-CDR1), 50 and 56 (LCDR2), 89 and 97 (L-CDR3); The heavy chain CDRs are delimited by residues at positions 31 and 35b (H-CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3) using the numbering conventions established by Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda (NIH Publication No. 91-3242).

The framework region refers to the amino acid sequences located between the CDRs. These parts of the antibody hold the CDRs in the proper orientation to ensure antigen binding.

The specificity determining residue or SDR refers to the amino acid residues of an immunoglobulin that are directly involved in contact with the antigen.

The term constant region refers to the portion of an antibody molecule that provides effector functions. In the present invention, antibody variants include constant regions derived from human immunoglobulins. The constant region of the heavy chain may be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Heavy chains of different subclasses (e.g., the IgG heavy chain subclass) are responsible for different effector functions. Thus, by selecting the desired constant region of the heavy chain, humanized antibodies with the desired effector function can be obtained. The constant region of the light chain may be of the kappa or lambda type, preferably of the kappa type.

Effective amount or therapeutically effective amount: an amount of an agent sufficient to prevent, treat (including prophylaxis), reduce and/or alleviate the symptoms and/or underlying causes of any disorder or disease, such as preventing, inhibiting amyloidosis. In some embodiments, an effective amount is sufficient to reduce or eliminate a symptom of the disease. An effective amount can be administered one or more times. For example, an effective amount of a peptide is an amount sufficient to bind amyloid. The peptide can be effective, for example, when administered parenterally in amounts from about 1 μg per kg of body weight to about 30 mg/kg.

Expression control sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which they are operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, where appropriate, translation of the nucleic acid sequence. Thus, expression control sequences may include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) upstream of a protein-coding gene, a splicing signal for introns, maintenance of the correct reading frame of the gene to ensure proper translation of mRNA, and stop codons. The term regulatory sequences is intended to include, at a minimum, components whose presence may affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Expression control sequences may include a promoter.

A promoter is the minimal sequence sufficient to direct transcription. Also included are those promoter elements that are sufficient to make promoter-dependent gene expression cell-type-specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of a gene. Both constitutive and inducible promoters are included (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, inducible promoters such as lambda bacteriophage pL, plac, ptrp, ptac (a ptrp-lac hybrid promoter), and the like may be used in bacterial cloning systems. In one embodiment,

- 12 048681 promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the long terminal repeat of a retrovirus, the late promoter of an adenovirus, the 7.5 kDa promoter of a vaccinia virus) can be used for cloning in mammalian cell systems. Promoters obtained by recombinant DNA or synthetic methods can also be used to ensure transcription of nucleic acid sequences. The polynucleotide can be inserted into an expression vector containing a promoter sequence that promotes efficient transcription of the inserted host genetic sequence. The expression vector typically contains an origin of replication, a promoter, and specific nucleic acid sequences that provide phenotypic selection of transformed cells.

Inhibit: Reduce to a measurable degree. Inhibition, for example, does not require a complete loss of function or a complete cessation of the measurable aspect. For example, inhibiting plaque formation could mean stopping further plaque growth, slowing further plaque growth, or reducing plaque size.

Inhibition or treatment of a disease: Inhibition of the complete development of a disease or pathological condition, for example, inhibition of amyloidosis. The term treatment refers to a therapeutic intervention that alleviates a sign or symptom of a disease or pathological condition after it has begun to develop. The term alleviation, in relation to a disease or pathological condition, refers to any observable beneficial effect of the treatment. A beneficial effect can be evidenced, for example, by a delay in the onset of clinical symptoms of the disease in a susceptible subject, a decrease in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the general health or well-being of the subject, or other parameters well known in the art that are specific to a particular disease. Prophylactic treatment is a treatment that is administered to a subject that does not show signs of a disease or shows only early signs, in order to reduce the risk of developing a pathology.

With respect to the formation of amyloid deposits, inhibition refers to preventing a decrease in the formation of amyloid deposits, for example, compared to a control. For example, inhibition may result in a decrease of about 10%, 20%, 30%, 40%, 50%, 60% or more in amyloid deposits compared to a control.

A label refers to any detectable compound or composition that is directly or indirectly conjugated to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent labels, chemiluminescent labels, haptens, enzymatic linkages, and radioactive isotopes. A protein that is detectably labeled, for example, means that the presence of the protein can be determined by the label associated with the protein.

Isolated: An isolated biological component, such as a peptide (e.g., one or more peptides described herein), cell samples, nucleic acids, or serum, has been substantially separated, isolated, or purified from other biological components in a cell of the organism in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins. Thus, nucleic acids, peptides, and proteins that have been isolated include nucleic acids and proteins that have been purified by standard purification techniques. The term also includes nucleic acids, peptides, and proteins produced by recombinant expression in a cell, as well as chemically synthesized peptides and nucleic acids. The term isolated or purified does not require absolute purity; rather, it is a relative term. Thus, for example, an isolated peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein in its natural environment within the cell. Preferably, the preparation is purified such that the protein or peptide constitutes at least 50% of the total peptide or protein content of the preparation, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even at least 99% of the peptide or protein concentration.

Linking: As used herein, the term linking, linked, connected, or associated refers to any method known in the art for functionally linking proteins and/or protein domains. For example, one protein domain can be linked to another protein domain by a covalent bond, such as in a recombinant fusion protein, with or without intervening sequences or domains. The term linked also includes, for example, joining two sequences together, such as placing two nucleic acid sequences together on the same nucleic acid strand such that the sequences are expressed together.

Nucleic acid: A polymer consisting of nucleotide units (ribonucleotides, deoxyribonucleotides, related natural structural variants and their synthetic non-natural analogues) linked by phosphodiester bonds, related natural structural variants and their

- 13 048681 synthetic non-natural analogues. Thus, this term includes nucleotide polymers in which the nucleotides and the linkages therebetween include non-natural synthetic analogues such as, for example, but not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNA), etc. Such polynucleotides can be synthesized, for example, using an automatic DNA synthesizer. The term oligonucleotide generally refers to short polynucleotides, usually not exceeding about 50 nucleotides. It should be understood that when the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C), in which U replaces T.

A nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine, or their synthetic analogs, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

To describe nucleotide sequences, this document uses conventional notations: the left end of a single-stranded nucleotide sequence is the 5' end; the leftward direction of a double-stranded nucleotide sequence is called the 5' direction. The direction of addition of nucleotides from the 5' to the 3' end of nascent RNA transcripts is called the direction of transcription. The strand of DNA that has the same sequence as the mRNA is called the coding strand; the sequences on the DNA strand that have the same sequence as the mRNA transcribed from it and that are 5' to the 5' end of the RNA transcript are called upstream sequences; the sequences on the DNA strand that have the same sequence as the RNA and that are 3' to the 3' end of the coding RNA transcript are called downstream sequences.

cDNA refers to DNA that is complementary or identical to mRNA in single-stranded or double-stranded form.

The term coding refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes that have a specific nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a specific amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA produced by the gene results in the formation of the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to that of the mRNA and is usually provided in sequence listings, and the noncoding strand used as a template for transcription of a gene or cDNA, may be considered to encode a protein or other product of that gene or cDNA. Unless otherwise specified, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

The term recombinant nucleic acid refers to a nucleic acid having nucleotide sequences that are not naturally associated with one another. This includes nucleic acid vectors, such as adenovirus vectors, which contain amplified or assembled nucleic acid that can be used to transform a suitable host cell. A host cell containing a recombinant nucleic acid is called a recombinant host cell. A gene is then expressed in the recombinant host cell to produce, for example, a recombinant polypeptide. The recombinant nucleic acid may also serve a non-coding function (such as a promoter, origin of replication, ribosome binding site, etc.). A first sequence is antisense to a second sequence if the polynucleotide whose sequence is the first sequence specifically hybridizes to the polynucleotide whose sequence is the second sequence.

Pharmaceutically Acceptable Carriers: Pharmaceutically acceptable carriers that may be used are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, ^19th Edition (1995) describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins described herein.

In general, the nature of the carrier will depend on the particular route of administration employed. For example, parenteral formulations typically contain injection liquids that include pharmaceutically and physiologically acceptable liquids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like as a carrier. For solid compositions (e.g., in the form of a powder, pill, tablet, or capsule), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutically acceptable carriers may include

- 14 048681 pharmaceutical compositions may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents, etc., for example, sodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, with L-isomers being preferred. The terms polypeptide or protein as used herein encompass any amino acid sequence and include modified sequences such as glycoproteins. The term polypeptide is specifically intended to refer to naturally occurring proteins as well as proteins produced recombinantly or synthetically. In some examples, a peptide is one or more peptides described herein.

Purified: The term purified does not require absolute purity; rather, it is a relative term. Thus, for example, a purified protein preparation is one in which the protein in question is purer than the protein in its natural environment inside a cell or inside a manufacturing reaction chamber (as appropriate).

Recombinant: A recombinant nucleic acid is a nucleic acid that has a sequence that does not occur in nature, or has a sequence created by the artificial combination of two otherwise separated sequence segments. This artificial combination is often produced by chemical synthesis or, more commonly, by artificial manipulation of isolated nucleic acid segments, such as by genetic engineering techniques.

Sequence identity: The similarity between two nucleic acid sequences or two amino acid sequences is expressed in terms of the similarity between the sequences, otherwise called sequence identity. Sequence identity is often measured in terms of percent identity (or similarity, or homology); the higher the percentage, the more similar the two sequences are.

Methods for aligning sequences for comparison are well known in the art. Various alignment programs and 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), which provides a detailed discussion of sequence alignment techniques and homology calculations.

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 Biological Information (NCBI, Bethesda, MD) and on the Internet, for use with the sequence analysis programs blastp, blasm, blastx, tblastn, and tblastx.

Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, where necessary, to connect two protein-coding regions in the same reading frame.

Pharmaceutical agent: A chemical compound or composition capable of producing a desired therapeutic or prophylactic effect when properly administered to a subject or cell.

Vector: A nucleic acid molecule introduced into a host cell to form a transformed host cell. Recombinant DNA vectors are vectors containing recombinant DNA. The vector may include nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses. The term vector includes plasmids, linear nucleic acid molecules, and, as described elsewhere, adenoviral vectors and adenoviruses.

The subject is a vertebrate. The vertebrate may be a mammal, such as a human. The subject may be a human patient. The subject may be a patient suffering from a disease or condition or suspected of having a disease or condition and may be in need of treatment or diagnosis or may be in need of monitoring for progression of the disease or condition. The patient may also be receiving a therapeutic treatment whose effectiveness needs to be monitored. In some illustrations,

- In certain embodiments, the subject comprises a subject having amyloidosis, such as Alzheimer's disease, Huntington's disease, or prion diseases, or peripheral amyloidosis, such as that seen in patients with light chain amyloidosis (AL) and type 2 diabetes.

The terms treatment or treatment refer to a therapeutic intervention that alleviates a sign or symptom of a disease or pathological condition after it has begun to develop. The term alleviation, when applied to a disease or pathological condition, refers to any observable beneficial effect of the treatment. A beneficial effect may be evidenced, for example, by a delay in the onset of clinical symptoms of the disease in a susceptible subject, a decrease in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the general health or well-being of the subject, or other parameters well known in the art that are specific to the particular disease. Prophylactic treatment is a treatment that is administered to a subject that does not show signs of a disease or shows only early signs, in order to reduce the risk of developing a pathology.

Preferably, non-identical residue positions are distinguished by conservative amino acid substitutions. The term conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains are glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic hydroxyl side chains are serine and threonine; a group of amino acids having amide-containing side chains are asparagine and glutamine; a group of amino acids having aromatic side chains are phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains are lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-partic acid, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are considered to be encompassed by the present invention, so long as the variations in the amino acid sequence are conserved by at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are provided. Conservative substitutions are substitutions that occur within a family of amino acids linked in their side chains. Genetically encoded amino acids are typically divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) Non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, 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) serine and threonine, which belong to the family of aliphatic hydroxyl-containing amino acids; (ii) asparagine and glutamine, which belong to the amide-containing amino acid family; (iii) alanine, valine, leucine, and isoleucine, which belong to the aliphatic amino acid family; and (iv) phenylalanine, tryptophan, and tyrosine, which belong to the aromatic amino acid family. For example, it is reasonable to expect that the isolated substitution of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or a similar substitution of an amino acid with a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not affect an amino acid in a framework site. It can be readily determined whether an amino acid change results in a functional peptide by assaying the specific activity of the derived polypeptide. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino and carboxyl termini of the fragments or analogs are near the boundaries of the functional domains. Structural and functional domains can be identified by comparing nucleotide and/or amino acid sequence data to publicly available or proprietary sequence databases. It is preferred to use computerized comparison methods to identify sequence motifs or domains of predicted protein conformation that occur in other proteins of known structure and/or function. Methods are known for identifying protein sequences that fold into a known three-dimensional structure. (Bowie et al. Science 253:164 (1991). Thus, the above examples demonstrate that those skilled in the art can determine sequence motifs and structural conformations that can be used to determine structural and functional domains according to the invention.

Preferred amino acid substitutions are those that: (1) reduce sensitivity

- 16 048681 proteolytic activity, (2) reduce sensitivity to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (4) impart or modify other physicochemical or functional properties of such analogs. Analogs may include various muteins with a sequence different from the natural peptide sequence. For example, single or multiple amino acid substitutions (preferably, conservative amino acid substitutions) can be made in a native sequence (preferably, in a portion of the polypeptide outside the domain(s) that form intermolecular contacts. A conservative amino acid substitution should not substantially alter the structural characteristics of the original sequence (e.g., the substituting amino acid should not result in the disruption of a helix that occurs in the original sequence or in the disruption of other types of secondary structure that characterize the original sequence). Examples of art-recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

With the exception of CDR1 in VH, CDRs typically contain amino acid residues that form hypervariable loops. CDRs also contain specificity-determining residues, or SDRs, which are the residues that contact the antigen. SDRs are contained in regions of the CDRs, called CDRs or a-CDRs for short. Examples of a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, aCDR-H2, and a-CDR-H3) correspond to amino acid residues 31–34 L1, 50–55 L2, 89–96 L3, 31–35B H1, 50–58 H2, and 95–102 H3. (See Almagro and Fransson, Front. Biosci. 13: 1619–1633 (2008).)

The framework or FR refers to the residues of the variable domain other than the CDR residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences typically appear in the following sequence in VH (or VL): FR1 - H1(L1) - FR2 - H2(L2) - FR3 - H3(L3) - FR4; or FR1 - CDR-H1(L1) - FR2 - CDR-H2(L2) - FR3 CDR3-H3(L3) - FR4.

II. Modified immunoglobulins and antibody-peptide fusion proteins A. Modified immunoglobulins

In some exemplary embodiments, modified immunoglobulins that target amyloids are provided. Such modified immunoglobulins include, for example, amyloid-reactive peptides that are attached to an immunoglobulin (Ig), for example, by extending the N-terminus of an Ig light chain protein in a fragment, an antigen-binding region (Fab), or through the C-terminus of a heavy chain, thereby forming a peptide-Ig fusion. The modified immunoglobulins can be used to treat a subject suffering from amyloidosis, for example, by administering the modified immunoglobulins to the subject. In some embodiments, the modified immunoglobulin is a fusion protein comprising an antibody attached to an amyloid-reactive peptide.

In some embodiments, the modified immunoglobulin comprises an antibody attached to a peptide. In some embodiments, the modified immunoglobulin comprises an antibody that comprises one, two, three, four, five, or six CDRs of antibody 11-1F4. In some embodiments, the antibody comprises a VH and/or VL of antibody 11-1F4. In some embodiments, the antibody comprises a heavy chain and/or a light chain of antibody 11-1F4, wherein the antibody is attached to a peptide.

In a specific embodiment, the modified immunoglobulin comprises an antibody, wherein the antibody comprises a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, wherein the antibody is attached to a peptide.

In a specific embodiment, the modified immunoglobulin comprises an antibody, wherein the antibody comprises a VL that comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, wherein the antibody is attached to a peptide.

In one embodiment, the modified immunoglobulin comprises an antibody that comprises a VL comprising the amino acid sequence of SEQ ID NO: 16 and a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the antibody is attached to a peptide.

In another aspect, a modified immunoglobulin comprises an antibody, wherein said antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence of SEQ

- 17 048681

ID NO: 22, and wherein the antibody is attached to a peptide. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In another aspect, a modified immunoglobulin comprises an antibody, wherein the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH having the sequence set forth in SEQ ID NO: 15, and a VL CDR1, VL CDR2, of a VL having the sequence set forth in SEQ ID NO: 16; and wherein the antibody is attached to a peptide. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the modified immunoglobulin comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide. In some embodiments, the modified immunoglobulin comprises a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 without the C-terminal lysine residue and a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the antibody is attached to a peptide.

In another aspect, a modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified immunoglobulins comprise an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is attached to the N-terminus of the antibody light chain or to the C-terminus of the heavy chain. In some embodiments, the antibody also comprises a spacer amino acid sequence between the peptide and the N-terminus of the light chain or the C-terminus of the heavy chain. In some embodiments, the peptide is attached to the N-terminus of the light chain. In some embodiments, the peptide is attached to the N-terminus of the heavy chain.

In some embodiments, the modified immunoglobulin comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the modified immunoglobulins comprise an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified immunoglobulin comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the modified immunoglobulin comprises an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the modified immunoglobulin comprises an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified immunoglobulins comprise an antibody whose light chain comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the modified immunoglobulin comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO:

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NO: 16, wherein the light chain is attached to a peptide comprising any of the amino acid sequences from Table 1.

In some embodiments, the modified immunoglobulin comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide is attached to the light chain at the N-terminus.

In some embodiments, the modified immunoglobulin comprises an antibody, the heavy chain of which comprises a VH comprising the amino acid sequence of SEQ ID NO: 15, and the light chain of which comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is attached to the light chain at the N-terminus.

In some embodiments, the modified immunoglobulins described herein bind to amyloid deposits or fibrils. In some embodiments, the modified immunoglobulin binds to one or more amyloidogenic peptides in amyloids. In some embodiments, the amyloids associated with the modified immunoglobulins comprise amyloidogenic λ6 variable domain protein (V/.6Wil) or amyloidogenic immunoglobulin light chain (AL), AB(1-40) amyloid-like fibril or amyloidogenic AB precursor protein, or serum amyloid A protein (AA). In other embodiments, the amyloids associated with the modified immunoglobulin include amyloidogenic forms of immunoglobulin heavy chain (AH), _β2 -microglobulin ( _Ab2M ), transthyretin variants (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen a variants (AFib), cystatin variants (ACys), calcitonin (ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP), α-synuclein (AaSyn), Tay (ATau), atrial natriuretic factor (AANF) or IAAP, ALk4, AR1, other amyloidogenic peptides. The amyloidogenic peptides associated with the modified immunoglobulins may be a protein, a protein fragment, or a protein domain. In some embodiments, the amyloid deposits or amyloid fibrils comprise recombinant amyloidogenic proteins. In some embodiments, the amyloids are part of the disease pathology.

As understood by those skilled in the art, the antigen-binding fragment (or Fab region) is the head portion of an antibody that naturally interacts with a target antigen. Components of the Fab region, for example, allow antibodies to bind to specific ligands and, through this 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, consisting of 2 heavy chains and 2 light chains. Both the heavy and light chains are further subdivided into variable domains and constant domains - the light and heavy variable domains comprising the functional Fab region, and the heavy chains forming the crystallizable fragment (Fc) domains that interact with cellular receptors and complement. The Fc regions of Ig carry a highly conserved N-glycosylation site.

In certain exemplary embodiments, one or more of the peptides listed in Table 1 below can be linked to an Ig antibody or a functional fragment thereof via the N-terminus of a light chain protein or the C-terminus of a heavy chain, thereby forming a modified immunoglobulin. Thus, any of the sequences listed in Table 1 below can be linked to a heavy or light chain of an Ig antibody or a functional fragment thereof independently or simultaneously to form a peptide-Ig conjugate. For example, two amyloid-reactive peptides can be linked to a single Ig antibody, such as by linking the amino acid sequences of the amyloid-reactive peptide to the N-terminus of the Ig light chain proteins.

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Table 1

Examples of amyloid-reactive peptide sequences

PEPTIDE PRIMARY SEQUENCE: SEQ ID NO P5 KAQKA QAKQA KQAQK AQKAQ AKQAK Q SEQ Ш NO: 1 P5R RAQRA QARQA RQAQR AQRAQ ARQAR Q SEQ Ш NO: 2 P5G GAQGA QAGQA GQAQG AQGAQ AGQAG Q SEQ Ш NO: 3 P8 KAKAK AKAKA K A K A K SEQ Ш NO: 4 P9 KAQAK AQAKA QAKAQ AKAQA KAQAK AQAK SEQ Ш NO: 5 P19 KAQQA QAKQA QQAQK AQQAQ AKQAQ Q SEQ Ш NO: 6 P20 QAQKA QAQQA KQAQQ AQKAQ AQQAK Q SEQ Ш NO: 7 P31 KAQKA QAKQA KQAQK AQKAQ AKQAK Q SEQ Ш NO: 8 P37 KTVKT VTKVT KVTVK TVKTV TKVTK V SEQ Ш NO: 9 P42 VYKVK TKVKT KVKTK VKT SEQ Ш NO: 10 P43 AQAYS KAQKA QAKQA KQAQK AQKAQ AKAK Q SEQ Ш NO: 11 P44 AQAYA RAQRA QARQA RQAQR AQRAQ ARQAR Q SEQ Ш NO: 12 P5+14 KAQKA QAKQA KQAQK AQKAQ AKQAK QAQKA QKAQA KQAKQ SEQ Ш NO: 13 P5R+14 RAQRA QARQA RQAQR AQRAQ ARQAR QAQRA QRAQ A RQARQ SEQ Ш NO: 14

Without wishing to be bound by any particular theory, it is believed that the peptide domain of the peptide-Ig conjugate, when administered to a subject, targets the modified immunoglobulin to amyloid deposits. The Fc domain then triggers an immune response at the site of the amyloid, resulting in the removal of the amyloid, such as by opsonization. In addition, the modified immunoglobulin is believed to have a longer half-life than amyloid-reactive peptides alone. For example, the half-life of circulating human IgG is approximately 21 days, whereas the half-life of amyloid-reactive peptide alone in humans is approximately 11 hours. Thus, the Ig increases the half-life of the modified immunoglobulin in the circulation. In some exemplary embodiments, contacting the amyloid deposit with the modified immunoglobulin results in an increase in half-life by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to contacting the amyloid deposit with only the amyloid-reactive peptide. Thus, the modified immunoglobulin, when administered to a subject, can exert an immunostimulatory effect at the site of the amyloid deposit for a longer period of time, thereby enhancing the immune response at the site of the amyloid deposit.

In some embodiments, the amyloid-reactive peptides of the modified immunoglobulin peptides described herein comprise an amino acid sequence that has at least 80%, 85%, 90%, or more identity to the amino acid sequence set forth in any of SEQ ID NOs: 1-14, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any of SEQ ID NOs: 1-14. In some embodiments, the amyloid-reactive peptides associated with an Ig antibody or functional fragments thereof may comprise or consist of from about 10 to about 55 amino acids. The amyloid-reactive peptides of the present invention may, for example, comprise 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, which is incorporated herein in its entirety.

The amino acids that form all or part of the amyloid-reactive peptides associated with the Ig antibody or fragment thereof may be stereoisomers and modifications of natural amino acids, unnatural amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. The amino acids that form the peptides of the present invention may be one or more of the 20 common amino acids found in natural proteins, or one or more modified and unusual amino acids. The modified immunoglobulin may be produced by any method known to those skilled in the art, including chemical synthesis or recombinant methods using standard molecular biology techniques.

In some embodiments, recombinant DNA technology may be used, in which a nucleotide sequence encoding a peptide of the invention is cloned, fused

- 20 048681 with an Ig light chain into an expression vector, transformed or transfected into a suitable host cell and cultured under conditions suitable for expression (see Examples). The peptide-Ig light chain hybrid is then isolated. Advantageously, and as those skilled in the art will understand in connection with this disclosure, the methods described herein can be used to attach any peptide sequence to an Ig antibody. That is, although amyloid-reactive peptides are used as an example of a peptide attached to an Ig antibody, the method of attaching a peptide to an Ig antibody, such as to the N-terminal end of an Ig light chain protein and/or the N- and/or terminal end of an Ig heavy chain protein, can be used for a variety of different peptides to attach a peptide to Ig antibodies.

In some exemplary embodiments, several identical or different peptides may be attached to a single Ig antibody or functional fragment thereof. For example, a first expression vector may comprise a light chain nucleic acid sequence integrated with a nucleic acid sequence encoding peptide A, wherein the nucleic acid sequence for peptide A is positioned in the vector such that peptide A is expressed as attached to the N-terminus of a light chain protein. Furthermore, a second expression vector may comprise a heavy chain nucleic acid sequence integrated with a nucleic acid sequence encoding peptide B, wherein the nucleic acid sequence for peptide B is positioned in the vector such that peptide B is expressed as attached to the N-terminus of a light chain protein.

In such exemplary embodiments, when both expression vectors are expressed in the same cell, the resulting Ig protein may have a single peptide A sequence at the N-terminus of each light chain (for a total of two A peptides) and a peptide B at the N-terminus of the heavy chain. In certain exemplary embodiments, the vector may include a peptide C at the C-terminal end, resulting in an antibody having two peptide A sequences (one on each light chain), a peptide B sequence at the N-terminus of the heavy chain, and a peptide C sequence attached to the C-terminal end of the heavy chain. As such, and as will be understood by one of skill in the art based on this invention, the expression vectors may be adapted to modify the immunoglobulin to have the same or different combinations of proteins. As a specific example of the use of an amyloid-reactive peptide, the modified immunoglobulin may include two p5 protein sequences (SEQ ID NO: 1), i.e. one at the N-terminal end of each light chain. In other exemplary embodiments, the peptides attached to the immunoglobulin may have affinity for the ligand and therefore may be used to detect the ligand.

In some embodiments, the modified immunoglobulins may be obtained by isolation or purification. Protein purification methods include, at one level, homogenization and crude fractionation of cells, tissues, or organs into peptide and non-peptide fractions. Other protein purification methods include, for example, precipitation with ammonium sulfate, polyethylene glycol (PEG), antibodies, and the like, or heat denaturation followed by: centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite, and affinity chromatography; isoelectric focusing; gel electrophoresis, such as polyacrylamide gel electrophoresis; and combinations of these and other methods.

Various chromatographic methods include, but are not limited to, ion exchange chromatography, gel exclusion chromatography, affinity chromatography, immunoaffinity chromatography, and reversed phase chromatography. A particularly effective method for purifying peptides is fast-flow liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC). In some embodiments, the Fc domain can be attached to the amyloid-reactive peptide via a GGGYS linker sequence (SEQ ID NO: 27).

In certain embodiments, the modified immunoglobulins may include amino acid spacer sequences between the N-terminus of the light chain or the C-terminus of the heavy chain and the amyloid-reactive peptide. In certain embodiments, the peptide-Ig conjugates may include amino acid spacer sequences between the N-terminus of the peptide and the leader sequence necessary for secretion of the Ig peptide by cells expressing the reagent. In some embodiments, the spacer peptide may comprise or consist of from about 3 to about 55 amino acids. The spacer peptides of the present invention may comprise 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. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. For example, two nucleic acid sequences may be positioned adjacent to each other as described herein, but still include an intervening spacer sequence.

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B. Antibody-peptide fusion proteins

Also provided herein are antibody-peptide fusion proteins that target amyloids. Such antibody-peptide fusion proteins include, for example, amyloid-reactive peptides that are attached to immunoglobulin (Ig), for example, by extending the N-terminus of an Ig light chain protein in a fragment of the antigen-binding region (Fab) or through the C-terminus of a heavy chain, thereby forming a peptide-Ig fusion. The antibody-peptide fusion proteins can be used to treat a subject suffering from amyloidosis, for example, by administering the antibody-peptide fusion proteins to the subject.

In some embodiments, the antibody-peptide fusion protein comprises an antibody attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises an antibody that comprises one, two, three, four, five, or six CDRs of an antibody, as shown in Table 3.

In a specific embodiment, the antibody-peptide fusion protein comprises an antibody, wherein the antibody comprises a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, wherein the antibody is attached to a peptide.

In a particular embodiment, the antibody-peptide fusion protein comprises an antibody, wherein the antibody comprises a VL that comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, wherein the antibody is attached to a peptide.

In one embodiment, the antibody-peptide fusion protein comprises an antibody that comprises a VL comprising the amino acid sequence of SEQ ID NO: 16 and a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the antibody is attached to a peptide.

In another aspect, an antibody-peptide fusion protein comprises an antibody, wherein the antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and wherein the antibody is linked to a peptide. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In another aspect, an antibody-peptide fusion protein comprises an antibody, wherein said antibody comprises a VH CDR1, a VH CDR2, and a VH CDR3 of a VH having the sequence set forth in SEQ ID NO: 15, and a VL CDR1, a VL CDR2, of a VL having the sequence set forth in SEQ ID NO: 16; and wherein the antibody is attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises an antibody attached to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 without the C-terminal lysine residue and a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the antibody is attached to a peptide.

In another aspect, an antibody-peptide fusion protein comprises an antibody fused to an amyloid-reactive peptide. In some embodiments, the antibody-peptide fusion protein comprises an antibody fused to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody fused to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody fused to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is fused to the N-terminus of the light chain of the antibody or to the C-terminus of the heavy chain. In some embodiments

- 22 048681 embodiments, the antibody also comprises a spacer amino acid sequence between the peptide and the N-terminus of the light chain or the C-terminus of the heavy chain. In some embodiments, the peptide is attached to the N-terminus of the light chain.

In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is linked to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is linked to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises an antibody whose light chain comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is linked to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1.

In some embodiments, the antibody-peptide fusion protein comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide is attached to the light chain at the N-terminus.

In some embodiments, an antibody-peptide fusion protein comprises an antibody whose heavy chain comprises a VH comprising the amino acid sequence of SEQ ID NO: 15 and whose light chain comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is attached to the light chain at the N-terminus.

In some embodiments, the antibody-peptide fusion proteins described herein bind to amyloid deposits or fibrils. In some embodiments, the antibody-peptide fusion protein binds to one or more amyloidogenic peptides in amyloids. In some embodiments, the amyloids bound by the antibody-peptide fusion proteins comprise amyloidogenic λ6 variable domain protein (V/.6Wil) or amyloidogenic immunoglobulin light chain (AL), amyloid-like fibril αb(1-40) or amyloidogenic precursor protein αb, or serum amyloid A protein (AA). In other embodiments, the amyloids associated with the antibody-peptide fusion protein include amyloidogenic forms of immunoglobulin heavy chain (AH), _β2 -microglobulin ( _Ab2M ), transthyretin variants (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen a variants (AFib), cystatin variants (ACys), calcitonin (ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP), α-synuclein (AaSyn), Tau (ATau), atrial natriuretic factor (AANF) or IAAP, ALk4, AR1, others amyloidogenic peptides. The amyloidogenic peptides linked by the antibody-peptide fusion proteins may be a protein, a fragment of a protein, or a domain of a protein. In some embodiments, the amyloid deposits or amyloid fibrils comprise recombinant amyloidogenic proteins. In some embodiments, the amyloids are part of the disease pathology.

In some embodiments, the antibodies provided herein specifically bind to amyloid fibrils of light chains. In some embodiments, the amyloid-reactive peptide binds to various amyloid fibrils, such as amyloidogenic variable domain protein λ6 (V/.6Wil) or amyloidogenic immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibril or amyloidogenic precursor protein Aβ, or serum

- 23 048681 oral amyloid protein A (AA). In other embodiments, the amyloids associated with the antibody-peptide fusion protein include amyloidogenic forms of immunoglobulin heavy chain (AH), β2-microglobulin (Ab2M), transthyretin variants (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen a variants (AFib), cystatin variants (ACys), calcitonin (ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP), α-synuclein (AaSyn), Tau (ATau), atrial natriuretic factor (AANF) or IAAP, ALk4, AlX1, others amyloidogenic peptides. In some embodiments, the amyloid-reactive peptide binds to heparan sulfate glycosaminoglycans.

As understood by those skilled in the art, the antigen-binding fragment (or Fab region) is the head portion of an antibody that naturally interacts with a target antigen. Components of the Fab region, for example, allow antibodies to bind to specific ligands and, through this 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, consisting of 2 heavy chains and 2 light chains. Both the heavy and light chains are further subdivided into variable domains and constant domains - the light and heavy variable domains comprising the functional Fab region, and the heavy chains forming the crystallizable fragment (Fc) domains that interact with cellular receptors and complement. The Fc regions of Ig carry a highly conserved N-glycosylation site.

In certain exemplary embodiments, one or more of the peptides listed in Table 1 below can be linked to an Ig antibody or functional fragment thereof via the N-terminus of a light chain protein or the C-terminus of a heavy chain protein, thereby forming an antibody-peptide fusion protein. That is, any of the sequences listed in Table 1 below can be linked to the heavy or light chain of an Ig antibody or functional fragment thereof independently or simultaneously to form a peptide-Ig conjugate. For example, two amyloid-reactive peptides can be linked to a single Ig antibody, such as by linking the amino acid sequences of the amyloid-reactive peptides to the N-terminus of the Ig light chain proteins.

Without wishing to be bound by any particular theory, it is believed that the peptide domain of the peptide-Ig conjugate, when administered to a subject, targets the antibody-peptide fusion protein to amyloid deposits. The Fc domain then triggers an immune response at the site of the amyloid, resulting in the removal of the amyloid, such as by opsonization. In addition, the antibody-peptide fusion protein is believed to have a longer half-life than amyloid-reactive peptides alone. For example, the half-life of circulating IgG in humans is approximately 21 days, whereas the half-life of amyloid-reactive peptide alone in humans is approximately 11 hours. Thus, Ig increases the half-life of the antibody-peptide fusion protein in the circulation. In some embodiments, contacting the amyloid deposit with the antibody-peptide fusion protein results in an increase in half-life by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to contacting the amyloid deposit with only the amyloid-reactive peptide. Thus, the antibody-peptide fusion protein, when administered to a subject, can exert its immunostimulatory effects at the site of the amyloid deposit for a longer period of time, thereby enhancing the immune response at the site of the amyloid deposit.

In some embodiments, the amyloid-reactive peptides of the antibody-peptide fusion proteins described herein comprise an amino acid sequence that has at least 80%, 85%, 90%, or more identity to an amino acid sequence set forth as any of SEQ ID NOs: 1-14, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in any of SEQ ID NOs: 1-14. In some embodiments, the amyloid-reactive peptides linked to an Ig antibody or functional fragments thereof may comprise or consist of from about 10 to about 55 amino acids. The amyloid-reactive peptides of the present invention may, for example, comprise 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, which is incorporated herein in its entirety.

The amino acids that form all or part of the amyloid-reactive peptides associated with an Ig antibody or fragment thereof may be stereoisomers and modifications of naturally occurring amino acids, unnatural amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. The amino acids that form the peptides of the present invention may be one or more of the 20 common amino acids found in natural proteins, or one or more modified and unusual amino acids. If

- 24 048681 this antibody-peptide protein can be obtained by any method known to those skilled in the art, including chemical synthesis or recombinant methods using standard molecular biology techniques.

In certain exemplary embodiments, recombinant DNA technology may be used, wherein a nucleotide sequence encoding a peptide of the invention is cloned, fused to an Ig light chain in an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression (see the Examples). The peptide-Ig light chain hybrid is then isolated. Advantageously, and as will be understood by those skilled in the art in view of this disclosure, the methods described herein can be used to attach any peptide sequence to an Ig antibody. That is, although amyloid-reactive peptides are used as an example of a peptide attached to an Ig antibody, the method of attaching a peptide to an Ig antibody, such as to the N-terminal end of an Ig light chain protein and/or the N- and/or C-terminal end of an Ig heavy chain protein, can be used for a variety of different peptides to attach a peptide to Ig antibodies.

In some exemplary embodiments, several identical or different peptides may be attached to a single Ig antibody or functional fragment thereof. For example, a first expression vector may comprise a light chain nucleic acid sequence integrated with a nucleic acid sequence encoding peptide A, wherein the nucleic acid sequence for peptide A is positioned in the vector such that peptide A is expressed as attached to the N-terminus of a light chain protein. Furthermore, a second expression vector may comprise a heavy chain nucleic acid sequence integrated with a nucleic acid sequence encoding peptide B, wherein the nucleic acid sequence for peptide B is positioned in the vector such that peptide B is expressed as attached to the N-terminus of a light chain protein.

In such exemplary embodiments, where both expression vectors are expressed in the same cell, the resulting Ig protein may have one peptide A sequence at the N-terminus of each light chain (for a total of two peptide A's) and a peptide B at the N-terminus of the heavy chain. In certain exemplary embodiments, the vector may include a peptide C at the C-terminal end, resulting in an antibody having two peptide A sequences (one on each light chain), a peptide B sequence at the N-terminal end of the heavy chain, and a peptide C sequence attached to the C-terminal end of the heavy chain. As will be apparent from this disclosure, and as will be appreciated by one of skill in the art based on this invention, expression vectors may be adapted to modify an immunoglobulin to have the same or different combinations of proteins. As a specific example of the use of an amyloid-reactive peptide, an antibody-peptide fusion protein may include two p5 protein sequences (SEQ ID NO: 1), i.e. one at each N-terminal end of the light chain. In other exemplary embodiments, the peptides attached to the immunoglobulin may have affinity for the ligand and therefore may be used to detect the ligand.

In certain exemplary embodiments, the antibody-peptide fusion protein can be obtained by isolation or purification. Methods for purifying proteins include, at one level, homogenization and crude fractionation of cells, tissues, or organs into peptide and non-peptide fractions. Other methods for purifying proteins include, for example, precipitation with ammonium sulfate, polyethylene glycol (PEG), antibodies, and the like, or heat denaturation followed by: centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite, and affinity chromatography; isoelectric focusing; gel electrophoresis, such as polyacrylamide gel electrophoresis; and combinations of these and other methods.

Various chromatographic methods include, but are not limited to, ion exchange chromatography, gel exclusion chromatography, affinity chromatography, immunoaffinity chromatography, and reverse phase chromatography. A particularly effective method for purifying peptides is fast-flow liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC). In certain embodiments, the Fc domain can be attached to the amyloid-reactive peptide via a GGGYS linker sequence (SEQ ID NO: 27).

In certain embodiments, the antibody-peptide fusion protein may include amino acid spacer sequences between the N-terminus of the light chain or the C-terminus of the heavy chain and the amyloid-reactive peptide. In certain embodiments, the peptide-Ig conjugates may include amino acid spacer sequences between the N-terminus of the peptide and the leader sequence necessary for secretion of the Ig peptide by cells expressing the reagent. In some embodiments, the spacer peptide may comprise or consist of from about 3 to about 55 amino acids. The spacer peptides of the present invention may comprise 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. As used herein,

- 25 048681 document, a nucleic acid sequence or amino acid sequence is contiguous with 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 include an intervening spacer sequence.

III. Humanized antibodies that bind to human amyloid fibrils.

Provided herein are humanized antibodies that bind to human amyloid fibrils. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH) comprising one or more CDRs of a murine antibody. In some embodiments, the VH and/or VL are derived from human VH and/or VL sequences (e.g., human acceptor sequences). In some embodiments, the VH and/or VL comprise amino acid substitutions, such as in the VH and/or VL framework regions.

A. Humanized antibodies.

In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises one, two, three, four, five, or six CDRs of the antibody, as shown in Table 3. In some embodiments, the humanized antibody comprises a CDR-H1, CDR-H2, and CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 from a VH having the sequence set forth in SEQ ID NO: 15; and CDR-L1, CDR-L2 and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 16. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20 with one or more conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21 with one or more conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22 with one or more conservative amino acid substitutions, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, with one or more conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, with one or more conservative amino acid substitutions, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19, with one or more conservative amino acid substitutions. In some embodiments, the humanized antibody comprises one, two, three, four, five or six CDRs of an antibody as shown in Table 3, with one or more conservative amino acid substitutions. In some embodiments, the humanized antibody comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of a VH having the sequence set forth in SEQ ID NO: 15, with one or more conservative amino acid substitutions; and CDRL1, CDR-L2 and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 16, with one or more conservative amino acid substitutions.

In some embodiments, a humanized antibody that binds to human amyloid fibrils comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a framework region (FR) and the VH comprises a framework region (FR). In some embodiments, the framework region is FR1, FR2, FR3, or FR4. In some embodiments, the VL comprises FR1, FR2, FR3, and FR4. In some embodiments, the VL comprises OTN- To the C-terminus of FR1, CDR-L1, FR2, CDR-L2, FR3, CDR-L3, and FR4. In some embodiments, the VH comprises FR1, FR2, FR3, and FR4. In some embodiments, the VH comprises, from N- to C-terminus, FR1, CDR-H1, FR2, CDR-H2, FR3, CDR-H3, and FR4. In some embodiments, the humanized antibody comprises a VL and/or VH comprising an amino acid substitution at one or more framework region positions (e.g., FR1, FR2, FR3, or FR4) compared to a human CDR-grafted acceptor sequence (e.g., SEQ ID NO: 32 or SEQ ID NO: 43).

In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution in FR2, compared to a VL comprising an amino acid sequence,

- 26 048681 set forth in SEQ ID NO: 32. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of an amino acid substitution at position 36, an amino acid substitution at position 37, and an amino acid substitution at position 46, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of Tyr at position 36, Leu at position 37, and Leu at position 46, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution in FR3, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of an amino acid substitution at position 85 and an amino acid substitution at position 87, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of Leu at position 85 and Phe at position 87, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the humanized antibody comprises a VH comprising an amino acid substitution in FR2 compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of an amino acid substitution at position 37 and an amino acid substitution at position 48, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of Val at position 37 and Leu at position 48, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the humanized antibody comprises a VH comprising an amino acid substitution in FR3 compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of an amino acid substitution at position 67, an amino acid substitution at position 48, an amino acid substitution at position 71, an amino acid substitution at position 76, an amino acid substitution at position 78, an amino acid substitution at position 79, an amino acid substitution at position 80, an amino acid substitution at position 89, an amino acid substitution at position 93, and an amino acid substitution at position 94, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of Leu at position 67, Ser at position 48, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Phe at position 80, Thr at position 89, Val at position 93, and Thr at position 94, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the humanized antibody comprises a VL comprising one or more amino acid substitutions at one or more positions within the VL, as compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32, wherein the amino acid positions are numbered sequentially starting from the N-terminus of SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution at position 33, as compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution at position 34, as compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution at position 41, as compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments the humanized antibody comprises a VL comprising an amino acid substitution at position 42, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution at position 51, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution at position 90, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution at position 92, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, compared to with a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the humanized antibody comprises a VL comprising 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions compared to VL1, as shown in Table 6A.

In some embodiments, the humanized antibody comprises a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered starting from the N-terminus of the VL according to the numbering of SEQ ID NO: 32. In some

- 27 048 681 In some embodiments, the humanized antibody comprises a VL comprising a Ser, Gln, Glu, His, or Ala at position 33. In some embodiments, the humanized antibody comprises a VL comprising Ala or Val at position 34. In some embodiments, the humanized antibody comprises a VL comprising Tyr at position 41. In some embodiments, the humanized antibody comprises a VL comprising Leu at position 42. In some embodiments, the humanized antibody comprises a VL comprising Leu at position 51. In some embodiments, the humanized antibody comprises a VL comprising Leu at position 90. In some embodiments, the humanized antibody comprises a VL comprising Phe at position 92.

In some embodiments, a humanized antibody comprises a VH comprising one or more amino acid residues at one or more positions within the VH, wherein the amino acid positions are numbered starting from the N-terminus of the VH. In some embodiments, the humanized antibody comprises a VH comprising Val at position 37. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 48. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 67. In some embodiments, the humanized antibody comprises a VH comprising Ser at position 68. In some embodiments, the humanized antibody comprises a VH comprising Lys at position 71. In some embodiments, the humanized antibody comprises a VH comprising Ser at position 76. In some embodiments, the humanized antibody comprises a VH comprising Val at position 78. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 79. In some embodiments, the humanized antibody comprises a VH comprising Phe at position 80. In some embodiments, the humanized antibody comprises a VH comprising Thr at position 92. In some embodiments, the humanized the antibody comprises a VH comprising a Val at position 96. In some embodiments, the humanized antibody comprises a VH comprising a Thr at position 97.

In some embodiments, the humanized antibody comprises a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the humanized antibody comprises a VL comprising a Tyr at position 36. In some embodiments, the humanized antibody comprises a VL comprising a Leu at position 37. In some embodiments, the humanized antibody comprises a VL comprising a Leu at position 46. In some embodiments, the humanized antibody comprises a VL comprising a Leu at position 85. In some embodiments, the humanized antibody comprises a VL comprising a Phe at position 87.

In some embodiments, a humanized antibody comprises a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, a humanized antibody comprises a VL comprising Tyr at position 36 and Leu at position 37. In some embodiments, a humanized antibody comprises a VL comprising Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87. In some embodiments, a humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87.

In some embodiments, a humanized antibody comprises a VH comprising one or more amino acid residues at one or more positions within the VH, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the humanized antibody comprises a VH comprising Val at position 37. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 48. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 67. In some embodiments, the humanized antibody comprises a VH comprising Ser at position 68. In some embodiments, the humanized antibody comprises a VH comprising Lys at position 71. In some embodiments, the humanized antibody comprises a VH comprising Ser at position 76. In some embodiments, the humanized antibody comprises a VH comprising Val at position 78. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 79. In some embodiments, the humanized antibody comprises a VH comprising Phe at position 80. In some embodiments, the humanized antibody comprises a VH comprising Thr at position 89. In some embodiments, the humanized the antibody comprises a VH comprising a Val at position 93. In some embodiments, the humanized antibody comprises a VH comprising a Thr at position 94.

In some embodiments, a humanized antibody comprises a VH comprising one or more amino acid residues at one or more positions in the VH, wherein the amino acid positions

- 28 048681 acids are numbered according to the Kabat numbering system. In some embodiments, the humanized antibody comprises a VH comprising Val at position 37 and Leu at position 48. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 67, Ser at position 68, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the humanized antibody comprises a VH comprising Val at position 37, Leu at position 48, Leu at position 67, and Ser at position 68. In some embodiments, the humanized antibody comprises a VH comprising Val at position 37, Leu at position 48, Val at position 93, and Thr at position 94. In some embodiments, the humanized antibody comprises a VH comprising Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93 and Thr at position 94. In some embodiments, the humanized antibody comprises a VH comprising Lys at position 71, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94. In some embodiments, the humanized antibody comprises a VH comprising Lys at position 71, Ser at position 76, Val at position 93, and Thr at position 94. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94. In some embodiments, the humanized antibody comprises a VH comprising Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Val at position 93 and Thr at position 94.

In some embodiments, the humanized antibody comprises a VL comprising Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87, and a VH comprising Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94. In some embodiments, the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and A VH comprising Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Val at position 93, and Thr at position 94. In some embodiments, the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Lys at position 71, Ser at position 76, Val at position 93, and Thr at position 94. In some embodiments, the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Lys at position 71, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94.

In some embodiments, the humanized antibody comprises a VL amino acid sequence as shown in Table 6A. In some embodiments, the humanized antibody comprises 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, as shown in Table 6A. In some embodiments, the VL comprises an amino acid sequence set forth in the group consisting of SEQ ID NOs: 33-42.

In some embodiments, the humanized antibody comprises a VH amino acid sequence as shown in Table 6B. In some embodiments, the humanized antibody comprises a 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, VH9M64V, VH9-M64I, VH9-M64L, or VH9-M64A, as shown in Table 6B. In some embodiments, the VH comprises an amino acid sequence set forth in the group consisting of SEQ ID NOs: 44-63.

In some embodiments, the humanized antibody comprises a VL with VL4 as shown in Table 6A and a VH with VH9 as shown in Table 6B. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 35 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 51.

In some embodiments, the humanized antibody comprises a VL with VL3 as shown in Table 6A and a VH with VH6 as shown in Table 6B. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 34 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 48.

In some embodiments, the humanized antibody comprises a VL with VL4 as shown in Table 6A and a VH with VH10 as shown in Table 6B. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 35 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the humanized antibody comprises a VL with VL4 as shown in Table 6A and a VH with VH8 as shown in Table 6B. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 35 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 50.

- 29 048681 In some embodiments, the humanized antibody comprises a VL with VL4 as shown in Table 6A and a VH with VH7 as shown in Table 6B. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 35 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 69.

In some embodiments, the humanized antibody is a full-length antibody, a Fab fragment, or an scFv.

Humanized antibodies with CDR substitutions

In some embodiments, the humanized antibody comprises one or more substitutions in a CDR. In some embodiments, the substitution in a CDR is in CDR-L1. In some embodiments, the substitution in a CDR is in CDR-H2.

In some embodiments, the humanized antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20; 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, and VH comprising 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: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a VL comprising one or more amino acid residues selected from the group consisting of Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87; wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the humanized antibody comprises a VH comprising one or more amino acid residues selected from the group consisting of: Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Phe at position 80, Thr at position 89, Val at position 93, and Thr at position 94; wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 65, 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, and VH comprises CDRH1 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: 18, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 66, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 67, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1, comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a variable

- 30 048 681 light chain variable region (VL) and a heavy chain variable region (VH), wherein VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 68, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 69, 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, and VH comprises CDRH1 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: 18, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 70, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: NO: 21, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and 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: 18, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 71, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, 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, and VH comprises CDRH1 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: 72, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1, comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 75, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR

- 31 048681

H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 76, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 78, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 81, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the humanized antibody comprises one, two, three, four, five, or six CDRs of the antibody as shown in Table 6C and/or Table 6D.

In some embodiments, the humanized antibody comprises CDR-H1, CDR-H2, and CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDRH3 of VH having the sequence set forth in SEQ ID NOs: 53-63. In some embodiments, the humanized antibody comprises CDR-L1, CDR-L2, and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of VL having the sequence set forth in SEQ ID NOs: 36-42.

In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution in CDR-L1 compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the amino acid substitution in CDR-L1 is selected from the group consisting of an amino acid substitution at position 28 and an amino acid substitution at position 29, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-L1 is selected from the group consisting of Ser, Gln, Glu, His, or Ala at position 28, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-L1 is selected from the group consisting of Ala or Val at position 29, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the humanized antibody comprises a VH comprising an amino acid substitution in CDR-H2, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the amino acid substitution in CDR-H2 is selected from the group consisting of an amino acid substitution at position 54, position 55, or position 64, wherein the amino acid positions are numbered according to the Kabat numbering system.

- 32 048681

In some embodiments, the amino acid substitution in CDR-H2 is selected from the group consisting of Ser, Gln, Glu, Ala, or His at position 54, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDRH2 is selected from the group consisting of Ala or Val at position 55, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-H2 is selected from the group consisting of Val, Ile, Leu, or Ala at position 64, wherein the amino acid positions are numbered according to the Kabat numbering system.

B. Antibody-Peptide Fusion Proteins Comprising Humanized Antibodies Also provided herein are 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 a humanized antibody as described herein. In some embodiments, the humanized antibody is any of the humanized antibodies described above.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the humanized antibody comprises one, two, three, four, five, or six CDRs of the antibody as shown in Table 3. In some embodiments, the humanized antibody comprises CDRH1, CDR-H2, and CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 from a VH having the sequence set forth in SEQ ID NO: 15; and CDR-L1, CDRL2, and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 16. In some embodiments, the humanized antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20 with one or more conservative amino acid substitutions, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21 with one or more conservative amino acid substitutions, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22 with one or more conservative amino acid substitutions, and the VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, with one or more conservative amino acid substitutions, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, with one or more conservative amino acid substitutions, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19, with one or more conservative amino acid substitutions. In some embodiments, the humanized antibody comprises one, two, three, four, five or six CDRs of an antibody as set forth in Table 3, with one or more conservative amino acid substitutions. In some embodiments, the humanized antibody comprises a CDR-H1, CDR-H2 and CDRH3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of a VH having the sequence set forth in SEQ ID NO: 15, with one or more conservative amino acid substitutions; and CDR-L1, CDR-L2 and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 16, with one or more conservative amino acid substitutions.

In some embodiments, the humanized antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20; 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, and VH comprising CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising an amino acid substitution in

- 33 048681

FR2, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of an amino acid substitution at position 36, an amino acid substitution at position 37, and an amino acid substitution at position 46, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of Tyr at position 36, Leu at position 37, and Leu at position 46, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising an amino acid substitution in FR3 compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of an amino acid substitution at position 85 and an amino acid substitution at position 87, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of Leu at position 85 and Phe at position 87, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising an amino acid substitution in FR2 compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of an amino acid substitution at position 37 and an amino acid substitution at position 48, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR2 is selected from the group consisting of Val at position 37 and Leu at position 48, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising an amino acid substitution in FR3 compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of an amino acid substitution at position 67, an amino acid substitution at position 48, an amino acid substitution at position 71, an amino acid substitution at position 71, an amino acid substitution at position 76, an amino acid substitution at position 78, an amino acid substitution at position 79, an amino acid substitution at position 80, an amino acid substitution at position 89, an amino acid substitution at position 93, and an amino acid substitution at position 94, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in FR3 is selected from the group consisting of Leu at position 67, Ser at position 48, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Phe at position 80, Thr at position 89, Val at position 93, and Thr at position 94, wherein the amino acid positions are numbered according to the Kabat numbering system.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL that comprises one or more amino acid substitutions at one or more positions within the VL, compared to a VL that comprises the amino acid sequence set forth in SEQ ID NO: 32, wherein the amino acid positions are numbered starting from the N-terminus of SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL that comprises an amino acid substitution at position 33, compared to a VL that comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL that comprises an amino acid substitution at position 34, compared to a VL that comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the fusion protein the antibody-peptide comprises a humanized antibody, wherein the humanized antibody comprises a VL that comprises an amino acid substitution at position 41, compared to a VL that comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL that comprises an amino acid substitution at position 42, compared to a VL that comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL that comprises an amino acid substitution at position 51, compared to a VL that comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL that comprises an amino acid substitution at position 90, compared to a VL that comprises the amino acid sequence set forth in SEQ ID NO: 32. comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising an amino acid substitution at position 92,

- 34 048 681 compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions compared to VL1, as shown in Table 6A.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises one or more amino acid substitutions at one or more positions within the VH, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43, wherein the amino acid positions are numbered starting from the N-terminus of SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 37, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 48, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the fusion protein the antibody-peptide comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 67, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 68, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 71, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 76, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 78, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 79, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH that comprises an amino acid substitution at position 80, compared to a VH that comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising an amino acid substitution at position 92, as compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising an amino acid substitution at position 96, as compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising an amino acid substitution at position 97, as compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to VH1, as shown in Table 6B.

In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered starting from the N-terminus of the VL. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Ser, Gln, Glu, His, or Ala at position 33. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising an Ala or Val at position

- 35 048681 in section 34. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Tyr at position 41. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Leu at position 42. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Leu at position 51. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Leu at position 90. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Phe at position 92.

In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising one or more amino acid residues at one or more positions within the VH, wherein the amino acid positions are numbered starting from the N-terminus of the VH. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Val at position 37. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Leu at position 48. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Leu at position 67. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Ser at position 68. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Lys at position 71. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Ser at position 76. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Val at position 78. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Leu at position 79. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Phe at position 80. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Thr at position 92. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Val at position 96. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising a Thr at position 97.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising one or more amino acid substitutions at one or more positions in the VL, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Tyr at position 36. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Leu at position 37. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Leu at position 46. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Leu at position 85. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Phe at position 87.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Tyr at position 36 and a Leu at position 37. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Tyr at position 36, a Leu at position 37, a Leu at position 46, a Leu at position 85, and a Phe at position 87. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising a Leu at position 46 and a Phe at position 87.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising one or more amino acids

- 36,048,681 amino acid residues at one or more positions in VH, with the amino acid positions numbered according to the Kabat numbering system. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Val at position 37. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Leu at position 48. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Leu at position 67. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Ser at position 68. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Lys at position 71. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Ser at position 76. In some embodiments, the hybrid the antibody-peptide comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Val at position 78. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Leu at position 79. In some embodiments, the antibody-peptide hybrid comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Phe at position 80. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Thr at position 89. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Val at position 93. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Thr at position 94.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising one or more amino acid residues at one or more positions within the VH, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Val at position 37 and Leu at position 48. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Leu at position 67, Ser at position 68, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Val at position 37, Leu at position 48, Leu at position 67, and Ser at position 68. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Val at position 37, Leu at position 48, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Lys at position 71, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Lys at position 71, Ser at position 76, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VH comprising Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87, and a VH comprising Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position

- 37 048681 in 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Lys at position 71, Ser at position 76, Val at position 93, and Thr at position 94. In some embodiments, the antibody-peptide fusion protein comprises a humanized an antibody, wherein the humanized antibody comprises a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Lys at position 71, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL amino acid sequence as shown in Table 6A. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises 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, as shown in Table 6A. In some embodiments, the VL comprises an amino acid sequence set forth in the group consisting of SEQ ID NOs: 33-42.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VH amino acid sequence as shown in Table 6B. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a 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, VH9G55A, VH9-G55V, VH9-M64V, VH9-M64I, VH9-M64L, or VH9-M64A, as shown in Table 6B. In some embodiments, the VH comprises an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH9 as shown in Table 6B. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 51.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL with VL3 as shown in Table 6A and a VH with VH6 as shown in Table 6B. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 34 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 48.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH10 as shown in Table 6B. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH8 as shown in Table 6B. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH7 as shown in Table 6B. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 69.

In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide. In some embodiments, the amyloid-reactive peptide comprises one or more peptides listed in Table 1. In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14. In some embodiments, the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the amyloid-reactive peptide is positively charged.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a light chain. In some embodiments, the amyloid-reactive peptide is fused to the N-terminus of the light chain. In some embodiments, the

- 38 048681 an antibody-peptide fusion protein comprises a humanized antibody, wherein the humanized antibody comprises a light chain, wherein an amyloid-reactive peptide is fused to the N-terminus of the light chain using a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker comprises the amino acid sequence GGGYS. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the linker is uncharged.

In some embodiments, one or more of the peptides shown in Table 1 below can be linked to a humanized antibody or functional fragment thereof via the N-terminus of a light chain protein or the C-terminus of a heavy chain, thereby forming an antibody-peptide fusion protein comprising the humanized antibody. That is, any of the sequences shown in Table 1 below can be linked to the heavy or light chain of a humanized antibody or functional fragment thereof independently or simultaneously to form an antibody-peptide fusion protein. For example, two amyloid-reactive peptides can be linked to a single Ig antibody, such as by linking the amino acid sequences of an amyloid-reactive peptide to the N-terminus of a light chain of a humanized antibody.

In some embodiments, the antibody-peptide fusion protein comprises a light chain comprising, from N-terminus to C-terminus, an amyloid-reactive peptide and a light chain. In some embodiments, the light chain comprises, from N-terminus to C-terminus, VL and CL1. In some embodiments, VL is any of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain comprising, from N-terminus to C-terminus, VH, CH1, CH2 and CH3. In some embodiments, VH is any of the VHs described herein.

In some embodiments, the antibody-peptide fusion protein comprises a light chain comprising, from N-terminus to C-terminus, an amyloid-reactive peptide, a spacer peptide, and a light chain. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the light chain comprises, from N-terminus to C-terminus, VL and CL1. In some embodiments, the VL is any of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain comprising, from N-terminus to C-terminus, VH, CH1, CH2, and CH3.

In some embodiments, the antibody-peptide fusion protein comprises, from N-terminus to C-terminus, a secretory 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 light chain comprises, from N-terminus to C-terminus, VL and CL1. In some embodiments, VL is any of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain comprising, from N- to C-terminus, VH, CH1, CH2, and CH3. In some embodiments, VH is any of the VHs described herein. In some embodiments, the VH is any of the VHs described herein. In some embodiments, the antibody-peptide fusion protein has the structure shown in Fig. 11A or Fig. 11B.

In some embodiments, the antibody-peptide fusion protein comprises a spacer sequence of amino acids between the N-terminus of the light chain and the amyloid-reactive peptide. In some embodiments, the antibody-peptide fusion protein comprises a spacer sequence of amino acids between the N-terminus of the peptide and a leader sequence necessary for secretion of the antibody-peptide fusion protein from cells expressing the antibody-peptide fusion protein. In some embodiments, the spacer peptide is a flexible spacer peptide. In some embodiments, the spacer peptide is uncharged. In some embodiments, the spacer peptide is a glycine-serine linker. In some embodiments, the spacer peptide comprises a glycine-serine linker. In some embodiments, the spacer peptide comprises or consists of about 3 to about 55 amino acids. The spacer peptides of the present invention may comprise 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. In some embodiments, the spacer peptide is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, or 155 amino acids in length, including any value or range between those values. In some embodiments, the spacer peptide comprises 15 amino acids. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 24.

- 39 048681 In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antibody-peptide fusion protein does not comprise a spacer between the amyloid-reactive peptide and the antibody.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63 and a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63, without the C-terminal lysine residue, and a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the antibody is attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 without the C-terminal lysine residue and a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the antibody is attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 without the C-terminal lysine residue and a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the antibody is attached to a peptide.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NOs: 44-63, wherein the heavy chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 4463, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51, wherein the heavy chain is attached to a peptide comprising any from the amino acid sequences of Table 1. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SQ ID NO: 51, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SQ ID NO: 51, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SQ ID NO: 48, wherein the heavy chain is attached to a peptide comprising any of the amino acids of the sequence of Table 1. In some embodiments, an antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SQ ID NO: 48, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, an antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SQ ID NO: 48, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, an antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, an antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, an antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, an antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, an antibody-peptide fusion protein comprises a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide is attached to the light chain at the N-terminus.

In some embodiments, an antibody-peptide fusion protein comprises an antibody whose heavy chain comprises a VH comprising the amino acid sequence of SEQ ID NO: 51 and whose light chain comprises a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is attached to the light chain at the N-terminus.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1.

In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide is attached to the light chain at the N-terminus.

In some embodiments, an antibody-peptide fusion protein comprises an antibody whose heavy chain comprises a VH comprising the amino acid sequence of SEQ ID NO: 48 and whose light chain comprises a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is attached to the light chain at the N-terminus.

C. Humanized antibodies and antibody-peptide fusion proteins containing humanized antibodies

In some embodiments, a humanized antibody or antibody-peptide fusion protein comprising a humanized antibody of the present invention comprises an Fc region.

- 41 048681 in some embodiments, the Fc is of the IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the humanized antibody or antibody-peptide fusion protein comprising the humanized antibody promotes Fc-mediated antibody effector function. In some embodiments, the humanized antibody or antibody-peptide fusion protein comprising the humanized antibody promotes antibody-dependent cellular phagocytosis.

In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is less than about 100, 10, 1, 0.1, 0.01 μM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is about 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 therebetween. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is less than 500, 100, 10, or 1 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is less than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000, 2000, or 2200 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000, 2000, or 2200 nM, including any value or range therebetween. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is about 40-50 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is 40-50 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is less than 50 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) that is less than the Kd for binding of C11-1F4 to human amyloid fibrils.

In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a half-maximal binding at an antibody concentration ( _EC50 ) that is less than about 0.01, 0.1, or 1 μM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a half-maximal binding at an antibody concentration (EC50) that is about 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, or 10 μM, including any value or range therebetween. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a half-maximal binding at an antibody concentration (EC ₅₀ ) that is less than about 1, 10, 100, or 1000 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a half-maximal binding at an antibody concentration (EC ₅₀ ) that is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 100, 250, 500, 750, or 1000 nM, including any value or range therebetween. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a half-maximal binding at an antibody concentration (EC50) of about 17 nM, 7 nM, 16 nM, 75 nM, or 95 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a half-maximal binding at an antibody concentration ( _EC50 ) of less than about 10 nM, 20 nM, 80 nM, or 100 nM. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to human amyloid fibrils with a half-maximal binding at an antibody concentration (EC ₅₀ ) that is less than the EC ₅₀ for binding of c11-1F4 to human amyloid fibrils.

Methods for calculating dissociation constants and _EC50 are known in the art and include, for example, surface plasmon resonance and EuLISA (see, for example, Examples, Table 7, and Fig. 13A13G). In some embodiments, the dissociation constant is determined by measuring binding to a monomeric Len(1-22) peptide, for example, using surface plasmon resonance. In some embodiments, the _EC50 is determined using EuLISA. In some embodiments, the EC50 is determined using EuLISA to measure binding to rVL6Wil fibrils, Per125 wtATTR extract, Ken ATTR extract, SHI ALL liver extract, or TAL KAL liver extract.

In some embodiments, the humanized antibody or antibody fusion protein

- 42 048681 peptide is conjugated with a detectable label. In some embodiments, the detectable label is selected from the group consisting of radionuclides (e.g., I- ¹²⁵ , I- ¹²³ , I- ¹³¹ , Zr ^-89 , Tc ^-99m , Cu- ⁶⁴ , Br ⁷⁶ , F- ¹⁸ ); enzymes (horseradish peroxidase); biotin; fluorophores, etc. Any means known in the art for detectably labeling a protein can be used and/or adapted for use with the methods described herein. For example, humanized antibodies or antibody-peptide fusion proteins can be labeled with a radioactive isotope or labeled with a fluorescent label or a chemiluminescent label. Examples of radioisotopes include, for example, ¹⁸ F, ⁿ¹ In, ^99m Tc, and ¹²³ I, and ¹²⁵ I. These and other radioisotopes can be attached to a humanized antibody or antibody-peptide fusion protein using well-known chemistry, which may or may not include the use of chelating agents such as DTPA or DOTA, for example, covalently linked to the light chain protein of the humanized antibody or antibody-peptide fusion protein. Examples of fluorescent or chemiluminescent labels include fluorescein, Texas red, rhodamine, Alexa dyes, and luciferase, which can be conjugated to a humanized antibody or antibody-peptide fusion protein by reaction with lysine, cysteine, glutamic acid, and aspartic acid side chains. In one exemplary embodiment, the label is detected using a fluorescent microplate reader or fluorimeter using excitation and emission wavelengths appropriate to the label used. Radioactive labels can be detected, for example, using a gamma or scintillation counter, depending on the type of radioactive radiation and using windows for targeted energy transfer suitable for the precise detection of a particular radionuclide. However, any other suitable radioisotope detection technique can also be used to detect the label. In some embodiments, the detectable label is ¹²⁵ I.

In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to rVZ6Wil fibrils, Per125 wtATTR extract, KEN hATTR extract, SHI ΑΕλ liver extract, and/or TAL ALk liver extract. In some embodiments, the humanized antibodies or antibody-peptide fusion proteins described herein bind to amyloid deposits or fibrils. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to one or more amyloidogenic peptides in amyloids. In some embodiments, the amyloids bound by the humanized antibody or antibody-peptide fusion protein comprise amyloidogenic λ6 variable domain protein (V/.6Wil) or amyloidogenic immunoglobulin light chain (AL), Αβ(1-40) amyloid-like fibril or amyloidogenic Αβ precursor protein, or serum amyloid protein A (AA). In other embodiments, the amyloids associated with the humanized antibody or antibody-peptide fusion protein comprise amyloidogenic forms of immunoglobulin heavy chain (AH), _β2 -microglobulin ( _Αβ2M ), transthyretin variants (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AH (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen a variants (AFib), cystatin variants (ACys), calcitonin (ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP), α-synuclein (AaSyn), Tay (ATau), atrial natriuretic factor (AANF), or IAAP, ALk4, AlZ1, other amyloidogenic peptides. The amyloidogenic peptides associated with the humanized antibody or antibody-peptide fusion protein may be a protein, a protein fragment, or a protein domain. In some embodiments, the amyloid deposits or amyloid fibrils comprise recombinant amyloidogenic proteins. In some embodiments, the amyloids are part of the disease pathology.

In some embodiments, binding of a humanized antibody or antibody-peptide fusion protein to human amyloid promotes phagocytosis of human amyloid fibrils. In some embodiments, the humanized antibody or antibody-peptide fusion protein opsonizes human amyloid fibrils. In some embodiments, the humanized antibody or antibody-peptide fusion protein opsonizes r^6Wil fibrils. In some embodiments, contact of human amyloid fibrils with a humanized antibody or antibody-peptide fusion protein of the invention in the presence of macrophages promotes uptake of human amyloid fibrils by macrophages. In some embodiments, contact of human amyloid fibrils with a humanized antibody or antibody-peptide fusion protein of the invention in the presence of macrophages promotes opsonization of human amyloid fibrils. In some embodiments, binding of a humanized antibody or antibody-peptide fusion protein to human amyloid promotes phagocytosis of human amyloid fibrils to an equal or greater extent than a control antibody (e.g., mIgp5 and/or C11-1F4). In some embodiments, a humanized antibody or antibody-peptide fusion protein comprising a humanized antibody promotes antibody-dependent cellular phagocytosis.

This document also provides pharmaceutical compositions comprising any of

- 43 048 681 modified immunoglobulins, humanized antibodies or antibody-peptide fusion proteins described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

IV. Methods of diagnostics and detection.

In some exemplary embodiments, the modified immunoglobulins, humanized antibodies, and antibody-peptide fusion proteins may be labeled with various agents to allow for their detection in vivo and in vitro assays, such as after purification of the fusion peptides. Without limitation, this may include radionuclides (e.g., I- ¹²⁵ , I- ¹²³ , I- ¹³¹ , Zr ^-89 , Tc ^-99m , Cu- ⁶⁴ , Br ^-76 , F- ¹⁸ ); enzymes (horseradish peroxidase); biotin; fluorophores, etc. Any means known in the art for detectably labeling a protein may be used and/or adapted for use with the methods described herein. For example, Ig antibodies or fragments thereof and/or amyloid-reactive peptides can be radiolabeled or labeled with a fluorescent label or a chemiluminescent label. Examples of radioisotopes include, for example, ¹⁸ F, ¹¹¹ In, ^99m Tc, and ¹²³ I, and ¹²⁵ I. These and other radioisotopes can be attached to an isolated immunoglobulin light chain using well-known chemistry, which may or may not involve the use of a chelating agent such as DTPA or DOTA, for example covalently linked to the Ig antibody light chain protein. Examples of fluorescent or chemiluminescent labels include fluorescein, Texas red, rhodamine, Alexa dyes and luciferase, which can be conjugated to the protein by reaction with the side chains of lysine, cysteine, glutamic acid and aspartic acid. In one exemplary embodiment, the label is detected using a fluorescent microplate reader or fluorimeter using excitation and emission wavelengths appropriate for the label used. Radioactive labels can be detected, for example, using a gamma or scintillation counter depending on the type of radioactive radiation and using windows for targeted energy transfer suitable for the accurate detection of a particular radionuclide. However, any other suitable radioisotope detection technique can also be used to detect the label.

With respect to amyloidosis, such labeling can be used, for example, to diagnose the presence of amyloid, to determine amyloid protein load, to monitor the ability of a modified immunoglobulin, humanized antibodies, or antibody-peptide fusion proteins to bind amyloid in a particular subject, to monitor the progression of amyloidosis, and/or to observe the subject's response to treatment for amyloidosis (including treatment involving administration of modified immunoglobulins, humanized antibodies, or antibody-peptide fusion proteins to the subject). For example, modified immunoglobulins, including an amyloid-reactive peptide, humanized antibodies, or antibody-peptide fusion proteins, are labeled with a detectable label as described herein and then administered to a subject who has or is suspected of having an amyloid-associated disease (e.g., amyloidosis, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), or related plasma cell diseases). The subject can then be imaged, for example, to detect the presence of detectably labeled immunoglobulins, humanized antibodies, or antibody-peptide fusion proteins.

In some exemplary embodiments, signals from detectably labeled modified immunoglobulins, humanized antibodies, or antibody-peptide fusion proteins can be quantified, thereby providing an indication of the level of amyloid deposition in a subject. For example, signal intensity can be compared to a standard signal threshold above which amyloidosis is present, but below which amyloidosis is absent or at a low level. The subject can be diagnosed as having amyloid, in which case treatment can be administered, such as chemotherapy, corticosteroids (lenalidomide or thalidomide), and/or bortezomib (Velcade). Additionally or alternatively, modified immunoglobulins, humanized antibodies, or antibody-peptide fusion proteins described herein can be administered to a subject for the purpose of treating the subject as described herein. In certain embodiments, the subject may be divided into one or more groups, such as low amyloid load, moderate amyloid load, or high amyloid load, and then treated accordingly. To monitor the progress of treatment, the subject may be re-administered detectably labeled modified immunoglobulins, humanized antibodies, or antibody-peptide fusion proteins and, therefore, re-evaluated for their amyloid load.

V. Methods of treatment.

A. Methods using modified immunoglobulins.

In some exemplary embodiments, provided herein are methods of treating a subject suffering from amyloidosis. For example, an effective amount of a modified immunoglobulin as described herein is administered to the subject, thereby curing the subject or allowing amyloid deposits to be visualized. In certain exemplary aspects, provided is a method of clearing amyloid deposits in a subject. The method includes, for example, selecting a subject with amyloidosis and administering to the subject an effective amount of modified immunoglobulins, such as

- 44 048681 is described in this document. Modified immunoglobulins include, for example, an amyloid-reactive peptide that binds to amyloid deposits linked to an Ig antibody or fragment thereof via the N-terminus of the light chain protein or the C-terminus of the heavy chain of the Ig antibody or fragment thereof. Thus, administration of the amyloid-reactive Ig fusion peptide results in clearance of amyloid and, therefore, in a cure of the subject.

In some embodiments, the modified immunoglobulins bind amyloids in an individual. In some embodiments, the amyloid deposits may contribute to the development of a pathological condition. In other embodiments, the amyloid deposits may be indicative of amyloidosis or an amyloid-associated disease in an individual. In some embodiments, the modified immunoglobulins bind amyloids in an individual with amyloidosis. In some embodiments, the amyloidosis is localized to a particular tissue or organ system, such as the liver, heart, or central nervous system. In other embodiments, the amyloidosis is systemic amyloidosis. In some embodiments, the amyloidosis is familial amyloidosis. In other embodiments, the amyloidosis is sporadic amyloidosis. In some embodiments, the amyloidosis or amyloid-associated disease is AA amyloidosis, AL amyloidosis, AH amyloidosis, AB amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis type II diabetes, Alzheimer's disease, Down syndrome, hereditary cerebral hemorrhage with Dutch-type amyloidosis, cerebral beta-amyloid angiopathy, spongiform encephalopathy, thyroid tumors, Parkinson's disease, Lewis body dementia, tauopathy, Huntington's disease, senile systemic amyloidosis, familial amyloidosis, senile systemic aging, senile pituitary disease, iatrogenic syndrome, spongiform encephalopathies, reactive chronic inflammation, thyroid tumors, myeloma, or others forms of malignancy. In some embodiments, the modified immunoglobulins bind to amyloids associated with normal aging. In other embodiments, the modified immunoglobulins are used to diagnose, treat, or predict amyloidosis or an amyloid-associated disease in a subject.

In some exemplary embodiments, a method is provided for both diagnosing and treating a subject suffering from amyloidosis. Such a method includes administering to the subject a detectably labeled modified immunoglobulin comprising an amyloid-reactive peptide, and determining based on the administration of the labeled modified immunoglobulin that the subject suffers from amyloidosis. The subject may then be administered an effective amount of a drug for treating amyloidosis. For example, an effective amount of one or more modified immunoglobulins, including amyloid-reactive peptides, may be administered.

In some embodiments, the subject is a mammal, such as a primate, bovine, rodent, or pig. In some embodiments, the subject is a human.

B. Methods using humanized antibodies or antibody-peptide fusion proteins.

Also provided herein are methods of treating a subject suffering from an amyloid-associated disorder comprising administering to the subject an effective amount of a humanized antibody or antibody-peptide fusion protein of the present invention.

In some embodiments, the amyloid-associated disorder is selected from the group consisting of AL, AH, AB2M, ATTR, transthyretin, AA, AApoAI, AApoAII, AGel, ALys, ALEct2, AFib, ACys, ACal, AMed, AIAPP, APro, AIns, APrP, alpha-synuclein, au, or AB amyloidosis. In some embodiments, the amyloidosis or amyloid-associated disease is AA amyloidosis, AL amyloidosis, AH amyloidosis, AB amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis type II diabetes, Alzheimer's disease, Down syndrome, hereditary cerebral hemorrhage with Dutch-type amyloidosis, cerebral beta-amyloid angiopathy, spongiform encephalopathy, thyroid tumors, Parkinson's disease, Lewis body dementia, tauopathy, Huntington's disease, senile systemic amyloidosis, familial amyloidosis, senile systemic aging, senile pituitary disease, iatrogenic syndrome, spongiform encephalopathies, reactive chronic inflammation, thyroid tumors, myeloma, or other forms malignancy. In some embodiments, the amyloid-associated disorder is systemic amyloidosis. In some embodiments, the humanized antibody or antibody-peptide fusion protein binds to amyloids associated with normal aging. In other embodiments, the humanized antibody or antibody-peptide fusion protein is used to diagnose, treat, or prognosticate amyloidosis or an amyloid-associated disorder in a subject.

Also provided herein are methods for targeting amyloid deposits for clearance. In some embodiments, the method comprises contacting the amyloid deposit with a humanized antibody or antibody-peptide fusion protein of the present invention.

- 45 048681 n. In some embodiments, the amyloid deposit is removed. In some embodiments, the amyloid deposit is purified. In some embodiments, the amyloid deposit is opsonized by a humanized antibody or an antibody-peptide fusion protein. In some embodiments, binding of the humanized antibody or antibody-peptide fusion protein to human amyloid fibrils promotes phagocytosis of human amyloid fibrils and removal of amyloid deposits. In some embodiments, the humanized antibody or antibody-peptide fusion protein opsonizes human amyloid fibrils, thereby removing the amyloid deposit. In some embodiments, the humanized antibody or antibody-peptide fusion protein opsonizes rVZ6Wil fibrils. In some embodiments, binding of a humanized antibody or antibody-peptide fusion protein to human amyloid fibrils promotes phagocytosis and/or opsonization of human amyloid fibrils to an equal or greater extent than a control antibody (e.g., mIgp5 and/or C11-1F4).

In some embodiments, provided herein is a method of treating an amyloid-associated disorder comprising administering a modified immunoglobulin or antibody-peptide fusion protein conjugated to a detectable label, detecting the label, and administering to the subject an amyloidosis treating agent if a signal is detected. In some embodiments, the detectable label is a radiolabel. In some embodiments, the detectable label is an ^I125 , ^Tc99 label. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the detectable label is an enzymatic label. In some embodiments, the label is horseradish peroxidase or alkaline phosphatase. Labels further include chemical moieties, such as biotin, that can be detected by binding to a specific related detectable moiety, such as labeled avidin. In some embodiments, the amyloid deposit is identified in the liver, spleen, or blood of the subject. In some embodiments, the treatment of amyloidosis comprises a modified immunoglobulin or antibody-peptide fusion protein as provided herein.

Also provided herein is a method for detecting amyloid deposits in a subject, comprising administering a modified immunoglobulin or an antibody-peptide fusion protein, wherein the modified immunoglobulin or the antibody-peptide fusion protein is conjugated to a detectable label. In some embodiments, the method comprises detecting a signal from the modified immunoglobulin or the antibody-peptide fusion protein. In some embodiments, the detectable label is a radiolabel. In some embodiments, the detectable label is an I ¹²⁵ , Tc ⁹⁹ label. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the detectable label is an enzymatic label. In some embodiments, the label is horseradish peroxidase or alkaline phosphatase. Labels further include chemical moieties, such as biotin, that can be detected by binding to a specific, related detectable moiety, such as labeled avidin. In some embodiments, amyloid deposition is identified in the liver, spleen, or blood of the subject.

In some embodiments, provided herein is a method of detecting a ligand, comprising contacting the ligand with a modified immunoglobulin or a fusion antibody and a peptide conjugated to a detectable label and detecting a signal from the detectable label. In some embodiments, the detectable label is a radiolabel. In some embodiments, the detectable label is an ^I125 , ^Tc99 label. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the detectable label is an enzymatic label. In some embodiments, the label is horseradish peroxidase or alkaline phosphatase. Labels further include chemical moieties, such as biotin, that can be detected by binding to a specific cognate detectable moiety, such as labeled avidin. In some embodiments, the detection is performed in vitro. In some embodiments, the detection is performed in vivo.

VI. Nucleic acids, vectors, host cells and methods for producing antibodies.

A. Nucleic acids encoding a modified immunoglobulin or antibody-peptide fusion protein.

Also provided herein is a nucleic acid encoding a modified immunoglobulin. In some embodiments, the nucleic acid encodes any of the modified immunoglobulins described herein.

In some embodiments, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a VH and/or VL of antibody 11-1F4, wherein the antibody is attached to a peptide. In some embodiments, the nucleic acids encode an antibody comprising a heavy chain and/or a light chain of antibody 11-1F4, wherein the antibody is attached to a peptide.

- 46 048681

In a specific embodiment, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a VH comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, wherein the antibody is attached to a peptide.

In a specific embodiment, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a VL that comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, wherein the antibody is attached to a peptide.

In one embodiment, the nucleic acid encodes a modified immunoglobulin antibody comprising a VL comprising the amino acid sequence of SEQ ID NO: 16 and a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the antibody is attached to a peptide.

In another aspect, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a VL comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, wherein the antibody is linked to a peptide. In some embodiments, the modified immunoglobulin comprises an antibody linked to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In another aspect, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a VH CDR1, VH CDR2, and VH CDR3 of a VH having the sequence set forth in SEQ ID NO: 15; and a VL CDR1, VL CDR2, and VL CDR3 of a VL having the sequence set forth in SEQ ID NO: 16, wherein the antibody is linked to a peptide. In some embodiments, the modified immunoglobulin comprises an antibody linked to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified immunoglobulin comprises an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising an antibody heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and an antibody light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide. In some embodiments, the modified immunoglobulin comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 without the C-terminal lysine residue and a light chain comprising the amino acid sequence of SEQ ID NO: 16, wherein the antibody is attached to a peptide.

In another aspect, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a VH as in any of the embodiments above and a VL as in any of the embodiments above, and wherein the antibody is attached to a peptide.

In some embodiments, the nucleic acid encodes an antibody linked to an amyloid-reactive peptide. In some embodiments, the nucleic acid encodes an antibody linked to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the nucleic acid encodes an antibody linked to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody comprising a light chain linked to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is linked to the N-terminus of the light chain of the antibody or to the C-terminus of the heavy chain. In some embodiments, the nucleic acid encodes an antibody that further comprises a spacer amino acid sequence between the peptide and the N-terminus of the light chain of the antibody or the C-terminus

- 47 048681 heavy chain. In some embodiments, the peptide is attached to the N-terminus of the light chain of the antibody.

In some embodiments, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 linked to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the nucleic acid encodes an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 linked to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 linked to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16 linked to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the nucleic acid encodes an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16 linked to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16 linked to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes a sequence the modified immunoglobulin comprises an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1.

In some embodiments, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the nucleic acid encodes a modified immunoglobulin comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

Also provided herein is a nucleic acid(s) encoding an antibody-peptide fusion protein. In some embodiments, the nucleic acid encodes any of the antibody-peptide fusion proteins described herein.

In a specific embodiment, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody, wherein the antibody comprises a VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, wherein the antibody is attached to the peptide.

In a specific embodiment, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody, wherein the antibody comprises a VL that comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, wherein the antibody is attached to the peptide.

In one embodiment, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a VL comprising the amino acid sequence of SEQ ID NO: 16 and a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the antibody is attached to a peptide.

In another aspect, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody, wherein the antibody comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and wherein the antibody is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising any of the amino acids

- 48,048,681 of the sequences listed in Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In another aspect, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody, wherein the antibody comprises a VH CDR1, a VH CDR2, and a VH CDR3 of a VH having the sequence set forth in SEQ ID NO: 15, and a VL CDR1, a VL CDR2, of a VL having the sequence set forth in SEQ ID NO: 16; and wherein the antibody is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 without a C-terminal lysine residue and a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the antibody is attached to a peptide.

In another aspect, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising any of the amino acid sequences listed in Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody attached to an amyloid-reactive peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is attached to the N-terminus of the light chain of the antibody or to the C-terminus of the heavy chain. In some embodiments, the antibody also comprises a spacer amino acid sequence between the peptide and the N-terminus of the light chain or the C-terminus of the heavy chain.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody whose light chain comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes an antibody fusion protein.

- 49 048681 a peptide comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising any of the amino acid sequences from Table 1.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising an antibody whose heavy chain comprises a VH comprising the amino acid sequence of SEQ ID NO: 15 and whose light chain comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

B. Nucleic acids encoding a humanized antibody or an antibody-peptide fusion protein containing a humanized antibody.

Also provided herein is a nucleic acid(s) encoding a humanized antibody or antibody-peptide fusion protein of the present invention. The humanized antibody or antibody-peptide fusion protein may be any humanized antibody or antibody-peptide fusion protein described herein.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and the VH comprises a CDRH1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the nucleic acid encodes a humanized antibody comprising one, two, three, four, five, or six CDRs antibodies as shown in Table 3. In some embodiments, the nucleic acid encodes a humanized antibody comprising a CDR-H1, CDR-H2, and CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 from a VH having the sequence set forth in SEQ ID NO: 15; and a CDR-L1, CDR-L2, and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 from a VL having the sequence set forth in SEQ ID NO: 16.

In some embodiments, the nucleic acid encodes a humanized antibody, wherein the humanized antibody comprises one or more substitutions in the CDRs. In some embodiments, the humanized antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VH comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the nucleic acid encodes a humanized antibody, wherein the humanized antibody comprises a VL comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20; 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, and VH comprising 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: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising one or more amino acid substitutions at one or more positions within the VL, as compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32, wherein the amino acid positions are numbered starting from the N-terminus of SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid substitution at position 33, as compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid substitution at position 34, as compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid replacement at position 41, compared to VL,

- 50 048 681 comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid substitution at position 42, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid substitution at position 51, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid substitution at position 90, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid substitution at position 92, compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions compared to a VL comprising the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions compared to VL1, as shown in Table 6A

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising one or more amino acid substitutions at one or more positions within the VH, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43, wherein the amino acid positions are numbered starting from the N-terminus of SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 37, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 48, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid a substitution at position 67, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 68, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 71, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 76, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 78, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 79, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 80, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 92, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 96, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid substitution at position 97, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, compared to a VH comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, compared to a VH1, as shown in Table 6B.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered starting from the N-terminus of the VL. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising

- 51 048681 having Ser, Gln, Glu, His, or Ala at position 33. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Ala or Val at position 34. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Tyr at position 41. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Leu at position 42. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Leu at position 51. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Leu at position 90. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Phe at position 92.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising one or more amino acid residues at one or more positions within the VH, wherein the amino acid positions are numbered starting from the N-terminus of the VH. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 37. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 48. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 67. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Ser at position 68. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Lys at position 71. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Ser at position 76. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 78. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 79. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Phe at position 80. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Thr at position 92. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 96. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Thr at position 97.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising a Tyr at position 36. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising a Leu at position 37. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising a Leu at position 46. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising a Leu at position 85. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising a Phe at position 87.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising one or more amino acid residues at one or more positions within the VL, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Tyr at position 36 and Leu at position 37. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Leu at position 46 and Phe at position 87.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising one or more amino acid residues at one or more positions within the VH, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 37. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 48. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 67. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Ser at position 68. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Lys at position 71. In some embodiments, the nucleic acid encodes a humanized

- 52 048 681 a humanized antibody comprising a VH comprising a Ser at position 76. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising a Val at position 78. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising a Leu at position 79. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising a Phe at position 80. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising a Thr at position 89. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising a Val at position 93. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising a Thr at position 94.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising one or more amino acid residues at one or more positions within the VH, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 37 and Leu at position 48. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 67, Ser at position 68, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 37, Leu at position 48, Leu at position 67, and Ser at position 68. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 37, Leu at position 48, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Lys at position 71, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Lys at position 71, Ser at position 76, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Tyr at position 36, Leu at position 37, Leu at position 46, Leu at position 85, and Phe at position 87, and a VH comprising Val at position 37, Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Thr at position 89, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising an antibody comprising a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Leu at position 48, Leu at position 67, Ser at position 68, Lys at position 71, Ser at position 76, Val at position 78, Leu at position 79, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Lys at position 71, Ser at position 76, Val at position 93, and Thr at position 94. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising Leu at position 46 and Phe at position 87, and a VH comprising Lys at position 71, Val at position 78, Leu at position 79, Val at position 93 and Thr at position 94.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL amino acid sequence as shown in Table 6A. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL selected from the group consisting of VL2, VL3, VL4, VL4-N33S, VL4-N33Q, VL4-N33E, VL4-N33A, VL4-N33H, VL4G34A, or VL4-G34V, as shown in Table 6A. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NOs: 33-42.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH amino acid sequence as shown in Table 6B. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH selected from the group consisting of VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH9-D54S, VH9-D54Q,

- 53 048681

VH9-D54E, VH9-D54A, VH9-D54H, VH9-G55A, VH9-G55V, VH9-M64V, VH9-M64I, VH9-M64L, or VH9-M64A, as shown in Table 6B. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NOs: 44-63.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH9 as shown in Table 6B. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 51.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL with VL3 as shown in Table 6A and a VH with VH6 as shown in Table 6B. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 34 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 48.

In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH10 as shown in Table 6B. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH8 as shown in Table 6B. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VH comprising the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, the nucleic acid encodes a humanized antibody comprising a VL with VL4 as shown in Table 6A and a VH with VH7 as shown in Table 6B. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 35 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 69.

Also provided herein is a nucleic acid(s) encoding an antibody-peptide fusion protein comprising a humanized antibody of the present invention. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising any of the humanized antibodies of the present invention.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63 and a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63 without a C-terminal lysine residue and a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the antibody is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 without the C-terminal lysine residue and a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the antibody is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 without the C-terminal lysine residue and a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the antibody is attached to a peptide.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NOs: 44-63, wherein the heavy chain is joined to a peptide comprising any of the amino acid sequences of Table 1. ...

- 54 048 681 an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 44-63, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 4463, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51, wherein the heavy chain is attached to a peptide comprising any of the amino acid sequences from Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51, wherein the heavy chain is joined to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51, wherein the heavy chain is joined to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48, wherein the heavy chain is joined to a peptide comprising any from the amino acid sequences of Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48, wherein the heavy chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is joined to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a light chain comprising a VL comprising an amino acid sequence set forth in the group consisting of SEQ ID NO: 33-42, wherein the light chain is joined to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising any of the amino acid sequences from Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is joined to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is joined to a peptide comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is joined to a peptide comprising any of the amino acid sequences in Table 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody that comprises a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to

- 55 048681 peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody, the heavy chain of which comprises a VH comprising the amino acid sequence of SEQ ID NO: 51, and the light chain of which comprises a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising any of the amino acid sequences of Table 1.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody comprising a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the nucleic acid encodes an antibody-peptide fusion protein comprising a humanized antibody, the heavy chain of which comprises a VH comprising the amino acid sequence of SEQ ID NO: 48, and the light chain of which comprises a VL comprising the amino acid sequence of SEQ ID NO: 34, wherein the light chain is attached to a peptide comprising the amino acid sequence of SEQ ID NO: 2.

C. Vectors, host cells.

In some embodiments, the nucleic acid provided herein is in one or more vectors. For example, in some embodiments, provided herein is a vector comprising a heavy chain and a light chain of a modified immunoglobulin, wherein the light chain is attached to a peptide. In some embodiments, the heavy chain and the light chain attached to the peptide are in different vectors.

In some embodiments, the vector comprises nucleic acid(s) encoding a humanized antibody or antibody-peptide fusion protein of the present invention.

To produce antibodies, the heavy chain and light chain attached to the peptide expression vectors can be introduced into appropriate producer cell lines known in the art. The introduction of the expression vectors can be accomplished by cotransfection via electroporation or any other suitable transformation technology available in the art. The antibody-producing cell lines can then be selected and expanded, and the antibodies purified. The purified antibodies can then be analyzed by standard methods such as SDS-PAGE.

Also provided is a host cell comprising a nucleic acid encoding any of the modified immunoglobulins described herein. Host cells suitable for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, the modified immunoglobulin may be produced by bacteria, particularly where glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody linked to the peptide can be recovered from the bacterial cell biomass in a soluble fraction and can be further purified.

In some embodiments, the host cell comprises a vector comprising nucleic acid(s) encoding a humanized antibody or antibody-peptide fusion protein of the present invention.

Host cells suitable for expression of glycosylated antibody can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified,

- 56 048681 which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines are the SV40-transformed CV1 monkey kidney line (COS-7); the human embryonic kidney line (293 or 293 cells, as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli kidneys (TM4 cells, described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells, as described, for example, in Mather et al, Annals N Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

D. Methods for producing antibodies or antibody-peptide fusion proteins.

Also provided herein are methods for producing a modified immunoglobulin, a humanized antibody, or an antibody-peptide fusion protein of the present invention. In some embodiments, the method comprises culturing a host cell of the present invention under conditions suitable for expressing a vector encoding the modified immunoglobulin, the humanized antibody, or the antibody-peptide fusion protein, and isolating the modified immunoglobulin, the humanized antibody, or the antibody-peptide fusion protein.

VII. Method for humanizing a mouse antibody.

Provided herein are methods for humanizing a mouse antibody. In some embodiments, the mouse antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises CDR-L1, CDR-L2, and CDR-L3, and the VH comprises CDR-H1, CDR-H2, and CDR-H3, wherein the VH and VL comprise one or more framework residues. In some embodiments, CDR-L1 of the mouse antibody comprises the amino acid sequence set forth in SEQ ID NO: 20, CDR-L2 of the mouse antibody comprises the amino acid sequence set forth in SEQ ID NO: 21, and CDR-L3 of the mouse antibody comprises the amino acid sequence set forth in SEQ ID NO: 22, and CDR-H1 of the mouse antibody comprises the amino acid sequence set forth in SEQ ID NO: 17, CDR-H2 of the mouse antibody comprises the amino acid sequence set forth in SEQ ID NO: 18, and CDR-H3 of the mouse antibody comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the mouse antibody comprises one, two, three, four, five, or six CDRs of antibody 11-1F4, as shown in Table 3. In some embodiments, the murine antibody comprises a CDR-H1, CDR-H2, and CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 from a VH having the sequence set forth in SEQ ID NO: 15; and a CDR-L1, CDR-L2, and CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 from a VL having the sequence set forth in SEQ ID NO: 16.

In some embodiments, a method of humanizing a mouse antibody comprises: i. performing homology modeling to obtain a modeled structure of the mouse antibody; ii. calculating the solvent accessible surface area of framework residues in the modeled structure of the mouse antibody; iii. determining whether the framework residues are buried residues, wherein a buried residue is a residue with a solvent accessible surface area of less than about 15%; iv. providing a VH and VL of a human; v. introducing CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 of a mouse antibody into the human VH and VL, respectively, to obtain a grafted antibody; and vi. introducing a back mutation at a position of the grafted antibody, wherein the position to be back mutation is a buried residue. In some embodiments, step vi is repeated. In some embodiments, step vi is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

In some embodiments, the backmutation position is in close proximity to the CDR. In some embodiments, the backmutation position is separated from the CDR by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the backmutation position is separated from the CDR by no more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 angstroms, including any range or value therebetween.

In some embodiments, a reverse mutation is introduced into a human VL, wherein the

- 57,048,681 amino acid sequences are numbered according to the Kabat numbering system. In some embodiments, a back mutation is introduced into a human VL, wherein the back mutation comprises Tyr at position 36. In some embodiments, a back mutation is introduced into a human VL, wherein the back mutation comprises Leu at position 37. In some embodiments, a back mutation is introduced into a human VL, wherein the back mutation comprises Leu at position 46. In some embodiments, a back mutation is introduced into a human VL, wherein the back mutation comprises Leu at position 85. In some embodiments, a back mutation is introduced into a human VL, wherein the back mutation comprises Phe at position 87.

In some embodiments, a back mutation is introduced into a human VH, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Val at position 37. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Leu at position 48. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Leu at position 67. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Ser at position 68. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Lys at position 71. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Ser at position 76. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Val at position 78. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Leu at position 79. In some embodiments, a back mutation is introduced into a human VH, wherein the back mutation comprises Phe at position 80. In some embodiments, the back mutation is introduced into a human VH, wherein the back mutation comprises Thr at position 89. In some embodiments, the back mutation is introduced into a human VH, wherein the back mutation comprises Val at position 93. In some embodiments, the back mutation is introduced into a human VH, wherein the back mutation comprises Thr at position 94.

In some embodiments, the method further comprises introducing one or more amino acid substitutions into a CDR of the grafted antibody. In some embodiments, the humanized antibody comprises a VL comprising an amino acid substitution in CDR-L1 compared to the VL of the grafted antibody. In some embodiments, the amino acid substitution in CDR-L1 is selected from the group consisting of an amino acid substitution at position 28 and an amino acid substitution at position 29, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-L1 is selected from the group consisting of Ser, Gln, Glu, His, or Ala at position 28, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-L1 is selected from the group consisting of Ala or Val at position 29, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the humanized antibody comprises a VH comprising an amino acid substitution in CDR-H2 compared to the VH of the grafted antibody. In some embodiments, the amino acid substitution in CDR-H2 is selected from the group consisting of an amino acid substitution at position 54, position 55, or position 64, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-H2 is selected from the group consisting of Ser, Gln, Glu, Ala, or His at position 54, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-H2 is selected from the group consisting of Ala or Val at position 55, wherein the amino acid positions are numbered according to the Kabat numbering system. In some embodiments, the amino acid substitution in CDR-H2 is selected from the group consisting of Val, Ile, Leu, or Ala at position 64, wherein the amino acid positions are numbered according to the Kabat numbering system.

Embodiments of the invention

Embodiment 1. A modified immunoglobulin comprising an amyloid-reactive peptide, wherein the amyloid-reactive peptide comprises an amino acid sequence that has at least 85% identity to any of the amino acid sequences set forth in SEQ ID NO: 1-14; and an Ig antibody or a functional fragment thereof.

Embodiment 2. A modified immunoglobulin according to embodiment 1, characterized in that the amyloid-reactive peptide and the Ig antibody or a functional fragment thereof are linked together at the N-terminus of the Ig light chain protein or at the C-terminus of the Ig heavy chain protein.

Embodiment 3. A modified immunoglobulin according to embodiments 1 or 2, characterized in that the modified immunoglobulin comprises a spacer sequence between the amyloid-reactive peptide and the Ig antibody or a functional fragment thereof.

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Embodiment 4. The modified immunoglobulin of embodiments 1-3, wherein the modified immunoglobulin comprises at least two amyloid-reactive peptides, wherein the amyloid-reactive peptides are the same peptide or different peptides.

Embodiment 5. A method of treating a subject suffering from amyloidosis, comprising administering to the subject an effective amount of a modified immunoglobulin as specified in any one of embodiments 1-4.

Embodiment 6. A method of targeting an amyloid deposit for clearance, comprising contacting the amyloid deposit with a modified immunoglobulin as set forth in any one of embodiments 1-4.

Embodiment 7. The method of embodiment 6, wherein targeting the amyloid deposit for clearance of the amyloid deposit.

Embodiment 8. The method according to embodiment 6 or 7, characterized in that the clearance is a result of opsonization of the amyloid deposit.

Embodiment 9. The method according to any one of embodiments 1-8, characterized in that the amyloid-reactive peptide attached to the Ig antibody or functional fragment thereof binds to one or more types of amyloid deposits, including AA, AL, AH, ATTR, Ae2M, ALect2, wild type, TTR, AApoAI, AApoAII, AGel, ALys, ALect2, Afib, ACys, ACal, AMedin, AIAPP, APro, AIns, APrP, or Av.

Embodiment 10. The method according to any one of embodiments 6-8, characterized in that contacting the amyloid deposit with an amyloid-reactive peptide attached to an Ig antibody or a functional fragment thereof increases the half-life of the amyloid-reactive Igp5 conjugate by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to the amyloid-reactive peptide alone.

Embodiment 11. A modified immunoglobulin comprising:

a peptide; and an Ig antibody or a functional fragment thereof, wherein the peptide and the Ig antibody or a functional fragment thereof are linked together at the N-terminus of an Ig light chain protein and/or at the N- and/or C-terminus of an Ig heavy chain protein.

Embodiment 12. A modified immunoglobulin according to embodiment 11, characterized in that the modified immunoglobulin comprises a spacer sequence between the peptide and the Ig antibody or a functional fragment thereof.

Embodiment 13. The modified immunoglobulin of any one of embodiments 1-3, wherein the modified immunoglobulin comprises at least two peptides, wherein the peptides are the same peptide or different peptides.

Embodiment 14. A method for producing a modified immunoglobulin, comprising:

providing a first expression vector and a second expression vector, wherein the first expression vector comprises a first nucleic acid sequence encoding a light chain of an Ig antibody or a functional fragment thereof;

wherein the second expression vector comprises a second nucleic acid sequence encoding a heavy chain of an Ig antibody or a functional fragment thereof; and wherein the first expression vector and/or the second expression vector comprise a third nucleic acid sequence that encodes a first peptide, wherein the third nucleic acid sequence is located adjacent to the first nucleic acid sequence and/or the second nucleic acid sequence; and inserting the first and second expression vectors into a cell, wherein expression of the first and second expression vectors in the cell results in the formation of an immunoglobulin attached to the first peptide.

Embodiment 15. The method according to embodiment 14, characterized in that the first expression vector and/or the second expression vector comprise a fourth nucleic acid sequence that encodes a second peptide, wherein the fourth nucleic acid sequence is located adjacent to the first nucleic acid sequence and/or the second nucleic acid sequence.

Embodiment 16. The method according to embodiment 15, characterized in that the expression of the first and second expression vectors in the cell results in the formation of an immunoglobulin attached to the first peptide and the second peptide.

Embodiment 17. The method according to embodiment 14, characterized in that the spacer nucleic acid sequence is located between the third nucleic acid sequence and the first nucleic acid sequence and/or the second first nucleic acid sequence.

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Embodiment 18. The method of embodiment 14, wherein the first peptide comprises an amino acid sequence that has at least 85% identity to any of the amino acid sequences set forth in SEQ ID NOs: 1-14.

Embodiment 19. A modified immunoglobulin obtained using the method according to any one of embodiments 14-19.

Embodiment 20. A method of treating a subject who has or is suspected of having an amyloid-associated disease, comprising:

determining the presence of amyloid deposits in a subject by:

Detectably labeling a modified immunoglobulin according to any one of embodiments 1-4, administering the modified immunoglobulin to a subject, determining whether a signal associated with the detectable label can be detected in the subject; and, if the signal is detected, administering to the subject an agent for treating amyloidosis.

Embodiment 21. The method of embodiment 20, wherein if the signal is not detected, the subject is monitored for subsequent development of amyloid deposits.

Embodiment 22. The method of embodiment 21, further comprising determining the signal intensity and comparing the signal to a threshold value, above which the subject is determined to have amyloid deposits.

Embodiment 23. The method according to any one of embodiments 20-22, characterized in that the treatment of amyloidosis comprises administering to the subject a modified immunoglobulin according to any one of claims 1-4.

Embodiment 24. The method of embodiment 23, wherein administering the modified immunoglobulin results in clearance of amyloid deposits in the subject.

Embodiment 25. A method for detecting amyloid deposits in a subject, comprising detectably labeling a modified immunoglobulin of any one of embodiments 1-4, administering the modified immunoglobulin to the subject, and detecting a signal from the modified immunoglobulin.

Embodiment 26. The method of any one of embodiments 20-25, wherein it is determined that the subject lacks amyloid or has monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), or one or more related plasma cell diseases.

Embodiment 27. A method for detecting a ligand, comprising:

detectably labeling a modified immunoglobulin according to any one of embodiments 11-13, wherein the modified immunoglobulin peptide has a binding affinity for the ligand;

contacting a ligand with the modified immunoglobulin; and, detecting a signal from a detectable label, thereby detecting the ligand. Embodiment 28. A modified immunoglobulin comprising an amyloid-reactive peptide, wherein the amyloid-reactive peptide comprises any of the amino acid sequences set forth in SEQ ID NOs: 1-14; and an antibody or immunologically active fragment thereof, wherein the antibody comprises a heavy chain variable domain comprising CDRH1 set forth in SEQ ID NO: 17, CDRH2 set forth in SEQ ID NO: 18, CDRH3 set forth in SEQ ID NO: 19;

and a light chain variable domain comprising CDRL1 as set forth in SEQ ID NO: 20, CDRL2 as set forth in SEQ ID NO: 21, and CDRL3 as set forth in SEQ ID NO: 22.

Embodiment 29. The modified immunoglobulin of claim 28, wherein the amyloid-reactive peptide comprises SEQ ID NO: 1 or SEQ ID NO: 2.

Embodiment 30. The modified immunoglobulin of embodiments 28 or 29, wherein the amyloid-reactive peptide is attached to the N-terminus of the light chain of the antibody or an immunologically active fragment thereof.

Embodiment 31. A modified immunoglobulin according to any one of embodiments 28-30, characterized in that the amyloid-reactive peptide is attached to the N-terminus of the light chain of the antibody or an immunologically active fragment thereof via a linker.

Embodiment 32. A modified immunoglobulin according to any one of embodiments 28-31, characterized in that the antibody or immunologically active fragment thereof comprises an Fc region.

Embodiment 33. The modified immunoglobulin of any one of embodiments 28-32, wherein the antibody is chimeric or humanized.

Embodiment 34. The modified immunoglobulin of any one of embodiments 28-32, wherein the antibody comprises human framework sequences.

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Embodiment 35. A method of treating amyloidosis in a subject comprising administering the modified immunoglobulin of any one of embodiments 28-34 to a subject in need thereof.

Embodiment 36. A method for producing a modified immunoglobulin according to any one of embodiments 28-34, comprising transforming a host cell with one or more nucleic acids encoding the modified immunoglobulin and culturing the cell under conditions that ensure production of the modified immunoglobulin.

Embodiment 37. The modified immunoglobulin of any one of embodiments 1-4, 11-13, or 28-34, wherein the modified immunoglobulin binds to rV^6Wil, AB, AP(1-40), IAAP, ALk4, AR1, or ATTR fibrils.

Embodiment 38. A nucleic acid encoding a modified immunoglobulin according to any one of embodiments 1-4, 11-13, or 28-34.

Embodiment 39. A host cell comprising the nucleic acid of embodiment 38.

Embodiment 40. The host cell according to claim 39, characterized in that the host cell is a CHO cell.

Examples

The following examples further illustrate the invention, but should not be construed as limiting its scope in any way. In light of the present description and the general level of skill in the art, those skilled in the art will recognize that the following examples are intended for illustration only and that numerous changes, modifications and variations may be made without departing from the scope of the presently disclosed subject matter. The accompanying drawings should be considered an integral part of the disclosure and description of the invention.

The following abbreviations are used in this document: eq (equivalents); M (molar); µM (micromolar); N (normal); mol (moles); mmol (millimoles); µmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg (kilograms); µg (micrograms); L (liters); ml (milliliters); µL (microliters); cm (centimeters); mm (millimeters); µm (micrometers); nm (nanometers); °C (degrees Celsius); h (hours); min (minutes); sec (seconds); ms (milliseconds).

Example 1. Production and use of immunoglobulin fused with amyloidophilic peptides for targeting and removing amyloid deposits.

Introduction.

Immunoglobulin-peptide fusions are synthetic biomolecules consisting of an Ig that incorporates another protein or peptide. The Ig confers two major advantages to the peptide of interest; primarily, an increase in the plasma half-life of the amyloid-reactive peptide through interaction of the Ig with the neonatal Fc receptor and, second, the ability to recruit and activate effector immune cells through interactions via membrane-bound Fc receptors and complement fixation. To generate peptide-antibody fusions for targeting and removing (e.g., clearing) amyloid deposits, we synthesized a murine Ig-peptide fusion construct that incorporates the synthetic amyloidophilic peptide p5 (SEQ ID NO: 1) with murine IgG2a by fusing the peptide amino acid sequence and spacer elements to the N-terminus of the Ig light chain protein (Fig. 2).

As will be appreciated by those skilled in the art based on this disclosure, this construct may include any number of amyloid-reactive peptides that we have developed or disclosed and any number of spacer sequences as described herein (including those listed in Table 1; see also WO2016032949). The mouse Igp5 fusion was produced in HEK 293T/17 cells and used to generate proof-of-principle data using a panel of in vitro binding and phagocytosis assays, and was shown to localize to systemic amyloid deposits using mouse models of amyloidosis. Collectively, these data demonstrate that the Ig-peptide fusion is capable of: (1) binding multiple forms of amyloid deposits, (2) specifically binding amyloid in vivo, and; (3) opsonize amyloid extracts and synthetic fibrils in vitro using a cultured macrophage assay system. We propose that the mode of action is that the Ig-peptide hybrid binds to hypersulfated heparan sulfate proteoglycans and/or protein fibrils found in all amyloid deposits via an amyloid-reactive peptide. The Fc region can then fix complement and recruit macrophages and other cells expressing Fc receptors, which can then remove the amyloid. A schematic of this process is shown in Fig. 3.

Materials and methods.

For the Igp5 heavy chain construct, the pFUSE-mIgG2A-Fc vector encoding the CH2 (heavy chain constant domain 2) and CH3 (heavy chain constant domain 3) domains of the mouse IgG2a heavy chain was purchased from InvivoGen (San Diego, CA). cDNA for the home

- 61,048,681 new mouse heavy chain VH (heavy chain variable domain) and CH1 (heavy chain constant domain 1) derived from the mouse 11-1F4 Ig sequence (Genscript, Piscataway, NJ) and cloned in frame into a vector with the 5' secretory leader and CH2/CH3 domains of the Fc region. For the Igp5 light chain construct, cDNA for the p5 peptide (SEQ ID NO: 1) with a 5' secretory leader, a 5' spacer of 15 amino acids (SEQ ID NO: 23), and a 3' spacer of 5 amino acids (SEQ ID NO: 24), followed by sequences encoding the murine 11-1F4 VL (variable domain of the light chain) and CL (constant domain of the light chain) regions were synthesized (Genscript) and cloned into the pcDNA3.1-Hygro(+) vector. The amino acid sequence of the Igp5 light chain construct is shown in SEQ ID NO: 25. The vectors were transiently transfected into the HEK293T/17 cell line cultured in Ig-depleted serum. Secreted Igp5 was purified from the medium using protein A-conjugated matrix. Binding of purified Igp5 to synthetic amyloid fibrils was demonstrated using an affinity adsorption assay. In vivo reactivity with murine AA amyloid was assessed by microautoradiography, tissue biodistribution measurements, and small animal SPECT/CT imaging using iodine-125 ( ¹²⁵ I)-labeled Igp5. Igp5-mediated phagocytosis of synthetic fibrils was measured in vitro using human THP-1 macrophages in a quantitative fluorescence assay using pHrodo-red.

Results.

The Fcp5 fusion was expressed in both HEK cell lines at a concentration of ~1-4 μg/ml culture medium. For affinity adsorption analysis, Igp5 was labeled with ¹²⁵ I. Gel electrophoresis under reducing and non-reducing conditions was used to show the presence of both heavy and light chain in the Igp5 construct (Fig. 4).

Using an affinity adsorption assay, ¹²⁵ I-Igp5 was shown to bind synthetic fibrils composed of the recombinant amyloidogenic variable domain protein λ6 (rVX6Wil; Table 2). Reactivity was >70%, as evidenced by the presence of radiolabeled material in the fibril pellet.

Table 2

Binding of ¹²⁵ I-Igp5 to amyloid fibrils of the synthetic light chain (rVλ6Wil)

Sample ¹²⁵ I-Igp5 bound to fibril precipitate (% added material) rVZ6Wil 73

In AA-amyloid mice, ¹²⁵ I-Igp5 specifically bound to amyloid deposits in the liver and spleen (the sites of greatest amyloid deposition in these mice), as evidenced by tissue biodistribution measurements (Fig. 5). Notably, in healthy mice without amyloid deposits, ¹²⁵ Igp5 was not retained in these organs (Fig. 5). Microautoradiography revealed uptake by small amyloid deposits in all organs and tissues (not shown). SPECT/CT imaging of AA mice and healthy controls without amyloid deposits 20 h after i.v. injection confirmed that ¹²⁵ I-Igp5 was taken up by the liver and spleen of AA mice; however, in WT mice, it was shown to be present only in the blood pool (Fig. 6).

Finally, Igp5 efficiently mediated the phagocytosis of pHrodo Green-labeled synthetic light chain amyloid fibrils in vitro (Fig. 7).

Discussion.

Based on these data, we demonstrate that it is possible to generate functional Ig molecules with improved ligand binding properties by fusing functional peptides at the N-terminus of the Ig light chain. Furthermore, our data demonstrate that fusing amyloid-reactive peptides to the Ig light chain can generate an Ig-peptide hybrid that specifically binds amyloid in vitro and in vivo and can serve as an opsonizing reagent and enhance amyloid uptake by macrophages. The Igp5 hybrid or similar constructs using other amyloidophilic peptides may provide a novel reagent for targeting an immunologically active biomolecule (Ig) to amyloid deposits and thereby accelerating amyloid clearance in patients by immune cells (macrophages and neutrophils). These reagents can provide pan-amyloid reactivity and benefit patients with many, if not all, forms of amyloid-associated disease. In addition, these reagents, if labeled with the appropriate radionuclides, can also be used to detect amyloid deposits in patients using SPECT and PET imaging and be used to monitor response to therapy or stratify patients prior to therapy using unlabeled Ig-peptide fusion reagents.

Example 2. Binding of Ig-peptide hybrid to amyloid fibrils.

This example shows Igp5 binding to various amyloid-associated substrates. This example also demonstrates a peptide-Ig fusion in mouse tissue.

Materials and methods.

A peptide-Ig hybrid containing the IgK LC sequence fused to the p5 peptide was expressed

- 62 048681 in vector. The IgHC sequence containing the variable domains of IgG1, the CH1 domains of IgG1, and the CH2 and CH3 domains of IgG2a was expressed in a second vector system. The vectors were co-transfected into HEK 293T/17 cells and the Ig product was isolated using protein A-conjugated particles. Igp5 and control Ig were radiolabeled with ^125I and analyzed by gel electrophoresis. Binding of ^125I -Igp5 and control Ig to synthetic amyloid fibrils and amyloid extracts was assessed by affinity adsorption assays. To assess tissue retention, ¹²⁵ I-Igp5 was injected intravenously into healthy mice (WT) and mice with severe systemic serum albumin A-associated amyloidosis (AA) (H2/IL-6 transgenic). Tissue localization ^{of 125} IIgp5 was then assessed by automicroradiography (ARG).

Results.

Igp5 was transiently expressed in HEK cells, purified from cell culture supernatant using protein A beads, and radiolabeled with ^125I for preliminary analysis. ^125I -labeled Igp5 was analyzed by SDS-PAGE under both reducing and non-reducing conditions. Murine 11-1F4 (IgG1K) was used as an Ig control. The p5 peptide alone was also analyzed for comparison. Figure 8 shows the results of SDS-PAGE analysis of purified ^125I -Igp5. Purified Igp5 represented both intact heavy and light chain Ig after gel electrophoresis.

Binding of ¹²⁵ I-Igp5 and control Ig to synthetic amyloid fibrils and amyloid extracts was assessed by affinity adsorption assays. As shown in Fig. 9, ¹²⁵ Igp5 bound a variety of amyloid-associated substrates. ¹²⁵ I-m11-1F4 bound κ4-peptide-coated particles but not rV^6Wil fibrils, whereas ¹²⁵ I-Igp5 bound both substrates. ¹²⁵ I-Igp5 bound rV^6Wil and AB(1-40) amyloid-like fibrils by approximately 64% (21-fold higher than the control Ig, mouse 11-1F4). Binding to amyloid extracts was also 10-30-fold higher and positively correlated with ¹²⁵ I-p5 binding (r=0.9, p=0.01). Binding of ¹²⁵ I-Igp5 to various synthetic amyloid fibrils and amyloid extracts was significantly enhanced compared to m11-1F4, with reactivity correlating with that of the p5 peptide alone, indicating that binding is peptide-driven.

To assess IgP5 retention in vivo, ¹²⁵ I-Igp5 was injected intravenously into healthy mice (WT) and mice with severe systemic serum albumin A-associated amyloidosis (AA) (H2/IL-6 transgenic). Localization of ¹²⁵ I-Igp5 in various tissues was assessed by microautoradiography (ARG). Congo red staining was used to detect amyloid deposits. As shown in panel A of Fig. 10, ARG and Congo red staining demonstrate specific retention ^{of 125} I-Igp5 in AA amyloid of mouse liver and spleen, as well as amyloid binding in other tissues. No specific reactivity to tissues without amyloid deposits was observed in wild-type mice, in which the blood pool was the only identified source of ¹²⁵ I-Igp5, as shown in panel B of Fig. 10

These results demonstrate that Igp5 binds multiple forms of amyloid and specifically targets amyloid in vivo.

Example 3. Humanization of antibodies to amyloid.

The following example describes the production of humanized antibodies to amyloid.

Materials and methods.

Amino acid sequences of the original antibody and peptide.

The murine antibody m11-1F4 was used as a parent antibody to generate humanized antibodies. The parent murine antibody with the p5 peptide fused at the N-terminus of the light chain was named mIgG-p5. From the N-terminus to the C-terminus, the light chain of mIgG-p5 included a secretory leader sequence that was fused to an amino acid spacer sequence, a p5 peptide sequence, another amino acid spacer sequence, and the light chain of the antibody (see Fig. 11A and Fig. 11B).

The amino acid sequence of p5 is shown in SEQ ID NO: 1 (see Table 1).

Humanization of antibodies.

Humanized antibodies were prepared as follows. First, the sequences of the variable domains of m11-1F4 were analyzed. The complementarity determining regions (CDRs), hypervariable loops, and framework regions (FRs) were identified. The amino acid sequences of the complementarity determining regions of m11-1F4 are presented in Table 3 below. The amino acid sequences of the CDRs, FRs, and residues at the VH-VL interface are also shown in Fig. 12.

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Table 3

Amino acid sequences of CDR m11-1F4

11-1F4 CDR Amino acid sequence SEQ ID NO CDR-L1 RSSQSLVHRNGNTYLH 20 CDR-L2 KVSNRFS 21 CDR-L3 FQTTYVPNT 22 CDR-H1 GFSLSSYGVS 17 CDR-H2 VIWGDGSTNYHPNLMS 18 CDR-H3 LDY 19

Homology modeling was then performed to obtain the modeled structure of the mouse antibody. Homology models were built using the dedicated Build Homology Models protocol. Disulfide bridges were identified and connected. Loops were optimized using the discrete optimization of protein energy (DOPE) method.

The solvent accessible surface area of the framework residues was calculated. Based on this result, cryptic framework residues were identified. Residues with a solvent accessible surface area of less than 15% were considered cryptic residues.

One human acceptor for each of the VH and VL sequences was then selected that had the highest sequence identity with the mouse counterparts (see Tables 4A, 4B). The mouse antibody CDRs were directly grafted onto the human acceptor scaffolds to produce the grafted antibody sequence without any back mutations. These sequences are designated VH1 and VL1 (see Tables 6A, 6B below).

Table 4A Sequences of parental and human VLs used to generate humanized antibodies

IgG Description Amino acid sequence of VL SEQ ID NO 11-1F4 VL Initial mouse antibody DVVMTQTPLSLPVSLGDQASISCRSSQSLVHR NGNTYLHWYLQKPGQSPKLLIYKVSNRFSGV PDRFSGSGSGTDFTLKISRVEAEDLGLYFCFQ TTYVPNTFGGGTKLEIK 16 IGKV2- 30*02 Human Germline Sequence DVVMTQSPLSLPVTLGQPASISCRSSQSLVHS DGNTYLNWFQQRPGQSPRRLIYKVSNRDSGV PDRF SSGSGSGTDFTLKISRVEAED VGVYYCM QGTHWPP 28 Acceptor VL Human acceptor sequence DVVMTQSPLSLPVTLGQPASISCRSSQSLVHS DGNTYLNWFQQRPGQSPRRLIYKVSNRDSGV PDRF SSGSGSGTDFTLKISRVEAED VGVYYCFQ TTYVPNTFGGGTKLEIK 29

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Table 4B

Native and human VH sequences used to create humanized antibodies

IgG Description Amino acid sequence of VH SEQ ID NO 11-1F4 VH Initial mouse antibody QVQLKESGPGLVAPSQSLSITCTVSGFSLSSY GVSWVRQPPGKGLEWLGVIWGDGSTNYHPN LMSRLSISKDISKSQVLFKLNSLQTDDTATYY CVTLDYWGQGTSVTVSS 15 IGHV4- 4*08 Human Germline Sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISSY YWSWIRQPPGKGLEWIGYIYTSGSTNYNPSL KSRVTISVDTSKNQFSLKLSSVTAADTAVYY CAR 30 Acceptor VH Human acceptor sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISSY YWSWIRQPPGKGLEWIGYIYTSGSTNYNPSL KSRVTISVDTSKNQFSLKLSSVTAADTAVYY CARLDYWGQGTSVTVSS 31

The critical residues identified in step 5 were then compared between the grafted VH1 and VL1 and mouse VH and VL sequences. All critical residues that differed between the grafted and mouse antibody backbone sequences (i.e., putative back mutation sites) were identified.

One or more backmutations were then introduced into the grafted antibody sequence in a stepwise manner according to the following guidelines: (1) cryptic residues that were less likely to elicit an immune response were selected for backmutation; (2) cryptic residues that were also in close proximity to a CDR were selected for backmutation with higher priority; and (3) other cryptic FR residues were selected for backmutation with lower priority.

Finally, additional mutations were introduced into the CDRs of the humanized heavy chain variable region VH9 at residues D54, G55, or M64 and into the CDRs of the humanized light chain variable region VL4 at residues N33 or G34, counted from the N-terminus of VH or VL. These mutations were designed to remove post-translational modification sites and are listed in Tables 6A, 6B below.

Surface plasmon resonance (SPR).

The binding affinity of humanized anti-amyloid antibodies to the monomeric peptide VK4Len(1-22) (Len(1-22) monomer), the natural ligand of the m11-1F4 antibody, was measured using the Biacore 8K surface plasmon resonance (SPR) system. The parent antibody m11-1F4 binds to an epitope present at the N-terminus of denatured kappa-4 light chain proteins. Len(1-22) monomer is a peptide containing this epitope as described in International Application No. PCT/US2017/015905. The binding affinities of anti-amyloid antibodies comprising the humanized heavy chain region VH9 and the humanized light chain region VL4, including VH9 and VL4 variants with additional mutations, were tested (Table 5).

All SPR data were processed using Biacore 8K Evaluation software version 1.1. Flow cell 1 and the blank injections in each run were used as a double standard for subtracting response units. Additional experimental conditions for the SPR experiments are presented in Table 3 below.

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Table 5

Experimental conditions of SPR for binding of Len(1-22) monomer to amyloid antibody

Immobilization Ligand Antibodies Immobilization level (Ru) -7000 Association and dissociation Contact time of association (s) 120 Contact time of dissociation (s) 360 Flow rate (µl/min) 30 Sample concentrations (nM) 6.25, 12.5, 25, 50, 100, 200, 400 Surface regeneration Buffer for regeneration 10 mM glycine-HO Contact time (s) 30 Flow rate (µl/min) 30

Results.

Antibodies with humanized VH and VL sequences were generated from m11-1F4. The humanized VL sequences were designated VL1-VL4, and the humanized VH sequences were designated VH1-VH10. The amino acid sequences of the humanized VH and VL regions are presented below in Tables 6A-6B. In Tables 6A-6B, the CDR sequences are underlined, and the backmutated residues and additional mutations that were introduced into VL4 and VH9 are shown in bold and italics. Additional mutations that were introduced into VL4 and VH9 are listed in the IgG column of Tables 6A and 6B; these mutations are numbered relative to the N-terminus of VL or VH. VL4 and VH9 variants were generated with modified CDRs, and these CDR sequences are presented in Table 6A-6B. 6C and 6D compared to VL4 and VH9.

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Table 6A

Amino acid sequences of humanized light chain variable region sequences

IgG Amino acid sequence of VL SEQ ID NO VLI DVVMTOSPLSLPVTLGOPASISCRSSOSLVHRNGNTYLHWFOQR PGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG VYYCFQTTYVPNTFGGGTKLEIK 32 VL2 DVVMTOSPLSLPVTLGOPASISCRSSQSLVHRNGNTYLHWFLOR PGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG VYYCFQTTYVPNTFGGGTKLEIK 33 VL3 DVVMTOSPLSLPVTLGOPASISCRSSQSLVHRNGNTYLHWFLOR PGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGZ YFCFQTTYVPNTFGGGTKLEIK 34 VL4 DVVMTOSPLSLPVTLGOPASISCRSSOSLVHRNGNTYLHWFOQR PGQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 35 VL4- N33S DVVMTOSPLSLPVTLGOPASISCRSSOSLVHRSGNTYLHWFOQRP GQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 36 VL4- N33Q DVVMTOSPLSLPVTLGOPASISCRSSOSLVHROGNTYLHWFOQR PGQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 37 VL4- N33E DVVMTOSPLSLPVTLGOPASISCRSSOSLVHREGNTYLHWFOQR PGQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 38 VL4- N33A DVVMTOSPLSLPVTLGOPASISCRSSOSLVHRAGNTYLHWFOQR PGQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 39 VL4- N33H DVVMTOSPLSLPVTLGOPASISCRSSOSLVHRHGNTYLHWFOQR PGQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 40 VL4- G34A DVVMTOSPLSLPVTLGOPASISCRSSOSLVHRAGNTYLHWFOQR PGQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 41 VL4- G34V DVVMTOSPLSLPVTLGOPASISCRSSQSLVHRVGNTYLHWFOOR PGQSPRZLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YFCFQTTYVPNTFGGGTKLEIK 42

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Table 6B

Amino acid sequences of the humanized heavy chain variable region

IgG Amino acid sequence of VH SEQ ID NO VH1 QVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWIGVIWGDGSTNYHPNLMSRVTISVDTSKNOF SLKLS S VTA ADTAVYYCARLDYWGQGTSVTVSS 43 VH2 OVOLOESGPGLVKPSETLSLTCTVSGFSLSSYGVSWFRQPPGK GLEWLGVIWGDGSTNYHPNLMSRVTIS VDTSKNQF SLKLS S VT AADTAVYYCARLDYWGQGTS VTVS S 44 VH3 OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWIGVIWGDGSTNYHPNLMSRLVISVDTSKNQFSLKLSSVTA ADTA7YYC L7LDYWGOGTS VTVSS 45 VH4 OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWFROPPGK GLEWLGVIWGDGSTNYHPNLMSRLMSVDTSKNQFSLKLSSVT AADTAVYYCARLDYWGQGTS VT VS S 46 VH5 OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWEROPPGK GLEWLGVIWGDGSTNYHPNLMSRLMSVDTSKNOFSLKLSSVT A ADT AVY YC F7LDYWGQGT S VT VS S 47 VH6 OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWEROPPGK GLEWLGVIWGDGSTNYHPNLMSRLMSXDTSKNOF SLKLS SVT AADTA7YYC E7LDYWGQGTS VTVSS 48 VH7 OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWIGVIWGDGSTNYHPNLMSRVTISADTSKNOELLKLSSVTA ADT AVYYC F7LDYWGQGT SVT VS S 49 VH8 QVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWIGVIWGDGSTNYHPNLMSRVTISADTSK.SOF SLKLS S VTA ADT AVYYC F7LDYWGQGT SVT VS S 50 VH9 QVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDGSTNYHPNLMSRVTISVDTSK5Q ELFKLS S VT AADTAVYYC A7LDYWGQGTS VT VS S 51 VH10 OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDGSTNYHPNLMSRLMSXDTSKAQ ELLKLS S VTA ADT AVYYC E7LDYWGQGT SVT VS S 52 VH9-D54S OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGSGSTNYHPNLMSRVTISVDTSKyQ ELFKLSS VTA 53

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ADTAVYYC A7LDYWGQGTS VT VS S VH9-D54Q OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWG0GSTNYHPNLMSRVTISVDTSK50ELFKLSSVT A ADTAVYYC A7LDYWGQGTS VT VS S 54 VH9-D54E OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGEGSTNYHPNLMSRVTIS VDTSK5Q EEFKLS S VTA ADTAVYYC A7LDYWGQGTSVT VS S 55 VH9-D54A OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGAGSTNYHPNLMSRVTISVDTSKAQ EC H<LS S VT AADTAVYYC A7LDYWGQGTS VT VS S 56 VH9-D54H OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGHGSTNYHPNLMSRVTISVDTSK50ELFKLSSVT AADTAVYYC A7LDYWGQGTSVT VS S 57 VH9-G55A OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDASTNYHPNLMSRVTISVDTSKAQ EC H<LS S VT AADTAVYYC A7LDYWGQGTSVT VS S 58 VH9-G55V OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDVSTNYHPNLMSRVTISVDTSKAQ ELFKLS S VT AADTAVYYC A7LDYWGQGTSVT VS S 59 VH9- M64V OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDGSTNYHPNLVSRVTISVDTSK50 ELFKLSS VTA ADTAVYYC A7LDYWGQGTSVT VS S 60 VH9-M64I OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDGSTNYHPNLISRVTIS VDTSK5Q ELFKLS S VTA ADTAVYYC A7LDYWGQGTSVT VS S 61 VH9- M64L OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDGSTNYHPNLLSSRVTISVDTSKAQ ELFKLS S VTA ADTAVYYC A7LDYWGQGTSVT VS S 62 VH9- M64A OVOLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIROPPGKG LEWLGVIWGDGSTNYHPNLASRVTISVDTSKAQ ELFKLSS VTA ADTAVYYC A7LDYWGQGTSVT VS S 63

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Table 6C

Amino acid sequence of CDR VL4

IgG CDR-L1 CDR-L2 CDR-L3 Amino acid SEQ Amino acids SEQ Amino acid SEQ ID t sequen ce ID NO sequence ID NO subsequence NO VL4 RSSQSLVH RNGNTYLH 20 KVSNRFS 21 FQTTYVPNT 22 VL4- N33S RSSQSLVH RSGNTYLH 64 KVSNRFS 21 FQTTYVPNT 22 VL4- N33Q RSSQSLVH RQGNTYLH 65 KVSNRFS 21 FQTTYVPNT 22 VL4- N33E RSSQSLVH REGNTYLH 66 KVSNRFS 21 FQTTYVPNT 22 VL4- N33A RSSQSLVH RAGNTYLH 67 KVSNRFS 21 FQTTYVPNT 22 VL4- N33H RSSQSLVH RHGNTYLH 68 KVSNRFS 21 FQTTYVPNT 22 VL4- G34A RSSQSLVH RAGNTYLH 69 KVSNRFS 21 FQTTYVPNT 22 VL4- G34V RSSQSLVH RVGNTYLH 70 KVSNRFS 21 FQTTYVPNT 22

Table 6D

Amino acid sequence of CDR VH9

IgG CDR-H1 CDR-H2 CDR-H3 Amino acid sequence SEQ ID NO Amino acid sequence SEQ ID NO Amino acid sequence SEQ ID NO VH9 GFSLSSYG VS 17 VIWGDGSTNYHPN LMS 18 LDY 19 VH9- D54S GFSLSSYG VS 17 VIWGSGSTNYHPN LMS 71 LDY 19 VH9- D54Q GFSLSSYG VS 17 VIWGQGSTNYHPN LMS 72 LDY 19 VH9- D54E GFSLSSYG VS 17 VIWGEGSTNYHPN LMS 73 LDY 19 VH9- GFSLSSSYG 17 VIWGAGSTNYHPN 74 LDY 19

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D54A VS LMS VH9- D54H GFSLSSYG VS 17 VIWGHGSTNYHPN LMS 75 LDY 19 VH9- G55A GFSLSSYG VS 17 VIWGDASTNYHPN LMS 76 LDY 19 VH9- G55V GFSLSSYG VS 17 VIWGDVSTNYHPN LMS 77 LDY 19 VH9- M64V GFSLSSYG VS 17 VIWGDGSTNYHPN LVS 78 LDY 19 VH9- M64I GFSLSSYG VS 17 VIWGDGSTNYHPN LIS 79 LDY 19 VH9- M64L GFSLSSYG VS 17 VIWGDGSTNYHPN LLS 80 LDY 19 VH9- M64A GFSLSSYG VS 17 VIWGDGSTNYHPN LAS 81 LDY 19

The amino acid sequence identities of the humanized and wild-type VL sequences were compared by sequence alignment. Of the 112-residue VL sequence, VL4 had 91.1% identity with m11-1F4, VL3 had 93.8% identity with m11-1F4, and VL3 had 98.1% identity with VL4.

Binding affinity of humanized antibodies to amyloid.

Table 7 presents the results of SPR assays measuring the binding of humanized anti-amyloid antibodies to the monomeric Len(1-22) peptide. Specifically, humanized VH9 and VL4 sequences were tested with or without additional amino acid substitutions as indicated in the Ligand column of Table 7.

Table 7

Analysis of binding of monomeric peptide Len(1-22) by SPR method

Ligand Analyte X ² (RU ² ) ka (1/M*s) k _d (1/s) K _D (M) Rm ah (RU) VH9- D54S+VL4 Monomeric peptide Len 1-22 2,29E- 02 2.30E+0 3 1,39E- 04 6.05E- 08 9.8 VH9- D54Q+VL4 Monomeric peptide Len 1-22 5,00E- 03 3.90E+0 3 9.97E- 04 2,56E- 07 21.8 VH9- D54E+VL4 Monomeric peptide Len 1-22 1,04E- 02 8.28E+0 3 1,3 5E- 03 1,63E- 07 26.8 VH9- D54A+VL4 Monomeric peptide Len 1-22 3.06E- 02 3.56E+0 2 7.78E- 04 2,18E- 06 10.7

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VH9- D54H+VL4 Monomeric Len 1-22 peptide 1,97E- 02 5.93E+0 3 1,3 9E- 03 2,3 5E- 07 15.1 VH9- G55A+VL4 Monomeric Len 1-22 peptide 7.41E- 02 2.82E+0 4 4.84E- 04 1.72E- 08 21.3 VH9- G55V+VL4 Monomeric Len 1-22 peptide 8.29E- 02 3.38E+0 1,63E- 03 4.83E- 07 9.8 VH9- M64V+VL4 Monomeric Len 1-22 peptide 6.24E- 02 2.38E+0 4 5,20E- 04 2,18E- 08 12.8 VH9- M64I+VL4 Monomeric Len 1-22 peptide 1,07E- 01 9.29E+0 4 1,92E- 03 2,07E- 08 6.5 VH9- M64L+VL4 Monomeric Len 1-22 peptide 1,16E- 01 9.30E+0 4 2,08E- 03 2,24E- 08 7,8 VH9- M64A+VL4 Monomeric Len 1-22 peptide 1,50E- 01 7.79E+0 4 1,04E- 03 1,ZZE- 08 10,5 VH9+VL4- N33S Monomeric Len 1-22 peptide 1,10E- 01 1.43E+0 4 9.54E- 04 6.69E- 08 23.8 VH9+VL4- N33Q Monomeric Len 1-22 peptide 6.49E- 02 1,ЗЗЕ+0 4 1,04E- 03 7.84E- 08 21.7 VH9+VL4- N33E Monomeric Len 1-22 peptide 5.27E- 02 1.23E+0 4 1,31E- 03 1,06E- 07 15.4 VH9+VL4- N33A Monomeric Len 1-22 peptide 5.56E- 02 1DZE+0 4 1,00E- 03 8.85E- 08 17.4 VH9+VL4- N33H Monomeric Len 1-22 peptide 7,20E- 02 1.31E+0 4 7.43E- 04 5.66E- 08 17.6 VH9+VL4- G34A Monomeric Len 1-22 peptide 9.04E- 01 5.95E+0 4 1,81E- 03 3.04E- 08 24.9 VH9+VL4- G34V Monomeric Len 1-22 peptide 5.83E- 01 4D8E+0 4 1,46E- 03 3.50E- 08 29.7 VH9+VL4 Monomeric Len 1-22 peptide 1.88E+ 00 6.04E+0 4 2,60E- 03 4.3 0E- 08 47.2

Example 4. Binding of anti-amyloid antibodies fused with p5 peptide to amyloid fibrils and cell extracts.

The following example describes enzyme-linked immunosorbent assays and affinity adsorption experiments that test the ability of humanized anti-amyloid antibodies to bind amyloid fibrils and human AL and ATTR amyloid extracts.

Materials and methods.

Amyloid antibodies tested.

Humanized amyloid antibodies were used as described in Example 3. In addition, p5 peptide variants with lysine (p5) or arginine (p5R) residues were fused to the N-terminus of the light chain to generate VH6/VL3-p5, VH6/VL3-p5R, VH9/VL4-p5, and VH9/VL4-p5R. The amino acid sequence of p5R is shown in SEQ ID NO: 2 (see Table 1).

Europium immunosorbent assays (EuLISA).

For europium immunosorbent assays (EuLISA), suspensions of rVX6Wil fibrils (sonicated) were prepared at a concentration of 0.83 μM in phosphate-buffered saline (PBS). Fibrils were applied to the wells of a 96-well microplate by adding 50 μL to each well. The plates were dried at 37°C overnight. The wells of the microplate were blocked by adding Superblock (Thermofisher) (SB) at a rate of 200 μL per well and left at 37°C for 1 h. Primary (test) antibody (i.e., humanized antibody) prepared in SB + 0.05% Tween-20 (SBT) was added in a 1:2 dilution series, starting from 1 μM, using 100 μL/well. The plates were then incubated for 1 hour at 37°C. After a washing step (the plates were washed three times using PBS+0.05% Tween-20), the secondary antibody was added as a 1:3000 dilution of biotinylated goat anti-human IgG (Sigma) in SBT at 100 μl/well. The plates were incubated for 1 hour at 37°C. After another washing step, 100 μl/well of europium/streptavidin dilution (Perkin

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Elmer) 1:1000 in SBT. The plates were incubated for 1 hour at 37°C. After the last wash step, 100 μl/well of europium enhancement solution (Perkin Elmer) were added. Time-resolved fluorescence emission was recorded using a microplate reader (Wallac).

Affinity adsorption binding assay.

To prepare synthetic amyloid fibrils from rV/6Wil, a 1 mL volume containing 1 mg/mL monomer in phosphate-buffered saline (PBS), 0.01% w/v NaN3, pH 7.5, was filtered through a 0.2 mm pore size filter, added to a 15 mL conical polypropylene tube (BD BioSciences, Bedford, MA) and shaken at 45° at 225 rpm for 3-5 days at 37°C until the reaction mixture became opaque.

Purified human amyloid tissue extracts were prepared using autopsy tissues from patients with light chain (AL) or transthyretin (ATTR) amyloidosis using the water flotation method as described by Pras et al. (Pras, M. et al. J Clin Invest. 1968 Apr; 47(4): 924-933) without modification. Purified amyloid material recovered by water washing and the amyloid-rich pellet were collected and stored lyophilized at room temperature until use.

For affinity adsorption assays, twenty-five microliters of 1 mg/mL AL extract or synthetic rV/6Wil variable domain fibrils (Wall, J. et al. Biochemistry 38 (1999) 14101–14108) were centrifuged in a 0.5-mL microcentrifuge tube at 21,000 x g for 5 min. The supernatant was removed and the pellet was resuspended in 200 μL PBS with 0.05% Tween-20 (PBST). Ten microliters of a 1:100 dilution of ¹²⁵ I-p5+14 stock solution (~100,000 counts/minute (cpm); ~5 ng peptide) was added to the suspension. The mixture was rotated at room temperature for 1 h. The samples were then centrifuged twice at 15,000 x g for 10 min. The supernatants and pellets were separated after each step and the radioactivity in each was measured using a Cobra II gamma counter (Perkin Elmer) with data recording for 1 min. The percentage of ¹²⁵ I-p5+14 bound to the pellet was determined as follows: CPM pellet/(CPM pellet + CPM supernatant) x 100

Results.

EuLISA.

The ability of humanized anti-amyloid antibodies to bind rV/6Wil fibrils was tested by EuLISA (Fig. 13A). Based on the data shown in Fig. 13A, c11-1F4 bound with an _EC50 value of ~72 nM, VH10/VL4 bound with an _EC50 value of 17 nM, VH9/VL4 bound with an _EC50 value of 7 nM, VH8/VL4 bound with an _EC50 value of 16 nM, VH7/VL4 bound with an _EC50 value of 75 nM and VH6/VL3 bound with an _EC50 value of 95 nM. VH9/VL4 showed increased binding compared to c11-1F4 (Fig. 13A). VH9/VL4 antibody bound amyloid fibrils to a greater extent than VH6/VL3 (Fig. 13A). VH9/VL4 and VH6/VL3 were selected for further refinement.

As shown in Fig. 13B, the addition of the p5 and p5R peptides to the N-terminus of the VH6/VL3 light chain enhanced binding to rV/_6Wil fibrils by approximately 30-fold (based on _EC50 ). Based on these data presented in Fig. _13B , VH6/VL3-p5 bound with an _EC50 of 3 nM, VH6/VL3-p5R bound with an EC50 of 3 nM, c11-1F4 bound with an _EC50 of ~100 nM, and VH6/VL3 bound with an _EC50 of 95 nM.

Overall, the p5 arginine variant (p5R) variants were superior to the p5 variants (Fig. 13C and Fig. 13D). This result is consistent with previous studies of the peptides alone (Wall, J.S. et al. PLoS One. 2013 Jun 4;8(6):e66181).

As shown in Fig. 13E, VH9/VL4 had the same reactivity towards rV/6Wil fibrils as the mouse parent antibody.

As shown in Fig. 13E and Fig. 13F, both VH6/VL3-p5 and VH6/VL3-p5R exhibited binding to hATTR-amyloid extracts. Based on the data in Fig. 13E and Fig. 13F, VH6/VL3-p5 bound to Sno hATTR extract with an EC ₅₀ value of 50 nM and Ken ATTR extract with an EC ₅₀ value of 90 nM, and VH6/VL3-p5R bound to Sno ATTR extract with an EC ₅₀ value of 47 nM, Ken ATTR extract with an EC ₅₀ value of 70 nM, and Per125 wtATTR with an EC ₅₀ value of 85 nM.

Table 8 below presents the results for ELISAs measuring the ability of mIgp5, hIgGl, c11-1F4, m11-1F4, VH6/VL3-p5, VH9/VL4-p5, and VH9/VL4-p5R to bind rV/6Wil fibrils, Per125 wtATTR extract, KEN hATTR extract, SHI ALA liver extract, and TAL ALk liver extract. For each antibody and substrate combination, the log-transformed _EC50 , EC50, and maximum binding in the assay are shown. Conditions marked n/a were not tested.

As shown in Table 8, humanized anti-amyloid antibodies fused to p5 or p5R were able to bind a variety of amyloid fibrils and amyloid extracts. VH6/VL3-p5, VH9/VL4-p5, and VH9/VL4-p5R bound all fibrils and extracts tested with higher affinity (based on EC ₅₀ measurements) than m1-1F4 and all other control antibodies. VH9/VL4-p5R generally exhibited lower EC ₅₀ values than VH9/VL4-p5, and VH9/VL4-p5 generally exhibited lower EC ₅₀ values than VH6/VL3-p5.

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Table 8

EuLISA data_______________________

mlgp5 Substrate LogEC ₅₀ ec ₅₀ Max. rVZ6Wil 8,541 2,88E-09 58.5 Perl25 wtATTR 8,205 6,24E-09 31.7 KEN HATTR 8,448 3,56E-09 19.9 SHI ΑΕλ liver 8,532 2,94E-09 24,0 TAL ALk liver 8,472 3,37E-09 24.6 hlgGl Substrate LogEC ₅₀ ec ₅₀ Max. rVZ6Wil 5,894 l,28E-06 39.3 Perl25 wtATTR -7,917 n/a - 8,378 KEN HATTR -9,061 n/a -0.8 SHI ΑΕλ liver -7,878 n/a 2,5 TAL ALk liver -3,001 n/a -2217 C11-1F4 Substrate LogEC ₅₀ yes ₅₀ Max. rVZ6Wil 6,536 2,91E-07 63.9 Perl25 wtATTR 6,419 3.81E-07 71,12 KEN HATTR --1,360 n/a - 19728 SHI ΑΕλ liver 6,592 2,56E-07 51.31 TAL ALk liver - 0.5994 n/a - 17788

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mll-lF4 Substrate LogECso EC ₅ o Max. rVZ6Wil 6,265 5,43E-07 183.6 Perl25 wtATTR 5,725 l,88E-06 208.8 KEN HATTR -2,386 n/a - 294290 SHI ΑΕλ liver - 4,447 n/a - 1506 TAL ALk liver -3,028 n/a -35143 VH6/VL3-p5 Substrate LogECso ECso Max. rVZ6Wil 8,457 3.49E-09 65.3 Perl25 wtATTR 7,404 3.94E-08 33.9 KEN HATTR 7.18 6,61E-08 16.9 SHI ΑΕλ liver 7,303 4.98E-08 29.8 TAL ALk liver 7,314 4.85E-08 29,5 VH9/VL4-p5 Substrate LogECso ESeo Max. rVZ6Wil 8,393 4.05E-09 71,02 Perl25 wtATTR 7,611 2,45E-08 58.06 KEN HATTR 7,357 4,40E-08 37.79 SHI ΑΕλ liver 7,709 1,95E-08 41.93 TAL ALk liver 7,613 2,44E-08 60,05 VH9/VL4-p5R Substrate LogECso EU ₅ o Max. rVX6Wil 8,408 3.91E-09 82.68 Perl25 wtATTR 7,855 1.40E-08 55.74 KEN HATTR 7,683 2.07E-08 26.64 SHI ΑΕλ liver 7,823 1.50E-08 40 TAL ALk liver 7,901 1.26E-08 45.28

Affinity adsorption experiments.

The ability of anti-amyloid antibodies to immunoprecipitate substrates was studied. mIgG-p5 demonstrated excellent binding to Wil fibrils and amyloid extracts in an affinity adsorption assay (Fig. 14).

The ability of the parent VH9/VL4 protein and its variants to affinity adsorb substrates was significantly reduced compared to mIgp5, as shown in Table 9 below. In Table 9, values reported represent percentage binding, and cells with no data represent antibody/substrate combinations that were not tested.

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Table 9

Summary of Affinity Adsorption Experiments _______

Substrate mlgp5 VH9/VL4-p5 VH9/VL4-p5R VH9/VL4 VH6/VL3-p5 Synthetic fibrils rVZ6Wil 71 24.73 22.62 10.45 22.53 Fibrils Αβ(1-40) 7.38 20.57 Fibril hlAPP 2.46 4.82 Particles with Vk4(LEN(1-22) 54.44 50.17 57.44 54.87 HIGH ALKI 10 0.19 0.51 TAL ALk 37 0.72 0.94 SHI ΑΕλ 34 1.97 1,22 0.68 0,60 TYL ΑΕλ 21 0.56 0.69 CAB ALk4 2.43 2.80 SNO hATTR 12 0.41 0.49 KEN HATTR 15 0.61 0.79 wtATTR-PER125 31 1.33 1,12 1.36 1,00 wtATTR-PER253 17 0.80 1.58

Example 5. Anti-amyloid antibodies fused to p5 peptide and ex vivo phagocytosis.

The following example describes experiments to test the ability of humanized amyloid antibodies to act as opsonins. In particular, the level of phagocytosis of amyloid fibrils was measured ex vivo in the presence of amyloid antibodies.

Materials and methods.

Phagocytosis ex vivo.

For the solid-phase uptake assay of Wil fibrils, 24-well tissue culture plates were coated with rat collagen type I (75 μg/ml in 20 mM acetic acid, 0.4 ml) for 2 h at room temperature, washed with 0.5 ml PBS, and coated with 20 μg/well pHrodo Red-labeled rVX6 fibrils (30% labeled fraction) overnight in 0.5 ml PBS at 4°C. Wells were washed with 0.5 ml PBS, and 0.5 ml serum- and phenol red-free RPMI 1640 was added. Opsonin antibodies were added at 5 μg per well, followed immediately by the addition of RAW 264.7 cells or uninduced THP-1 cells in serum-free, phenol red-free RPMI 1640 (1.2 x 10 ⁶ in 0.5 ml) for 4 h of incubation at 37°C. For absorbance measurements, cells from each well were transferred to triplicate wells of a black, clear-bottomed 96-well microplate (Corning) for fluorescence measurement in a BioTek SynergyHT-1 microplate reader at 530/25 nm excitation and 645/40 nm emission in well scanning mode. Background values from wells incubated in 1 ml of medium only were subtracted to obtain relative fluorescence units.

Results.

The ability of humanized amyloid antibodies to act like opsonins against amyloid fibrils (i.e., to stimulate phagocytosis of amyloid fibrils) was tested.

VH9/VL4-p5 and VH9/VL4-p5R stimulated rVX6Wil fibril uptake better than VH6/VL3p5 and VH6/VL3-p5R, consistent with the difference in ELISA binding data described above (see Fig. 15A and Fig. 15B). VH9/VL4 without peptide was approximately as effective as VH6/VL3 with attached p5 or p5R, and many-fold better than VH6/V13 without peptide, as shown in Fig. 15C.

Only VH9/VL4 was a more effective opsonin than C11-1F4 (Fig. 15B).

VH6/VL3-p5 and VH6/VL3-p5R promoted equivalent fibril uptake levels compared to mIgp5 and performed better than C11-1F4 (Fig. 15A). VH9/VL4-p5 and VH9/VL4-p5R also promoted equivalent fibril uptake levels compared to mIgp5 and performed better than C11-1F4 (Fig. 15B).

Surprisingly, humanized anti-amyloid antibodies conjugated to p5 or p5R peptides provided significantly better opsonization than c11-1F4.

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Example 6. Measurement of pharmacokinetic parameters for VH9/VL4 in wild-type mice.

The following example describes the evaluation of the pharmacokinetics of VH9/VL4 administered to wild-type mice. Materials and Methods.

Introduction of VH9/VL4.

VH9/VL4 was labeled with ^125I and injected into wild-type mice.

Results.

^125I -labeled VH9/VL4 was injected into wild-type mice and the level of ^125I -labeled VH9/VL4 present over time was measured (Fig. 16).

By fitting the linear elimination component (larger dots in Fig. 16) to the curve, the half-life was calculated as (-0.693/-slope): t _1/2 = 173.5 h (7 days).

By fitting the entire curve (the smaller dots with the dashed line in Fig. 16) to a double exponential decay, the half-life was calculated as (-0.693/-k ₂ ): t _1/2 = 86.6 h (3.6 days).

Example 7. Biodistribution of VH9/VL4 in wild-type mice.

The following example describes the biodistribution assessment of VH9/VL4 conjugated to p5 or p5R administered to wild-type mice.

Materials and methods.

Wild-type mice were injected with VH9/VL4-p5 or VH9/VL4-p5R, and antibody levels were measured in muscle, liver, pancreas, spleen, left and right kidneys, stomach, upper and lower intestines, heart, lungs, and blood 48 hours after injection (Fig. 17A and Fig. 17B).

Results.

There was no evidence of specific accumulation of VH9/VL4-p5 (Fig. 17A) or VH9/VL4-p5R (Fig. 17B) in wild-type mice. The high blood pool at 48 h was consistent with pharmacokinetic assessment of baseline IgG. The high lung activity was due to blood infiltration at the time of euthanasia.

Example 8. Testing peptide integrity and plasma stability

The following example describes SDS-PAGE and peptide capture ELISA experiments to assess the integrity and stability of humanized anti-amyloid antibodies conjugated to p5 and p5R in plasma.

Materials and methods.

Peptide capture ELISA.

To measure the stability of VH6/VL3-p5 in mouse plasma, a peptide capture ELISA was performed. 100 nM VH6/VL3-p5 was added to either PBS at 4°C, PBS at 37°C, EDTA plasma at 37°C, or heparin plasma at 37°C (Fig. 18), and the amount of intact VH6/VL3-p5 present was measured over 72 h. VH6/VL3-p5 was captured using an anti-human Fc antibody and detected with a biotinylated mAb against the p5 peptide. The signal was detected using streptavidin-conjugated HRPO and quantified using a plate reader.

Results.

In addition, binding of monoclonal antibodies to the p5 peptide and heparin binding studies demonstrated that the peptide is present on IgG (data not shown).

In a mouse plasma stability assay, VH6/VL3-p5 levels were unchanged over 72 h in fresh mouse plasma (Fig. 18).

Sequences

All polynucleotide sequences are shown in the 5'^3 direction. All polypeptide sequences are shown in the N-terminal to C-terminal direction.

VH sequence 11-1F4 (SEQ ID NO: 15)

QVQLKESGPGLVAPSQSLSITCTVSGFSLSSYGVSWVRQPPGKGLEWLGVIWGD

GSTNYHPNLMSRLSISKDISKSQVLFKLNSLQTDDTATYYCVTLDYWGQGTSVTVSS

Sequence VL 11-1F4 (SEQ ID NO:16)

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIY

KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCFQTTYVPNTFGGGTKLEIK

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CDR-H1 sequence 11-1F4 (SEQ ID NO: 17)

GFSLSSYGVS

CDR-H2 sequence 11-1F4 (SEQ ID NO: 18)

VIWGDGSTNYHPNLMS

CDR-H3 sequence 11-1F4 (SEQ ID NO: 19)

LDY

CDR-L1 sequence 11-1F4 (SEQ ID NO:20)

RSSQSLVHRNGNTYLH

CDR-L2 sequence 11-1F4 (SEQ ID NO:21)

KVSNRFS

CDR-L3 sequence 11-1F4 (SEQ ID NO:22)

FQTTYVPNT

5' spacer sequence (SEQ ID NO:23)

AQAGQAGQAQGGGYS

3' spacer sequence (SEQ ID NO:24)

VTPTV

Igp5 light chain construct (SEQ ID NO:25)

AQAGQAGQAQGGGYSKAQKAQAKQAKQAQKAQKAQAKQAKQVTPTVDVVM TQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAEDLGLYFCFQTTYVPNTFGGGTKLEIK

p5-3'cneficep-VL 11-1F4 sequence (SEQ ID NO:26)

KAQKAQAKQAKQAQKAQKAQAKQAKQVTPTVDVVMTQTPLSLPVSLGDQASI SCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDLGLYFCFQTTYVPNTFGGGTKLEIK

Linker sequence (SEQ ID NO:27)

GGGYS

IGKV2-30*02- human germline sequence (SEQ ID NO:28)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYK VSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPP

Human VL acceptor sequence (SEQ ID NO:29) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQTTYVPNTFGGGTKLEIK

IGHV4-4*08- human germline sequence (SEQ ID NQ:30)

QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYTSGS TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

Human VL acceptor sequence (SEQ ID NO:31)

QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYTSGS TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDYWGQGTSVTVSS

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VL1 (SEQ ID NO:32)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRRLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQTTYVPNTFGGGTKLEIK

VL2 (SEQ ID NO:33)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWYLQRPGQSPRRLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQTTYVPNTFGGGTKLEIK

VL3 (SEQ ID NO:34)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWYLQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGLYFCFQTTYVPNTFGGGTKLEIK

VL4 (SEQ ID NO:35)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VL4-N33S (SEQ ID NO:36)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRSGNTYLHWFQQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VL4-N33Q (SEQ ID NO:37)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRQGNTYLHWFQQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VL4-N33E (SEQ ID NO:38)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHREGNTYLHWFQQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VL4-N33A (SEQ ID NO:39)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRAGNTYLHWFQQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VL4-N33H (SEQ ID NQ:40)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRHGNTYLHWFQQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VL4-G34A (SEQ ID NO:41)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRAGNTYLHWFQQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VL4-G34V (SEQ ID NO:42)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRVGNTYLHWFQQRPGQSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCFQTTYVPNTFGGGTKLEIK

VH1 (SEQ ID NO:43)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWIGVIWGDG STNYHPNLMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDYWGQGTSVTVSS

VH2 (SEQ ID NO:44)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWVRQPPGKGLEWLGVIWGD GSTNYHPNLMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDYWGQGTSVTVSS

VH3 (SEQ ID NO:45)

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QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWIGVIWGDG STNYHPNLMSRLSISVDTSKNQFSLKLSSVTAADTATYYCVTLDYWGQGTSVTVSS

VH4 (SEQ ID NO:46)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWVRQPPGKGLEWLGVIWGD GSTNYHPNLMSRLSISVDTSKNQFSLKLSSVTAADTAVYYCARLDYWGQGTSVTVSS

VH5 (SEQ ID NO:47)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWVRQPPGKGLEWLGVIWGD GSTNYHPNLMSRLSISVDTSKNQFSLKLSSVTAADTAVYYCVTLDYWGQGTSVTVSS

VH6 (SEQ ID NO:48)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWVRQPPGKGLEWLGVIWGD GSTNYHPNLMSRLSISKDTSKNQFSLKLSSVTAADTATYYCVTLDYWGQGTSVTVSS

VH7 (SEQ ID NO:49)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWIGVIWGDG STNYHPNLMSRVTISKDTSKNQVLLKLSSVTAADTAVYYCVTLDYWGQGTSVTVSS

VH8 (SEQ ID NO:50)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWIGVIWGDG STNYHPNLMSRVTISKDTSKSQFSLKLSSVTAADTAVYYCVTLDYWGQGTSVTVSS

VH9 (SEQ ID NO:51)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGDG STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH10 (SEQ ID NO:52)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGDG STNYHPNLMSRLSISKDTSKSQVLLKLSSVTAADTAVYYCVTLDYWGQGTSVTVSS

VH9-D54S (SEQ ID NO:53)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGSG STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-D54Q (SEQ ID NO: 54)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGQG STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-D54E (SEQ ID NO: 55)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGEG STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-D54A (SEQ ID NO: 56)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGAG STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-D54H (SEQ ID NO:57)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGHG STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-G55A (SEQ ID NO:58)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGDA

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STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-G55V (SEQ ID NO:59)

Q VQLQESGPGLVKP SETLSLTCTVSGF SLS S YGVSWIRQPPGKGLEWLGVIWGD V

STNYHPNLMSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-M64V (SEQ ID NQ:60)

Q VQLQESGPGLVKP SETLSLTCTVSGF SLS SYGVSWIRQPPGKGLEWLGVIWGDG

STNYHPNLVSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-M64I (SEQ ID NO:61)

Q VQLQESGPGLVKP SETLSLTCTVSGF SLS SYGVSWIRQPPGKGLEWLGVIWGDG

STNYHPNLISRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-M64L (SEQ ID NO:62)

Q VQLQESGPGLVKP SETLSLTCTVSGF SLS SYGVSWIRQPPGKGLEWLGVIWGDG

STNYHPNLLSSRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

VH9-M64A (SEQ ID NO:63)

Q VQLQESGPGLVKP SETLSLTCTVSGF SLS SYGVSWIRQPPGKGLEWLGVIWGDG

STNYHPNLASRVTISVDTSKSQVLFKLSSVTAADTAVYYCATLDYWGQGTSVTVSS

CDR-L1 VL4-N33S (SEQ ID NO: 64)

RSSQSLVHRSGNTYLH

CDR-L1 VL4-N33Q (SEQ ID NO: 65)

RSSQSLVHRQGNTYLH

CDR-L1 VL4-N33E (SEQ ID NO: 66)

RSSQSLVHREGNTYLH

CDR-L1 VL4-N33A (SEQ ID NO: 67)

RSSQSLVHRAGNTYLH

CDR-L1 VL4-N33H (SEQ ID NO: 68)

RSSQSLVHRHGNTYLH

CDR-L1 VL4-G34A (SEQ ID NO: 69)

RSSQSLVHRAGNTYLH

CDR-L1 VL4-G34V (SEQ ID NO: 70)

RSSQSLVHRVGNTYLH

CDR-H2 VH9-D54S (SEQ ID NO: 71)

VIWGSGSTNYHPNLMS

CDR-H2 VH9-D54Q (SEQ ID NO: 72)

VIWGQGSTNYHPNLMS

CDR-H2 VH9-D54E (SEQ ID NO: 73)

VIWGEGSTNYHPNLMS

CDR-H2 VH9-D54A (SEQ ID NO: 74)

VIWGAGSTNYHPNLMS

CDR-H2 VH9-D54H (SEQ ID NO: 75)

VIWGHGSTNYHPNLMS

CDR-H2 VH9-G55A (SEQ ID NO: 76)

VIWGDASTNYHPNLMS

CDR-H2 VH9-G55V (SEQ ID NO: 77)

VIWGDVSTNYHPNLMS

CDR-H2 VH9-M64V (SEQ ID NO: 78)

VIWGDGSTNYHPNLVS

CDR-H2 VH9-M64I (SEQ ID NO: 79)

VIWGDGSTNYHPNLIS

CDR-H2 VH9-M64L (SEQ ID NO: 80)

VIWGDGSTNYHPNLLS

CDR-H2 VH9-M64A (SEQ ID NO: 81)

VIWGDGSTNYHPNLAS

N-terminus of Ig light chain (SEQ ID NO: 82)

DVVMTQTP

-

Claims (26)

CLAUSE OF INVENTION 1. Modified immunoglobulin containing: (i) an amyloid-reactive peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14; and (ii) an Ig antibody or antigen-binding fragment thereof that binds to human amyloid fibrils, wherein the Ig antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein: a) VL comprises a CDR-L1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; or b) VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 20; 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, and VH comprising CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, CDRH2 comprising the amino acid sequence set forth in any of SEQ ID NOs: 71-81; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; c) VL comprises a CDR-L1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 64-70, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-81; and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19; and wherein the modified immunoglobulin is a fusion protein comprising an Ig antibody or an antigen-binding fragment thereof fused to an amyloid-reactive peptide. 2. A modified immunoglobulin according to claim 1, characterized in that the amyloid-reactive peptide and the Ig antibody or its antigen-binding fragment are linked together at the N-terminus of the Ig light chain or at the N-terminus of the Ig heavy chain. 3. A modified immunoglobulin according to item 2, characterized in that: a) VL comprises a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, and VH comprises a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19. 4. The modified immunoglobulin according to claim 3, characterized in that VL contains Leu at position 46 and Phe at position 87, and VH contains Leu at position 48, Ser at position 96, Val at position 78, Leu at position 79, Phe at position 80 and Thr at position 94. 5. The modified immunoglobulin according to claim 3, characterized in that VL comprises the amino acid sequence indicated in SEQ ID NO: 36, and VH comprises the amino acid sequence indicated in SEQ ID NO: 55. 6. A modified immunoglobulin according to any one of claims 1 to 5, characterized in that the modified immunoglobulin is humanized. 7. A modified immunoglobulin according to any one of claims 1 to 6, characterized in that the amyloid-reactive peptide is fused to the N-terminus of the light chain of the antibody or the C-terminus of the heavy chain of the antibody. 8. A modified immunoglobulin according to claim 7, characterized in that the amyloid-reactive peptide is fused to the N-terminus of the light chain of the antibody using a spacer or to the C-terminus of the heavy chain of the antibody using a spacer. 9. A modified immunoglobulin according to claim 8, characterized in that the spacer is a peptide spacer. 10. A modified immunoglobulin according to claim 9, characterized in that the spacer contains the amino acid sequence GGGYS. 11. A modified immunoglobulin according to any one of claims 1 to 10, characterized in that the antibody contains an Fc region. 12. A modified immunoglobulin according to any one of claims 1 to 11, characterized in that the Fc region is of the IgG1, IgG2, IgG3 or IgG4 isotype. 13. A modified immunoglobulin according to any one of the preceding claims, characterized in that the modified immunoglobulin binds to rVL6Wi1 fibrils, Per125 wtATTR extract, KEN hATTR extract, SHI ALL liver extract, TAL ALk liver extract, AB, Αβ(1-40), IAAP, ALk4, A1L1 or ATTR fibrils. - 82 048681 14. A pharmaceutical composition containing a modified immunoglobulin according to any one of claims 1-13. 15. Nucleic acid(s) encoding a modified immunoglobulin according to any one of claims 1 to 14. 16. A vector containing the nucleic acid(s) according to claim 15. 17. A host cell containing the vector according to claim 16. 18. A method for producing a modified immunoglobulin, comprising culturing the host cell according to claim 17 under conditions suitable for expressing a vector encoding the modified immunoglobulin. 19. A method of treating a subject having an amyloid-associated disorder, comprising administering an effective amount of a modified immunoglobulin according to any one of claims 1-13 to a subject having an amyloid-associated disorder. 20. The method according to claim 19, characterized in that the amyloid-associated disorder is amyloidosis. 21. The method according to claim 19, characterized in that the amyloid-associated disorder is selected from the group consisting of AL-, AH-, Av2M-, ATTR-, transthyretin, AA-, AApoAI-, AApoAII-, AGel-, ALys-, ALEct2-, AFib-, ACys-, ACal-, AMed-, AIAPP-, APro-, AIns-, APrP- or Av-amyloidosis. 22. The method according to any of paragraphs 19-21, characterized in that the subject is a human being. 23. A method of treating a subject who has or is suspected of having an amyloid-associated disease, comprising determining whether the subject has an amyloid deposit by detectably labeling a modified immunoglobulin according to any one of claims 1-13, administering the modified immunoglobulin to the subject, determining whether a signal associated with the detectable label can be detected in the subject; and, if the signal is detected, administering to the subject an agent for treating amyloidosis. 24. A method for detecting an amyloid deposit in a subject, comprising detectably labeling a modified immunoglobulin according to any one of claims 1-13, administering the modified immunoglobulin to the subject, and detecting a signal from the modified immunoglobulin. 25. A method of targeting an amyloid deposit for clearance, comprising contacting the amyloid deposit with a modified immunoglobulin according to any one of claims 1 to 13. 26. A method for detecting a ligand, comprising contacting the ligand with a modified immunoglobulin according to any one of claims 1 to 13, wherein the modified immunoglobulin comprises a detectable label, wherein the modified immunoglobulin peptide has a binding affinity for the ligand, and detecting a signal from the detectable label, thereby detecting the ligand. -