Immunoglobulin light chain antibodies and uses thereof

Abstract

The present disclosure is directed to antibodies or antigen-binding portions thereof that specifically bind immunoglobin free light chain (FLC), polynucleotides and vectors encoding them, and pharmaceutical compositions comprising them. Some embodiments of the present disclosure are directed to a method of measuring FLC in a biological sample, comprising contacting the biological sample with an anti-FLC antibody. Some embodiments of the present disclosure are directed to a method of treating a disease or disorder, comprising administering an anti-FLC antibody to a subject in need of treatment for the disease or disorder.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 125,281, filed December 14, 2020, the entirety of which is incorporated herein by reference. [Background technology]

[0002] AL amyloidosis is a widely underdiagnosed disease caused by misfolding of immunoglobulin free light chains (LC) that can be deposited as light chain amyloid (AL) in the heart, kidneys, or liver. The disease is caused by a causative plasma cell dyscrasia that results in plasma cells secreting a large excess of immunoglobulin free kappa (κ) and / or lambda (λ) light chains into the circulation. The disease is caused by the deposition of unique light chains secreted by a single clonal plasma cell population.

[0003] However, little is known about the origin of AL, and therefore there is a crucial need to develop tools to uncover the mechanisms underlying AL formation in order to design optimal therapeutic agents. Summary of the Invention

[0004] Described herein are specific epitopes of human light chains and antibodies that bind to said epitopes. The epitopes described herein are advantageous in that they allow for the production and / or screening of antibodies that bind to and promote the clearance of misfolded light chains, which are the etiological agents of light chain amyloidosis. One of the obstacles to the treatment of light chain amyloidosis is that different patients may produce light chains with different amino acid sequences that do not bind equally well by existing anti-light chain antibodies that target the variable region. The antibodies described herein target epitopes in the constant regions of kappa and lambda light chains, allowing for more consistent clinical outcomes and less variability in results between patients. Additionally, the use of the disclosed antibodies as part of a diagnostic test for light chain amyloidosis can provide the additional advantage of universality of binding. Furthermore, the antibodies described herein are specific for misfolded light chains and have little reactivity to properly folded light chains, thus reducing the potential for undesirable immunological side effects that result from low antibody levels.

[0005] Certain embodiments of the present disclosure are directed to antibodies that specifically bind to human immunoglobulin free light chains (FLC: "anti-FLC antibodies"), where the anti-FLC antibody cross-competes with a reference antibody for binding to a kappa light chain, and the reference antibody binds to an epitope on an immunoglobulin kappa light chain that includes or consists of the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1). In some embodiments, the antibody binds to a kappa light chain at an epitope that overlaps with all or a portion of the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1). In some embodiments, the antibody binds to an epitope on a kappa light chain that includes or consists of the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1).

[0006] Certain embodiments of the present disclosure are directed to an antibody that specifically binds to a human immunoglobulin free light chain ("anti-FLC antibody"), where the anti-FLC antibody cross-competes with a reference antibody for binding to a lambda light chain, and the reference antibody binds to an epitope on an immunoglobulin lambda light chain that comprises or consists of the amino acid sequence set forth in NKYAASSYLSL (SEQ ID NO:2). In some embodiments, the antibody binds to a lambda light chain at an epitope that overlaps with the amino acid sequence set forth in NKYAASSYLSL (SEQ ID NO:2). In some embodiments, the antibody binds to an epitope on a lambda light chain that comprises or consists of the amino acid sequence set forth in NKYAASSYLSL (SEQ ID NO:2).

[0007] In some embodiments, the antibody does not bind to a complex comprising an immunoglobulin light chain covalently bound to an immunoglobulin heavy chain. In some embodiments, the FLC comprises an aggregate of immunoglobulin light chain peptides. In some embodiments, the aggregate of immunoglobulin light chain peptides comprises one or more misfolded immunoglobulin light chain peptides.

[0008] In some embodiments, the antibody binds to light chain amyloid fibrils. In some embodiments, the amyloid fibrils are collected from a biological sample obtained from the subject. In some embodiments, the biological sample is selected from blood, serum, plasma, solid tissue, and any combination thereof.

[0009] In some embodiments, the FLC comprises a fragment of an immunoglobulin light chain peptide. In some embodiments, the FLC comprises a full-length immunoglobulin light chain peptide. In some embodiments, the antibody is a polyclonal or monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the antibody comprises an antigen-binding portion of an antibody selected from the group consisting of Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers thereof, minibodies, diabodies, multimers, and bispecific antibody fragments. In some embodiments, the antibody is a single chain antibody.

[0010] In some embodiments, the antibody is fused to or associated with a label, hi some embodiments, the label comprises a chemical label, a biological label, a fluorescent label, or any combination thereof.

[0011] In some embodiments, the antibody comprises a heavy chain and a light chain. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises a heavy chain variable region complementarity determining region (VH-CDR)1, a VH-CDR2, and a VH-CDR3, where (a) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, (b) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, (c) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, or (d) any combination of (a)-(c). In some embodiments, the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, where (a) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, (b) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, and (c) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the light chain variable region comprises light chain variable region complementarity determining region (VL-CDR)1, VL-CDR2, and VL-CDR3, where (a) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, (b) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, (c) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, or (d) any combination of (a)-(c). In some embodiments, the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3, where (a) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:11, (b) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:12, and (c) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:13.

[0012] In some embodiments, the antibody comprises (a) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (b) a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (c) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (d) a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, (e) a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and (f) a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0013] In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0014] Certain aspects of the present disclosure are directed to a polynucleotide or a set of polynucleotides that encode the antibody disclosed herein. Certain aspects of the present disclosure are directed to a vector or a set of vectors that comprise the polynucleotide or a set of polynucleotides disclosed herein. In some aspects, the vector further comprises one or more regulatory elements.

[0015] Certain aspects of the present disclosure are directed to a host cell comprising an antibody disclosed herein, a polynucleotide or set of polynucleotides disclosed herein, or a vector or set of vectors disclosed herein.

[0016] Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising an antibody disclosed herein, a polynucleotide or set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, or a host cell disclosed herein, and a pharma- ceutically acceptable excipient.

[0017] Certain embodiments of the present disclosure are directed to a method for producing an antibody that specifically binds to FLC, comprising culturing a host cell as disclosed herein under suitable conditions. In some embodiments, the method further comprises isolating the antibody.

[0018] Certain aspects of the present disclosure are directed to methods of diagnosing AL-mediated amyloidosis, comprising contacting a biological sample obtained from a subject with an antibody disclosed herein.

[0019] Certain aspects of the present disclosure are directed to a method of measuring FLC in a biological sample obtained from a subject, comprising contacting the biological sample with an antibody disclosed herein.

[0020] In some embodiments, the subject has one or more amyloid deposits. In some embodiments, the subject suffers from a disease or disorder characterized by amyloid deposits. In some embodiments, the biological sample is selected from blood, serum, plasma, solid tissue, and any combination thereof.

[0021] Certain aspects of the present disclosure are directed to methods of treating a subject having one or more amyloid deposits, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein.

[0022] Certain aspects of the present disclosure are directed to methods of treating a subject having a disease or condition characterized by amyloid deposits, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein.

[0023] In some embodiments, the subject has a plasma cell disorder. In some embodiments, the subject has systemic amyloidosis. In some embodiments, the subject has AL amyloidosis.

[0024] Certain aspects of the present disclosure are directed to methods of treating a plasma cell disorder in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein.

[0025] In some embodiments, the plasma cell disorder comprises a plasma cell dysplasia, hi some embodiments, the plasma cell dysplasia is selected from monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasma cell leukemia (PCL), and any combination thereof.

[0026] Certain aspects of the present disclosure are directed to a method of treating systemic amyloidosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein. In some embodiments, the systemic amyloidosis comprises AL amyloidosis.

[0027] In some embodiments, the anti-FLC antibody reduces the size of at least one amyloid deposit in the subject compared to the size of at least one amyloid deposit in the subject before administration.In some embodiments, the anti-FLC antibody reduces the number of amyloid deposits in the subject compared to the number of amyloid deposits in the subject before administration.In some embodiments, the anti-FLC antibody reduces the rate of formation of amyloid deposits in the subject compared to the rate of formation of amyloid deposits in the subject before administration.In some embodiments, the anti-FLC antibody stops the rate of formation of amyloid deposits in the subject compared to the rate of formation of amyloid deposits in the subject before administration.

[0028] In some aspects, disclosed herein are methods of making, screening, or selecting antibodies directed to a kappa light chain constant region, comprising contacting an antibody-producing cell or its conditioned medium, a polyclonal antibody mixture, or a phage display library with a polypeptide comprising the sequence of SEQ ID NO:1. In certain embodiments, the antibody-producing cell is a hybridoma. In certain embodiments, the polypeptide is less than 50 amino acids in length. In certain embodiments, the polypeptide is less than 25 amino acids in length. In certain embodiments, the polypeptide comprises a first portion and a second portion, wherein the first portion consists of the sequence of SEQ ID NO:1 and the second portion comprises an amino acid sequence heterologous to the kappa light chain constant region. In certain embodiments, the polypeptide consists of SEQ ID NO:1. In certain embodiments, the method further comprises selecting an antibody that binds to SEQ ID NO:1.

[0029] In some aspects, disclosed herein are methods for making, screening, or selecting antibodies directed to a lambda light chain constant region, comprising contacting an antibody-producing cell or its conditioned medium, an antibody mixture, or a phage display library with a polypeptide comprising the sequence of SEQ ID NO:2. In certain embodiments, the antibody-producing cell is a hybridoma. In certain embodiments, the polypeptide is less than 50 amino acids in length. In certain embodiments, the polypeptide is less than 25 amino acids in length. In certain embodiments, the polypeptide comprises a first portion and a second portion, where the first portion consists of the sequence of SEQ ID NO:2 and the second portion comprises an amino acid sequence heterologous to the lambda light chain constant region. In certain embodiments, the polypeptide consists of SEQ ID NO:2. In certain embodiments, the method further comprises selecting an antibody that binds to SEQ ID NO:2. [Brief description of the drawings]

[0030] [Figure 1A]Figures 1A-1B are schematic diagrams of pathology-specific epitopes present in both the kappa and lambda constant domain (CL) regions of full-length FL-LC. Figure 1A shows that pathology-specific CL epitopes are epitopes that are embedded within normal natively folded FL-LC homodimers but exposed in AL amyloidosis. [Figure 1B] 1A-1B are schematic diagrams of pathology-specific epitopes present in both the kappa and lambda constant domain (CL) regions of full-length FL-LC. Figure 1B shows that pathology-specific CL epitopes are exposed in misfolded amyloidogenic intermediates during the FL-LC amyloid (FL-AL) formation pathway, which can be captured using antibodies. [Figure 2A] 2A-2B are graphs showing detection of native and unfolded full-length light chain protein by indirect ELISA using a kappa light chain isotype rabbit polyclonal antibody described herein (FIG. 2A) or a commercially available pan-specific antibody (FIG. 2B). [Figure 2B] 2A-2B are graphs showing detection of native and unfolded full-length light chain protein by indirect ELISA using a kappa light chain isotype rabbit polyclonal antibody described herein (FIG. 2A) or a commercially available pan-specific antibody (FIG. 2B). [Figure 3A] Figures 3A-B show detection of unfolded full-length kappa light chain protein by denaturing Western blot using pooled kappa (KLC) and lambda (LLC) light chains from myeloma tumors. Figure 3A is a Western blot showing that the kappa light chain isotype rabbit polyclonal antibody disclosed herein exhibits specificity for kappa light chains but not lambda light chains. [Figure 3B]Figures 3A-B show detection of unfolded full-length kappa light chain protein by denaturing Western blot using pooled kappa (KLC) and lambda (LLC) light chains from myeloma tumors. Figure 3B is a Western blot showing that the commercially available antibodies have specificity for the indicated isotypes and show no binding to the secondary antibody alone. [Figure 4] Figure 4 is a plot of the signal detected using a κ light chain isotype rabbit polyclonal antibody showing pH-dependent aggregate binding. Grey bars show relative ThT fluorescence at the indicated pH (n=3, left y-axis), open circles show the ELISA readout of the κ light chain isotype rabbit polyclonal antibody showing the relationship between binding and aggregates (n=3, right y-axis), and closed circles show the readout of a commercial anti-κ antibody showing no pH-dependent relationship (n=3, right y-axis). [Diagram 5] Figure 5A-C are immunogold electron microscopy images using a κ light chain isotype rabbit polyclonal antibody, showing binding to kappa light chain aggregates. Figure 5A shows the results of an assay performed on aggregates prepared at pH 4.5 using a κ light chain isotype rabbit polyclonal antibody, showing binding to large aggregates approximately 100 nm in size. Figure 5B and C show the results of an assay performed on aggregates prepared at pH 6 using a κ light chain isotype rabbit polyclonal antibody, showing binding to large aggregates approximately 75 nm in size. [Figure 6A] 6A-6B are scatter plots showing that a rabbit polyclonal κ light chain isotype antibody enhances the uptake of kappa light chain aggregates by THP-1 monocytic cells. Monocytes phagocytosed pHrodo-labeled aggregates approximately three-fold more frequently in the presence of a rabbit polyclonal κ light chain isotype antibody (FIG. 6B) than in the absence of antibody (FIG. 6A). [Figure 6B]6A-6B are scatter plots showing that a rabbit polyclonal κ light chain isotype antibody enhances the uptake of kappa light chain aggregates by THP-1 monocytic cells. Monocytes phagocytosed pHrodo-labeled aggregates approximately three-fold more frequently in the presence of a rabbit polyclonal κ light chain isotype antibody (FIG. 6B) than in the absence of antibody (FIG. 6A). [Figure 7] Figures 7A-J show immunohistochemical staining of cardiac tissue from a patient with lambda AL amyloidosis. Congo red staining (Figures 7A-B), Congo red fluorescence (Figures 7C-D), and DAKO anti-lambda antibody (Figures 7E-F) all co-stain for amyloid, suggesting that this is a patient with lambda light chain cardiac amyloidosis. DAKO anti-kappa (Figures 7G-H) stains other areas of the tissue, and kappa light chain isotype rabbit polyclonal antibody (Figures 8I-J) shows little to no staining, indicating no cross-reaction with lambda amyloid. [Figure 8] Figures 8A-E show immunohistochemical staining of cardiac tissue from an ATTR cardiac amyloid patient. Congo red staining (Figure 8A) and Congo red fluorescence (Figure 8B) indicate the presence of amyloid. Amyloid is not bound by the DAKO anti-lambda antibody (Figure 8C), the DAKO anti-kappa antibody (Figure 8D), or the kappa light chain isotype rabbit polyclonal antibody (Figure 8E). [Figure 9] Figure 9 shows box plots of the signal detected using anti-CL antibodies using indirect ELISA of plasma samples from various patients. Plasma from patients with AL amyloidosis showed increased signal compared to HFpEF controls, such as patients with ATTR amyloidosis. [Figure 10A] 10A-10D are the amino acid sequence (FIGS. 10A-10B) of the LX-96 anti-kappa monoclonal antibody heavy chain (FIGS. 10A and 10C) and the nucleic acid sequence (FIGS. 10C-10D) of the LX-96 anti-kappa monoclonal antibody light chain (FIGS. 10B and 10D). The signal peptide sequence is in plain text. [Figure 10B]10A-10D are the amino acid sequence (FIGS. 10A-10B) of the LX-96 anti-kappa monoclonal antibody heavy chain (FIGS. 10A and 10C) and the nucleic acid sequence (FIGS. 10C-10D) of the LX-96 anti-kappa monoclonal antibody light chain (FIGS. 10B and 10D). The signal peptide sequence is in plain text. [Figure 10C] 10A-10D are the amino acid sequence (FIGS. 10A-10B) of the LX-96 anti-kappa monoclonal antibody heavy chain (FIGS. 10A and 10C) and the nucleic acid sequence (FIGS. 10C-10D) of the LX-96 anti-kappa monoclonal antibody light chain (FIGS. 10B and 10D). The signal peptide sequence is in plain text. [Figure 10D] 10A-10D are the amino acid sequence (FIGS. 10A-10B) of the LX-96 anti-kappa monoclonal antibody heavy chain (FIGS. 10A and 10C) and the nucleic acid sequence (FIGS. 10C-10D) of the LX-96 anti-kappa monoclonal antibody light chain (FIGS. 10B and 10D). The signal peptide sequence is in plain text. [Figure 11A] 11A-11C are line graphs showing the results of an indirect ELISA using LX-96 (FIG. 11A) or a commercial control antibody (FIG. 11B) to detect native or misfolded forms of kappa light chain. Various concentrations of native or misfolded AL09 bound to LX-96 (FIG. 11A) or the commercial control (FIG. 11B) and detection was performed using TMB chemistry with the appropriate HRP-conjugated secondary antibody. [Figure 11B] 11A-11C are line graphs showing the results of an indirect ELISA using LX-96 (FIG. 11A) or a commercial control antibody (FIG. 11B) to detect native or misfolded forms of kappa light chain. Various concentrations of native or misfolded AL09 bound to LX-96 (FIG. 11A) or the commercial control (FIG. 11B) and detection was performed using TMB chemistry with the appropriate HRP-conjugated secondary antibody. [Figure 11C]Figures 11A-11C are line graphs showing the results of an indirect ELISA using LX-96 (Figure 11A) or a commercial control antibody (Figure 11B) to detect native or misfolded forms of kappa light chains. Figure 11C shows binding to LX-96 to detect another kappa light chain variant, AL12, in native or misfolded forms. [Figure 12] Figure 12 is a graphical representation of the results of a competitive ELISA using misfolded or native AL09 as competitors for binding to LX-96. Binding is shown as a percentage, with 100% representing maximum binding between LX-96 and adsorbed AL09 (no competition). Increasing concentrations of the competitor misfolded AL09 successfully competed for binding with LX-96 (bottom / black), whereas native AL09 showed no competition (top / grey). [Figure 13A] Figures 13A-D are biolayer interferometry sensorgrams of the binding interaction between LX-96 and misfolded (Figure 13A,C) or native (Figure 13B,D) kappa light chain mutants. Binding of LX-96 to its targets at a range of target concentrations was measured by wavelength (nm) change over 2000 seconds (300 seconds association, 1700 seconds dissociation). Binding and dissociation curves were fitted using Octet Systems software to determine KD, KD error, X2 and R2 values ​​(table inset, Figure 13A-D). [Figure 13B] Figures 13A-D are biolayer interferometry sensorgrams of the binding interaction between LX-96 and misfolded (Figure 13A,C) or native (Figure 13B,D) kappa light chain mutants. Binding of LX-96 to its targets at a range of target concentrations was measured by wavelength (nm) change over 2000 seconds (300 seconds association, 1700 seconds dissociation). Binding and dissociation curves were fitted using Octet Systems software to determine KD, KD error, X2 and R2 values ​​(table inset, Figure 13A-D). [Figure 13C]Figures 13A-D are biolayer interferometry sensorgrams of the binding interaction between LX-96 and misfolded (Figure 13A,C) or native (Figure 13B,D) kappa light chain mutants. Binding of LX-96 to its targets at a range of target concentrations was measured by wavelength (nm) change over 2000 seconds (300 seconds association, 1700 seconds dissociation). Binding and dissociation curves were fitted using Octet Systems software to determine KD, KD error, X2 and R2 values ​​(table inset, Figure 13A-D). [Figure 13D] Figures 13A-D are biolayer interferometry sensorgrams of the binding interaction between LX-96 and misfolded (Figure 13A,C) or native (Figure 13B,D) kappa light chain mutants. Binding of LX-96 to its targets at a range of target concentrations was measured by wavelength (nm) change over 2000 seconds (300 seconds association, 1700 seconds dissociation). Binding and dissociation curves were fitted using Octet Systems software to determine KD, KD error, X2 and R2 values ​​(table inset, Figure 13A-D). [Figure 14] Figures 14A-B are images of native Western blots of misfolded, native, or amyloid species using a commercial control antibody (Figure 14A) or LX-96 (Figure 14B). Western blots were prepared by transfer from native SDS-PAGE and detection using the Licor system with appropriate secondary antibodies. Native (N) AL09 or misfolded (M) AL09 monomeric species appear as a band of approximately 25 kDa, while aggregates or large MW species (greater than 250 kDa) appear as a smear in the amyloid (A) and native lanes. [Figure 15]Figures 15A-E are images of immunohistochemical staining of biopsy-confirmed cases of kappa-AL amyloidosis. A liver case (Figure 15A-B) and two cardiac cases (Figure 15C-E) were stained (dark areas) using either a nonspecific anti-kappa antibody (Figure 15A and C) or LX-96 (Figure 15B, D, and E). Scale bars indicate 100 μm. [Figure 16] Figures 16A-I are images of immunohistochemical staining of biopsies of confirmed amyloidosis as pathological control tissues. ATTR cardiac tissue (Figure 16A-D), AL-lambda cardiac tissue (Figure 16E-H), or IAPP pancreatic tissue (Figure 16I) were stained using either Congo Red (Figure 16A and E: dark staining), nonspecific anti-lambda antibody (Figure 16B and F), nonspecific anti-kappa antibody (Figure 16C and G), or LX-96 (Figure 16D, H, and I). Positive antibody staining appears as black. LX-96 showed no staining in any of these tissues. Scale bars indicate 100 μm. [Figure 17] Figure 17 is a graphical representation of the inhibition of AL09 aggregation by LX-96 in vitro. Aggregation of FITC-labeled AL09 was measured at endpoint in the presence of various substoichiometric concentrations of LX-96. Aggregation was derived by normalization from FITC fluorescence of soluble material and shown as a percentage with a trend line as a guide for the eye. [Figure 18]Figure 18A-F are brightfield and fluorescence images of representative fields of RAW cells using brightfield (Figure 18A,C, and E) and pHrodo (Figure 18B,D, and F) channels. Cells were incubated with pHrodo-labeled AL09 for 3 hours in the presence of LX-96 (Figure 18E-F), isotype antibody (Figure 18C-D), or no antibody as a control (Figure 18A,B). PHrodo fluorescence signal (white) represents cells that have internalized AL09 amyloid by phagocytic uptake. Figure 18G is a bar graph showing uptake as quantified by mean pHrodo fluorescence per cell. p-values ​​were calculated using Student's t-test. [Figure 19] Figure 19 shows a fluorescent image of a mouse implanted with kappa light chain amyloid aggregates (amyloidoma; dotted line) and injected with a single dose of 10 mg / kg fluorescently labeled LX-96 antibody via tail vein injection. The white signal indicates increased accumulation of LX-96 in the amyloidoma within 60 minutes of injection. [Figure 20A] Figure 20A is a fluorescent image of mice transplanted with pHrodo-labeled kappa light chain amyloid, 3 hours after a single 10 mg / kg tail vein injection of LX-96 or isotype antibody control. Increased signal (white) represents increased phagocytosis. [Figure 20B] FIG. 20B is a graphical representation of quantification of phagocytosis using three antibody administration modes: co-mixing of amyloid and antibody prior to transplantation (co-implantation), subcutaneous injection (SubQ), and tail vein injection. [Figure 21A] Figure 21A shows fluorescent images of mice implanted with fluorescently labeled amyloidoma (top) or labeled amyloidoma premixed with 10 mg / kg LX-96 (bottom). Images were taken over a 3-day period. The white signal is an estimate of amyloid size. [Figure 21B] FIG. 21B tabulates the relative size of amyloid as quantified by fluorescent signal summation. [Figure 21C]FIG. 21C is a graph showing that administration of LX-96 enhanced the rate of reduction in amyloid size (3-fold) compared to untreated amyloidoma. [Figure 22] Figure 22 shows immunohistochemical staining of amyloidoma tissue excised 24 hours after injection from mice treated with either isotype control or LX-96. Representative fields are shown. Tissues were stained using a macrophage marker (F4 / 80) or a neutrophil marker (MPO). Positive staining is shown in black. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Certain aspects of the present disclosure are directed to antibodies that specifically bind to human immunoglobulin free light chains (FLC: "anti-FLC antibodies"). In some aspects, the anti-FLC antibodies bind to kappa light chains. In some aspects, the anti-FLC antibodies cross-compete with a reference antibody in binding to a kappa light chain, where the reference antibody binds to an epitope on an immunoglobulin kappa light chain comprising the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1). In some aspects, the anti-FLC antibodies bind to a kappa light chain at an epitope that overlaps with all or a portion of the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1). In some aspects, the anti-FLC antibodies bind to an epitope on a kappa light chain comprising the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1).

[0032] In some embodiments, the anti-FLC antibody binds to a lambda light chain. In some embodiments, the anti-FLC antibody cross-competes with a reference antibody in binding to a lambda light chain, where the reference antibody binds to an epitope on an immunoglobulin lambda light chain comprising the amino acid sequence shown in NKYAASSYLSL (SEQ ID NO: 2). In some embodiments, the anti-FLC antibody binds to a lambda light chain at an epitope that overlaps with all or part of the amino acid sequence shown in NKYAASSYLSL (SEQ ID NO: 2). In some embodiments, the anti-FLC antibody binds to an epitope on a lambda light chain comprising the amino acid sequence shown in NKYAASSYLSL (SEQ ID NO: 2).

[0033] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below, unless expressly stated otherwise herein. Additional definitions are set forth throughout this application.

[0034] It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0035] Furthermore, as used herein, "and / or" should be interpreted as a specific disclosure of each of two particular features or components, with or without the other feature. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (single), B (single), and C (single).

[0036] The term "about" is used herein to mean approximately, roughly, in the vicinity or within the region. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the set forth value by a variance of 10% above and below (higher or lower).

[0037] Whenever an embodiment is described herein with the word "comprising," it is understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by those skilled in the art in the field to which this disclosure relates.For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary for many of the terms used in this disclosure.

[0039] Units, prefixes, and symbols are shown in their International System of Units (SI) accepted format. Numeric ranges are inclusive of the numbers defining the range. Nucleotide sequences are written left to right in 5' to 3' orientation unless otherwise specified. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which aspects may be incorporated herein by reference in their entireties. Thus, the terms defined below are more fully defined herein by reference in their entireties.

[0040] As described herein, heterologous with respect to an amino acid or nucleic acid sequence refers to a sequence of different source or origin as compared to the amino acid or nucleic acid sequence to which the heterologous sequence is compared.

[0041] As used herein, an "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin or antigen-binding portion thereof that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region includes three constant domains, C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (referred to herein as V L The light chain constant region comprises one constant domain, C L Includes: V H and V L The regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L contains three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or elements, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab' ... 2 These include single chain antibody fragments, including fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain variable fragments (sFv or scFv), and single domain antibody (eg sdAb, sdFv, nanobody) fragments.

[0042] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids in antibody variable regions that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273:927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol, January 2003;27(1):55-77 (the "IMGT" numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, June 8, 2001, 309(3):657-70, (the "Aho" numbering scheme), and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB," Protein Eng. December 2000, 13(12):819-24 (the "AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0043] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. H and V L The variable domains of a single V domain generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). H Domain or V L The V domain may be sufficient to confer antigen-binding specificity. H Domain or V L Using the domains, respectively, the complementary V L Domain or V H Libraries of domains may be screened to isolate antibodies that bind a specific antigen (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0044] As used herein, an "intermediate" light chain refers to a light chain immunoglobulin polypeptide that is not a mature light chain immunoglobulin. In some aspects, an intermediate light chain is an unfolded light chain polypeptide. In some aspects, an intermediate light chain is a misfolded light chain polypeptide.

[0045] Immunoglobulins may be from any of the known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or portions, as well as single chain antibodies.

[0046] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from a non-human CDR and all or substantially all FR amino acid residues are derived from a human FR. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues of a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0047] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. However, an isolated antibody that specifically binds to an antigen may have cross-reactivity to other antigens, such as antigens from different species. Furthermore, an isolated antibody may also be substantially free of other cellular material and / or chemicals.

[0048] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules which are essentially identical in primary sequence and which display a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. mAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0049] The term "polyclonal antibody" ("pAb") refers to a non-naturally occurring preparation of antibody molecules of more than one molecular composition, i.e., antibody molecules which are not necessarily identical in primary sequence and which may exhibit different binding specificities and affinities for particular epitopes.

[0050] A "human antibody" refers to an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0051] "Humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR domain of a non-human antibody are replaced with the corresponding amino acids from human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domain are replaced with amino acids from human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions are left unchanged. Small additions, deletions, insertions, substitutions or modifications of such amino acids are permitted as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0052] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a murine antibody and the constant region is derived from a human antibody.

[0053] An "antigen" refers to any molecule, e.g., a peptide, that can be bound by an antibody or that elicits an immune response. An "epitope," as used herein, refers to a portion of a polypeptide that can be bound by an antibody or that elicits an immune response. Those skilled in the art will readily appreciate that virtually any macromolecule, including any protein or peptide, can function as an antigen. Antigens and / or epitopes can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed from exogenous DNA. Antigens and / or epitopes can be specific to a particular tissue, such as cancer cells, or can be broadly expressed. Additionally, fragments of larger molecules can act as antigens. In one embodiment, the antigen is a tumor antigen. Epitopes can be present within a longer polypeptide (e.g., within a protein), or epitopes can be present as fragments of a longer polypeptide. In some embodiments, epitopes are complexed with the major histocompatibility complex (MHC, also referred to herein as complexed with an HLA molecule, e.g., HLA class 1 molecule). In certain embodiments, epitopes include certain non-protein molecules (eg, sugars, lipids, phosphates, etc.) or non-protein molecules that modify amino acids of a protein molecule.

[0054] An "anti-antigen" antibody refers to an antibody that specifically binds to an antigen. For example, an anti-FLC antibody specifically binds to FLC.

[0055] An "antigen-binding portion" of an antibody (also called an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen bound by the whole antibody.

[0056] Percent (%) sequence identity to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways using available computer software. Suitable parameters for aligning sequences can be determined, including algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, CA), or may be compiled from its source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX Version 4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0057] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (alternatively, one may say a given amino acid sequence A has or contains a particular % amino acid sequence identity with or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, then the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0058] "Amyloidosis" as used herein refers to a disease or condition characterized by abnormal folding of immunoglobulin free light chains (LC) that can be deposited as light chain amyloid (AL) in the heart, kidney, or liver. The disease is caused by a causative plasma cell dyscrasia that results in plasma cells secreting a large excess of immunoglobulin free kappa (κ) and / or lambda (λ) light chains into the circulation. As used herein, "primary amyloidosis" or "AL amyloidosis" refers to the most common type of systemic amyloidosis, with an incidence of 9-14 cases per million population per year in the Western world.

[0059] "Plasma cell dysplasia" as used herein refers to a proliferative plasma cell disorder involving unregulated proliferation of a single abnormal plasma cell clone (terminally differentiated B cell) producing more than normal amounts of light chain protein. Plasma cell dysplasias are classified as either benign, as seen in monoclonal gammopathy of undetermined significance (MGUS), or malignant, as seen in multiple myeloma (MM) and plasma cell leukemia (PCL). Regardless of the type of abnormality involved, these secreted light chains can undergo misfolding events that cause them to aggregate and accumulate as amyloid in vital organs, including the heart, kidneys, and nervous system. Such infiltration can lead to nephrotic syndrome, hepatomegaly, peripheral neuropathy, restrictive cardiomyopathy, and ultimately death.

[0060] "Administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered by a route that is not parenteral, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more chronic periods.

[0061] As used herein, "treat," "treatment," or "treating" refers to the deliberate intervention in a physiological disease state that results in, for example, a decrease in the severity of a disease or condition, a decrease in the duration of a disease or condition, an improvement in or elimination of one or more symptoms associated with a disease or condition, or the provision of a beneficial effect to a subject having a disease or condition. Treatment does not necessarily require a cure of the underlying disease or condition.

[0062] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent is that amount of the agent, when used alone or in combination with another therapeutic agent, that protects a subject from developing a disease or promotes a shortening of the disease as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to the affliction of the disease. The ability of a therapeutic agent to promote a shortening of the disease can be evaluated using a variety of methods known to those skilled in the art, such as, for example, in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0063] The term "immunotherapy" refers to the treatment of a subject suffering from a disease, or at risk of suffering from a recurrence of a disease, by methods that involve inducing, enhancing, suppressing, or modifying an immune response. Examples of immunotherapy include, but are not limited to, administering an antibody or antigen-binding portion thereof that stimulates an immune response in a subject.

[0064] Various aspects of the disclosure are described in further detail in the following subsections.

[0065] II. Compositions of the Disclosure Certain aspects of the present disclosure are directed to antibodies that specifically bind to human immunoglobulin free light chains (FLC: "anti-FLC antibodies"). Immunoglobulins, by their nature, have highly variable amino acid sequences. Furthermore, typical AL deposits in amyloidosis contain aggregates of misfolded immunoglobulin free light chains (LC). As a result, targeting FLC requires the ability to recognize constants within the many variable sequences. To overcome this heterogeneity, the antibodies described herein are specifically designed to recognize epitopes in the constant region of the immunoglobulin light chain sequence.

[0066] In certain embodiments, the anti-FLC antibody binds to an immunoglobulin light chain polypeptide regardless of the antigen specificity of the immunoglobulin light chain. In some embodiments, the anti-FLC antibody binds to an epitope on an immunoglobulin light chain polypeptide, where the epitope is located within the constant region of the immunoglobulin light chain. In some embodiments, the epitope is located within a portion of the constant region of the immunoglobulin light chain that is conformationally inaccessible when the immunoglobulin light chain is in a complex with an immunoglobulin heavy chain. In some embodiments, the epitope is located within a portion of the constant region of the immunoglobulin light chain that is not surface-exposed when the immunoglobulin light chain is in a complex with an immunoglobulin heavy chain. In some embodiments, the anti-FLC antibody specifically binds to an epitope located within the constant region of the immunoglobulin light chain, where the anti-FLC antibody binds to the epitope only when the immunoglobulin light chain is not in a complex with an immunoglobulin heavy chain.

[0067] In certain embodiments, the anti-FLC antibody specifically binds to an immunoglobulin light chain that is not associated with an immunoglobulin heavy chain, hi some embodiments, the anti-FLC antibody does not bind to a complex that includes an immunoglobulin light chain that is covalently bound to an immunoglobulin heavy chain.

[0068] The anti-FLC antibodies disclosed herein can bind to immunoglobulin free light chains of any origin, with any configuration. In some embodiments, the anti-FLC antibodies disclosed herein bind to any immunoglobulin light chain, including all or a portion of the light chain constant region. In some embodiments, the FLC comprises a full-length immunoglobulin light chain peptide. In some embodiments, the FLC comprises a fragment of an immunoglobulin light chain peptide. In some embodiments, the fragment of an immunoglobulin light chain peptide comprises all or a portion of the light chain constant region. In some embodiments, the FLC comprises an aggregate of an immunoglobulin light chain peptide. In some embodiments, the aggregate comprises a full-length immunoglobulin light chain peptide. In some embodiments, the aggregate comprises a fragment of an immunoglobulin light chain peptide. In some embodiments, the aggregate comprises both a full-length immunoglobulin light chain peptide and a fragment of an immunoglobulin light chain peptide. In some embodiments, the aggregate of an immunoglobulin light chain peptide comprises one or more misfolded immunoglobulin light chain peptides.

[0069] In some embodiments, the anti-FLC antibody binds to one or more light chain amyloid fibrils. In some embodiments, the anti-FLC antibody can bind to light chain amyloid fibrils in vivo. In some embodiments, the anti-FLC antibody selectively binds to light chain fibrils more efficiently than it binds to naturally folded light chains. In some embodiments, the anti-FLC antibody selectively binds to light chain fibrils at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more efficiently than it binds to naturally folded light chains. In some embodiments, the anti-FLC antibody selectively binds to light chain fibrils more efficiently than it binds to naturally folded light chains. In some embodiments, the anti-FLC antibody selectively binds to light chain fibrils at least 2.0-fold more efficiently than it binds to naturally folded light chains. In some embodiments, the amyloid fibrils are collected from a biological sample obtained from the subject, hi some embodiments, the biological sample is selected from blood, serum, plasma, solid tissue, and any combination thereof.

[0070] In some embodiments, the anti-FLC antibody can inhibit the formation of light chain fibrils. In some embodiments, the anti-FLC antibody inhibits the formation of light chain fibrils from misfolded light chain intermediates in solution. In some embodiments, the anti-FLC antibody inhibits the formation of light chain fibrils from misfolded light chain intermediates in vivo.

[0071] In some embodiments, the anti-FLC antibody can reduce the formation of light chain fibrils. In some embodiments, the anti-FLC antibody reduces the formation of light chain fibrils from misfolded light chain intermediates in solution. In some embodiments, the anti-FLC antibody reduces the formation of light chain fibrils from misfolded light chain intermediates in vivo. In some embodiments, the anti-FLC antibody reduces light chain fibril formation by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to the formation of light chain fibrils under similar conditions but without the anti-FLC antibody.

[0072] In some embodiments, the anti-FLC antibody can induce an immune response in vivo. In some embodiments, the anti-FLC antibody binds to immunoglobulin light chains in a subject, such as immunoglobulin light chains in amyloid fibrils, thereby inducing and / or enhancing an immune response in the subject. In some embodiments, the immune response is a cellular immune response. In some embodiments, the immune response comprises eliminating, removing, and / or dissociating aggregates of immunoglobulin light chains, such as amyloid fibrils, in the subject. In some embodiments, the anti-FLC antibody enhances the immune response by at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times, compared to the immune response in a subject to which the anti-FLC antibody is not administered. In some embodiments, the anti-FLC antibody induces an immune response in a subject against immunoglobulin light chain aggregates, e.g., amyloid fibrils, wherein the subject would not have had an immune response against immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the absence of the anti-FLC antibody.

[0073] In some embodiments, the anti-FLC antibody can opsonize light chain immunoglobulin aggregates, such as amyloid fibrils, in a subject. In some embodiments, the anti-FLC antibody can induce clearance of light chain immunoglobulin aggregates, such as amyloid fibrils, in a subject.

[0074] In some embodiments, the anti-FLC antibody is a polyclonal antibody. In some embodiments, the anti-FLC antibody is a monoclonal antibody. In some embodiments, the anti-FLC antibody is a chimeric antibody. In some embodiments, the anti-FLC antibody is a humanized antibody. In some embodiments, the anti-FLC antibody is a human antibody.

[0075] In some embodiments, the anti-FLC antibody comprises an antigen-binding portion of an antibody. In some embodiments, the anti-FLC antibody comprises a Fab. In some embodiments, the anti-FLC antibody comprises a Fab'. In some embodiments, the anti-FLC antibody comprises a F(ab')2. In some embodiments, the anti-FLC antibody comprises an scFv. In some embodiments, the anti-FLC antibody comprises a dsFv. In some embodiments, the anti-FLC antibody comprises a ds-scFv. In some embodiments, the anti-FLC antibody comprises a dimer. In some embodiments, the anti-FLC antibody comprises a minibody. In some embodiments, the anti-FLC antibody comprises a diabody. In some embodiments, the anti-FLC antibody comprises a multimer of any of the above. In some embodiments, the anti-FLC antibody comprises a bispecific antibody fragment. In some embodiments, the anti-FLC antibody is a single chain antibody.

[0076] Immunoglobulin light chains are expressed from two loci in the human genome: the immunoglobulin kappa locus, which codes for kappa light chains, and the immunoglobulin lambda locus, which codes for lambda light chains. Each B lymphocyte expresses only one class of light chain, either from the kappa locus or the lambda locus. As a result, human serum contains a mixture of kappa and lambda light chain antibodies. Table 1 shows the standard amino acid sequences of kappa light chains (UniProt P0DOX7; SEQ ID NO: 5) and lambda light chains (UniProt P0DOX8; SEQ ID NO: 7).

[0077] [Table 1]

[0078] As noted above, each immunoglobulin light chain comprises a variable domain and a constant domain. The kappa light chain variable domain extends from amino acid residues 1 to 108 of SEQ ID NO:5, and the kappa light chain constant region extends from amino acid residues 109 to 214 of SEQ ID NO:5 (captured as SEQ ID NO:6). The lambda light chain variable domain extends from amino acid residues 1 to 112 of SEQ ID NO:7, and the lambda light chain constant region extends from amino acid residues 112 to 216 of SEQ ID NO:7 (captured as SEQ ID NO:8).

[0079] II.A. Anti-kappa light chain antibodies In some embodiments, the anti-FLC antibody specifically binds to a kappa light chain polypeptide. In some embodiments, the anti-FLC antibody specifically binds to a misfolded kappa light chain polypeptide. In some embodiments, the anti-FLC antibody specifically binds to a misfolded kappa light chain polypeptide and does not bind to a native kappa light chain polypeptide. In some embodiments, the anti-FLC antibody specifically binds to an unfolded kappa light chain polypeptide. In some embodiments, the anti-FLC antibody specifically binds to an unfolded kappa light chain polypeptide and does not bind to a natively folded kappa light chain polypeptide.

[0080] In some embodiments, the anti-FLC antibody binds to an epitope present in the constant region of a kappa light chain. In some embodiments, the epitope is presented on the surface of a misfolded kappa light chain polypeptide. In some embodiments, the epitope is presented on the surface of an unfolded kappa light chain polypeptide. In some embodiments, the epitope is not presented on the surface of a naturally folded kappa light chain polypeptide.

[0081] In some aspects, the anti-FLC antibody cross-competes with a reference antibody in binding to a kappa light chain polypeptide, where the reference antibody binds to one or more amino acids of an epitope comprising the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1). In some aspects, the anti-FLC antibody binds to a kappa light chain at an epitope that overlaps with all or a portion of the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1). In some aspects, the anti-FLC antibody binds to an epitope on a kappa light chain comprising the amino acid sequence set forth in STYSLSSTLT (SEQ ID NO: 1). In certain embodiments, the reference antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 14 and a light chain variable region amino acid sequence of SEQ ID NO: 10.

[0082] In some embodiments, the epitope comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten of the amino acids set forth in SEQ ID NO: 1. In some embodiments, the epitope comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten of the amino acids set forth in SEQ ID NO: 1. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , Thr 180 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 In some embodiments, the epitope comprises Thr, which corresponds to SEQ ID NO:5.172 In some embodiments, the epitope comprises the Tyr 173 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 174 In some embodiments, the epitope comprises Leu, which corresponds to SEQ ID NO:5. 175 , Ser 176 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 177 In some embodiments, the epitope comprises Thr, which corresponds to SEQ ID NO:5. 178 In some embodiments, the epitope comprises Leu, which corresponds to SEQ ID NO:5. 179 In some embodiments, the epitope comprises Thr, which corresponds to SEQ ID NO:5. 180 Includes.

[0083] In some embodiments, the epitope is Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises at least two of Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises at least three of the following: Ser, corresponding to SEQ ID NO:5; 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu179 , and Thr 180 In some embodiments, the epitope comprises at least four of Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises at least five of the following: Ser, corresponding to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises at least six of Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises at least seven of the following: Ser, corresponding to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises at least eight of the following: Ser, corresponding to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises at least nine of the following: Ser, corresponding to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 Includes.

[0084] In some embodiments, the epitope is Ser, which corresponds to SEQ ID NO:5. 171 and Thr 172 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , and Tyr 173 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , and Ser 174 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , and Leu 175 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , and Ser 176 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , and Ser177 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , and Thr 178 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 171 , Thr 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , and Leu 179 Includes.

[0085] In some embodiments, the epitope is Leu corresponding to SEQ ID NO:5. 179 and Thr 180 In some embodiments, the epitope comprises Thr, which corresponds to SEQ ID NO:5. 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises Leu, which corresponds to SEQ ID NO:5. 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:5. 174 , Leu 175 , Ser 176 , Ser177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises the Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 In some embodiments, the epitope comprises Thr, which corresponds to SEQ ID NO:5. 172 , Tyr 173 , Ser 174 , Leu 175 , Ser 176 , Ser 177 , Thr 178 , Leu 179 , and Thr 180 Includes.

[0086] In some embodiments, the anti-FLC antibody specifically binds to a kappa light chain polypeptide and comprises (i) a heavy chain and (ii) a light chain, wherein the heavy chain comprises a variable region and a constant region, the heavy chain variable region comprises a variable heavy complementarity determining region (VH-CDR)1, a VH-CDR2, and a VH-CDR3 of the heavy chain variable region, and the light chain variable region comprises a variable light chain (VL) CDR1, a VL-CDR2, and a VL-CDR3. In some embodiments, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0087] [Table 2]

[0088] In some embodiments, the anti-FLC antibody comprises (i) a heavy chain and (ii) a light chain, wherein the heavy chain comprises a variable region and a constant region, the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises variable light chain (VL) CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0089] In some embodiments, the anti-FLC antibody comprises (i) a heavy chain and (ii) a light chain, wherein the heavy chain comprises a variable region and a constant region, the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises variable light chain (VL) CDR1, VL-CDR2, and VL-CDR3, wherein VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0090] In certain embodiments, the anti-FLC antibody comprises (i) a heavy chain and (ii) a light chain, wherein the heavy chain comprises a variable region and a constant region, the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises variable light chain (VL) CDR1, VL-CDR2, and VL-CDR3, wherein VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0091] In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17.In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 98% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0092] In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, wherein the light chain variable region comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, wherein the light chain variable region comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, wherein the light chain variable region comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising an amino acid sequence having at least about 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, wherein the light chain variable region comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising an amino acid sequence having at least about 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, wherein the light chain variable region comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13.In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising an amino acid sequence having at least about 98% sequence identity with the amino acid sequence set forth in SEQ ID NO: 10, wherein the light chain variable region comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising an amino acid sequence having at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 10, wherein the light chain variable region comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-FLC antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0093] In some embodiments, the anti-FLC antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10.

[0094] In some aspects, the anti-FLC antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the antibody lacks secretory leader sequences of the heavy and light chains.

[0095] In some embodiments, the anti-FLC antibody competes with a reference antibody for binding to a kappa light chain polypeptide, wherein the reference antibody comprises (i) a heavy chain and (ii) a light chain, wherein the heavy chain comprises a variable region and a constant region, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein the light chain variable region comprises variable light chain (VL) CDR1, VL-CDR2, and VL-CDR3, wherein VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, wherein VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, and wherein VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the reference antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:10.

[0096] In some embodiments, the anti-kappa light chain antibodies provided herein have a dissociation constant (K D ) is about 1μM, 100nM, 50nM, 40nM, 30nM, 20nM, 15nM, 10nM, 5nM, 2nM, 1nM, 0.5nM, 0.1nM, 0.05nM, 0.01nM or less (e.g. 10 -8 M or less, e.g. 10 -8 M to 10 -13 Up to M, for example 10 -9 M to 10 -13 In some embodiments, the antibodies provided herein bind to misfolded kappa light chains with a dissociation constant (K D ) is about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or more (e.g., 10 -8 M or less, e.g. 10 -8 M to 10 -13 Up to M, for example 10 -9 M to 10 -13The antibody has a K between 100pM and 100nM, between 100pM and 50nM, between 100pM and 10nM, between 500pM and 100nM, between 500pM and 50nM, between 500pM and 10nM, between 1nM and 100nM, between 1nM and 50nM, between 1nM and 10nM. D They can be combined with K D can be measured by any suitable assay, including biolayer interferometry, as described herein.

[0097] In certain embodiments, the anti-kappa light chain antibody has a K between 500 picomolar and 25 nanomolar. D In certain embodiments, the anti-kappa light chain antibody binds to AL09 amyloid with a K between 500 picomolar and 10 nanomolar. D In certain embodiments, the anti-kappa light chain antibody binds to AL09 amyloid with a K between 1 picomolar and 10 nanomolar. D It binds to AL09 amyloid.

[0098] II.B. Anti-Lambda Light Chain Antibodies In some embodiments, the anti-FLC antibody binds to a lambda light chain. In some embodiments, the anti-FLC antibody cross-competes with a reference antibody in binding to a lambda light chain, and the reference antibody binds to an epitope on an immunoglobulin lambda light chain comprising the amino acid sequence shown. In some embodiments, the anti-FLC antibody binds to a lambda light chain at an epitope that overlaps with all or part of the amino acid sequence shown in NKYAASSYLSL (SEQ ID NO: 2). In some embodiments, the anti-FLC antibody binds to an epitope on a lambda light chain comprising the amino acid sequence shown in NKYAASSYLSL (SEQ ID NO: 2).

[0099] In some embodiments, the anti-FLC antibody specifically binds to a lambda light chain polypeptide. In some embodiments, the anti-FLC antibody binds to an epitope present in the constant region of the lambda light chain.

[0100] In some embodiments, the anti-FLC antibody cross-competes with a reference antibody for binding to a lambda light chain polypeptide, where the reference antibody binds to one or more amino acids of an epitope that includes the amino acid sequence set forth in NKYAASSYLSL (SEQ ID NO:2). In some embodiments, the anti-FLC antibody binds to a lambda light chain at an epitope that overlaps with all or a portion of the amino acid sequence set forth in NKYAASSYLSL (SEQ ID NO:2). In some embodiments, the anti-FLC antibody binds to an epitope on a lambda light chain that includes the amino acid sequence set forth in NKYAASSYLSL (SEQ ID NO:2).

[0101] In some embodiments, the epitope comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten of the amino acids set forth in SEQ ID NO: 2. In some embodiments, the epitope comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten of the amino acids set forth in SEQ ID NO: 2. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , Leu 184 In some embodiments, the epitope comprises Asn corresponding to SEQ ID NO:7. 174 In some embodiments, the epitope comprises Lys, which corresponds to SEQ ID NO:7. 175 In some embodiments, the epitope comprises the Tyr 176 In some embodiments, the epitope comprises Ala, which corresponds to SEQ ID NO:7. 177 In some embodiments, the epitope comprises Ala, which corresponds to SEQ ID NO:7. 178 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:7. 179 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:7. 180In some embodiments, the epitope comprises the Tyr 181 In some embodiments, the epitope comprises Leu, which corresponds to SEQ ID NO:7. 182 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:7. 183 In some embodiments, the epitope comprises Leu, which corresponds to SEQ ID NO:7. 184 Includes.

[0102] In some embodiments, the epitope is Asn corresponding to SEQ ID NO:7 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least two of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least three of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least four of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176, Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least five of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least six of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least seven of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least eight of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises at least nine of the Asn corresponding to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 Includes.

[0103] In some embodiments, the epitope is Asn corresponding to SEQ ID NO:7 174 and Lys 175 In some embodiments, the epitope comprises Asn, which corresponds to SEQ ID NO:7. 174 , Lys 175 , and Tyr 176 In some embodiments, the epitope comprises Asn, which corresponds to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , and Ala 177 In some embodiments, the epitope comprises Asn, which corresponds to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , and Ala 178 In some embodiments, the epitope comprises Asn, which corresponds to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , and Ser 179 In some embodiments, the epitope comprises Asn, which corresponds to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , and Ser 180In some embodiments, the epitope comprises Asn, which corresponds to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , and Tyr 181 In some embodiments, the epitope comprises Asn 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , and Leu 182 In some embodiments, the epitope comprises Asn, which corresponds to SEQ ID NO:7. 174 , Lys 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , and Ser 183 Includes.

[0104] In some embodiments, the epitope is Ser, which corresponds to SEQ ID NO:7. 183 and Leu 184 In some embodiments, the epitope comprises Leu, which corresponds to SEQ ID NO:7. 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises the Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:7. 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises Ser, which corresponds to SEQ ID NO:7. 179, Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises Ala, which corresponds to SEQ ID NO:7. 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises Ala, which corresponds to SEQ ID NO:7. 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises the Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 In some embodiments, the epitope comprises Lys, which corresponds to SEQ ID NO:7. 175 , Tyr 176 , Ala 177 , Ala 178 , Ser 179 , Ser 180 , Tyr 181 , Leu 182 , Ser 183 , and Leu 184 Includes.

[0105] II.C. Nucleic acid molecules Certain aspects of the present disclosure are directed to nucleic acid molecules encoding anti-FLC antibodies described herein. Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "made substantially pure" when they are purified away from other cellular components or other contaminants, such as other intracellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or intracellular proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel et al. (eds., 1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids described herein may be, for example, DNA or RNA, and may or may not include intron sequences. In some aspects, the nucleic acid is a cDNA molecule.

[0106] The nucleic acid described herein can be obtained using standard molecular biology techniques.For the antibody expressed by hybridoma (e.g., hybridoma prepared from genetically modified mouse with human immunoglobulin genes, as described further below), the cDNA encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.For the antibody obtained from an immunoglobulin gene library (e.g., using phage display techniques), the nucleic acid encoding the antibody can be retrieved from the library.

[0107] The method of producing the anti-FLC antibody disclosed herein may include expressing the heavy and light chains in a cell line that contains nucleotide sequences encoding the heavy and light chains with signal peptides. Host cells that contain these nucleotide sequences are encompassed herein.

[0108] After the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding the VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0109] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2 and / or CH3). Gene sequences of human heavy chain constant regions are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, e.g., an IgG1 region. In a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0110] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VH to another DNA molecule encoding the light chain constant region CL. Gene sequences of human light chain constant regions are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0111] To generate an ScFv gene, the VH and VL encoding DNA fragments are amplified to include a nucleic acid sequence encoding a flexible linker, e.g., the amino acid sequence (Gly 4 -Ser) 3 By operably linking the VH and VL sequences to another fragment encoding the VL and VH domains, they can be expressed as a contiguous single-chain protein in which the VL and VH domains are joined by a flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al. (1990) Nature 348:552-554).

[0112] II.D. Vector Certain aspects of the present disclosure are directed to vectors comprising the nucleic acid molecules disclosed herein. In some aspects, the vector is a viral vector. In some aspects, the vector is a viral particle or a virus. In some aspects, the vector is a mammalian vector. In some aspects, the vector is a bacterial vector.

[0113] In certain embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of adenoviral vector, lentivirus, Sendai virus, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, and adeno-associated virus (AAV) vector. In certain embodiments, the vector is an AAV vector. In some embodiments, the vector is a lentivirus. In certain embodiments, the vector is an AAV vector. In some embodiments, the vector is a Sendai virus. In some embodiments, the vector is a hybrid vector. Examples of hybrid vectors that can be used in the present disclosure can be found in Huang and Kamihira, Biotechnol. Adv. 31(2):208-23(2103), which is incorporated herein by reference in its entirety.

[0114] In some embodiments, the vector further comprises one or more regulatory elements.The regulatory elements useful in the vector disclosed herein include, but are not limited to, promoter, enhancer, polyA sequence, miRNA binding sequence, intron sequence, splice acceptor site, and any combination thereof.In some embodiments, the vector comprises tissue-specific promoter.In some embodiments, the vector comprises tissue-specific enhancer.

[0115] II.E. Pharmaceutical Compositions Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising the anti-FLC antibody disclosed herein and a pharmaceutically acceptable excipient.Some aspects of the present disclosure are directed to pharmaceutical compositions comprising the nucleic acid molecule encoding the anti-FLC antibody disclosed herein and a pharmaceutically acceptable excipient.Some aspects of the present disclosure are directed to pharmaceutical compositions comprising the vector or set of vectors disclosed herein and a pharmaceutically acceptable excipient.

[0116] In some embodiments, the composition of the present disclosure further comprises a bulking agent. The bulking agent can be selected from the group consisting of NaCl, mannitol, glycine, alanine, and any combination thereof. In other embodiments, the composition of the present disclosure comprises a stabilizing agent. The stabilizing agent can be selected from the group consisting of sucrose, trehalose, raffinose, arginine, or any combination thereof. In other embodiments, the composition of the present disclosure comprises a surfactant. The surfactant can be selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In certain embodiments, the composition further comprises a chelating agent. The chelating agent can be selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof. The pharmaceutical composition may comprise an antibody as described herein solubilized in a suitable isotonic buffer, such as 0.9% NaCl or 5% glucose, and may include other surfactants, pH buffers, or antioxidants.

[0117] In one embodiment, the composition further comprises NaCl, mannitol, pentetic acid (DTPA), sucrose, PS80, and any combination thereof.

[0118] As used herein, "pharmaceutical acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. In some embodiments, the carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibodies, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0119] The pharmaceutical compounds described herein may include one or more pharma- ceutically acceptable salts. A "pharma- ceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM et al. (1977) J.Pharm.Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include acid addition salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and acid addition salts derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0120] The pharmaceutical compositions described herein may also contain a pharma- ceutically acceptable antioxidant. Examples of pharma-ceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfate; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0121] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, such as vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0122] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial growth can be ensured by both the above-mentioned sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, prolonged absorption of the injectable pharmaceutical form may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0123] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Except where any conventional media or agent is incompatible with the active compound, its use is contemplated in the pharmaceutical compositions described herein.The pharmaceutical compositions may or may not contain preservatives.Supplementary active compounds may be incorporated into the composition.

[0124] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, the compositions may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions may be achieved by including in the composition an agent that delays absorption, for example, monostearate salts or gelatin.

[0125] Sterile injectable solutions can be prepared by combining the active compound in the required amount in a suitable solvent with one or a combination of the above listed components as required, and then performing sterilization microfiltration.Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the substances listed herein.For the preparation of sterile powders for preparing sterile injectable solutions, some of the preparation methods are vacuum drying and freeze-drying (lyophilization), which can obtain a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0126] The amount of active ingredient that can be mixed with a carrier material to create a single dosage form varies depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be mixed with a carrier material to create a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount ranges from about 0.01 percent to about 99 percent of active ingredient, or from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient combined with a pharma- ceutically acceptable carrier.

[0127] The compositions described herein may be formulated for administration via one or more routes.In some embodiments, pharmaceutical compositions are formulated for intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration, for example, by injection or infusion.The term "parenteral administration" as used herein refers to the mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0128] In some embodiments, the pharmaceutical compositions are formulated for parenteral routes, such as topical, epidermal, or mucosal routes of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical administration.

[0129] Anti-FLC antibody can be prepared with carriers that protect the compound from rapid release, such as sustained release preparations, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Many methods for preparing such preparations are patented or generally known to those skilled in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson (ed.), Marcel Dekker, Inc., New York, 1978.

[0130] In some embodiments, the anti-FLC antibody described herein is formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if desired), they may be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes may contain one or more moieties that selectively transport to specific cells or organs, thus enhancing targeted drug delivery (see, for example, V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.), mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090). See also K. Keinanen, ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion, IJ Fidler (1994) Immunomethods 4:273.

[0131] III.How to use The anti-FLC antibody used herein can be used for any purpose. Certain embodiments of the present disclosure are directed to a method for measuring FLC in a sample. Other embodiments of the present disclosure are directed to a method for treating a disease or condition of a subject in need of disease or disease treatment. In some embodiments, the anti-FLC antibody is modified and / or formulated for a particular use, for example as described herein.

[0132] III.A.Measurement method Certain embodiments of the present disclosure are directed to a method for measuring light chain immunoglobulin. The antibodies disclosed herein can be used to measure any immunoglobulin light chain or its aggregates. In some embodiments, anti-FLC antibodies are used to measure immunoglobulin light chain or its aggregates in vivo. In some embodiments, anti-FLC antibodies are used to measure immunoglobulin light chain or its aggregates in biological samples collected from subjects. In some embodiments, the biological sample is a plasma sample, a blood sample, a tissue sample (e.g., tissue biopsy), a urine sample, or any combination thereof.

[0133] In some embodiments, the method includes measuring light chain fibrils. Thus, the antibodies described herein can be used to identify subjects with a disease or condition characterized by the formation of immunoglobulin light chain deposits, e.g., amyloid fibril deposits. In some embodiments, the anti-FLC antibody is used to identify (e.g., diagnose) a subject as having amyloidosis. In some embodiments, the anti-FLC antibody is used to identify a subject as having systemic amyloidosis. In some embodiments, the anti-FLC antibody is used to identify a subject as having AL amyloidosis. In some embodiments, the anti-FLC antibody is used to identify a subject as having plasma cell dysplasia. In some embodiments, the anti-FLC antibody is used to identify a subject as having monoclonal gammopathy of undetermined significance (MGUS). In some embodiments, the anti-FLC antibody is used to identify a subject as having multiple myeloma (MM). In some embodiments, the anti-FLC antibody is used to identify a subject as having plasma cell leukemia (PCL).

[0134] In some embodiments, anti-FLC antibody comprises one or more labels. Any label known in the art can be used to increase the ability of anti-FLC to be detected and / or visualized. In some embodiments, the label comprises a chemical label. In some embodiments, the label comprises an enzyme label. In some embodiments, the label comprises a substrate that can be detected in the presence of a specific enzyme. In some embodiments, the label is a fluorescent label.

[0135] Certain embodiments of the present disclosure are directed to the method of measuring light chain immunoglobulin in vivo using anti-FLC antibody disclosed herein.In some embodiments, the anti-FLC antibody comprises a detectable probe.In some embodiments, the anti-FLC antibody is an antibody that binds to an imaging agent.In some embodiments, the method is a diagnostic medical imaging method, such as PET / CT, SPECT, MRI, or any combination thereof.

[0136] III.B. Treatment method Certain embodiments of the present disclosure are directed to a method of treating a disease or condition in a subject in need of such treatment, comprising administering an anti-FLC antibody as disclosed herein. In some embodiments, the disease or condition comprises amyloidosis. In some embodiments, the disease or condition comprises systemic amyloidosis. In some embodiments, the disease or condition comprises AL amyloidosis. In some embodiments, the disease or condition comprises plasma cell dysplasia. In some embodiments, the disease or condition comprises monoclonal gammopathy of undetermined significance (MGUS). In some embodiments, the disease or condition comprises multiple myeloma (MM). In some embodiments, the disease or condition comprises plasma cell leukemia (PCL).

[0137] Certain embodiments of the present disclosure are directed to a method of reducing and / or eliminating immunoglobulin light chain aggregates, e.g., amyloid fibrils, in a subject in need thereof, comprising administering an anti-FLC antibody as disclosed herein. In some embodiments, the subject has one or more immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the heart. In some embodiments, the subject has one or more immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the kidney. In some embodiments, the subject has one or more immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the tissues of the nervous system. In some embodiments, the subject has one or more immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the brain.

[0138] Certain aspects of the present disclosure are directed to a method of treating nephrotic syndrome in a subject in need of such treatment, comprising administering to the subject an anti-FLC antibody disclosed herein, wherein the nephrotic syndrome comprises one or more immunoglobulin light chain deposits, e.g., one or more amyloid fibril deposits. Certain aspects of the present disclosure are directed to a method of treating hepatomegaly in a subject in need of such treatment, comprising administering to the subject an anti-FLC antibody disclosed herein, wherein the hepatomegaly comprises one or more immunoglobulin light chain deposits, e.g., one or more amyloid fibril deposits. Certain aspects of the present disclosure are directed to a method of treating peripheral neuropathy in a subject in need of such treatment, comprising administering to the subject an anti-FLC antibody disclosed herein, wherein the peripheral neuropathy comprises one or more immunoglobulin light chain deposits, e.g., one or more amyloid fibril deposits. Certain aspects of the present disclosure are directed to a method of treating restrictive cardiomyopathy in a subject in need thereof, comprising administering to the subject an anti-FLC antibody disclosed herein, wherein the restrictive cardiomyopathy comprises deposits of one or more immunoglobulin light chains, e.g., deposits of one or more amyloid fibrils.

[0139] In some embodiments, administering an anti-FLC antibody to a subject results in the reduction and / or disappearance of one or more immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the subject. In some embodiments, the size of the immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the subject is reduced after administration of an anti-FLC antibody. In some embodiments, the size of the immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% after administration of an anti-FLC antibody. In some embodiments, immunoglobulin light chain aggregates, eg, amyloid fibrils, are completely dissociated or otherwise resolved in a subject following administration of an anti-FLC antibody.

[0140] In some embodiments, administering an anti-FLC antibody to a subject reduces or delays the formation of one or more immunoglobulin light chain aggregates, such as amyloid fibrils, in the subject. In some embodiments, the rate of formation of immunoglobulin light chain aggregates, such as amyloid fibrils, in the subject is reduced after administration of an anti-FLC antibody. In some embodiments, the size of immunoglobulin light chain aggregates, such as amyloid fibrils, in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% after administration of an anti-FLC antibody. In some embodiments, the rate of immunoglobulin light chain aggregate formation, e.g., the rate of amyloid fibril formation, in a subject is stopped following administration of an anti-FLC antibody, e.g., no further immunoglobulin light chain aggregates are formed.

[0141] In some embodiments, the anti-FLC antibody induces an immune response in the subject after administration. In some embodiments, the anti-FLC antibody opsonizes immunoglobulin light chains, e.g., immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the subject. In some embodiments, the immune response comprises inducing macrophages. In some embodiments, the immune response comprises recruiting macrophages. In some embodiments, the immune response comprises increasing the phagocytosis of immunoglobulin light chains, e.g., immunoglobulin light chain aggregates, e.g., amyloid fibrils, in the subject. In some embodiments, the immune response is enhanced at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold compared to the immune response before administration of the anti-FLC antibody.

[0142] IV. Antibody Production The anti-FLC antibodies described herein can be produced using a variety of known techniques, such as the standard somatic cell hybridization method described in Kohler and Milstein, Nature 256:495 (1975). Although the somatic cell hybridization procedure is preferred, essentially other techniques for producing monoclonal antibodies can also be used, such as viral or oncogenic transformation of B lymphocytes, phage display methods using libraries of human antibody genes.

[0143] An example of an animal system for preparing hybridomas is the murine system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine bone marrow cells) and fusion procedures are also known.

[0144] The chimeric or humanized anti-FLC antibody described herein can be prepared based on the sequence of mouse monoclonal antibody prepared as described above.The DNA encoding heavy and light chain immunoglobulin can be obtained from the mouse hybridoma of interest using standard molecular biology techniques and manipulated to include non-mouse (e.g., human) immunoglobulin sequence.For example, to make chimeric antibody, the mouse variable region can be linked to human constant region using methods known in the art (e.g., U.S. Patent No. 4,816,567 to Cabilly et al.).To make humanized antibody, the mouse CDR region can be inserted into human framework using methods known in the art (e.g., see U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 to Queen et al.).

[0145] In some embodiments, the anti-FLC antibody described herein is a human monoclonal antibody. Such human monoclonal antibodies directed to FLC can be produced using genetically modified mice or artificial chromosome-transduced mice that have human immune system parts rather than mouse systems. These genetically modified mice and artificial chromosome-transduced mice include those referred to herein as HuMAb mice (e.g., HuMAb mice® (Medarex, Inc.)) and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

[0146] In some embodiments, the anti-FLC antibody described herein is produced using a mouse with human immunoglobulin sequences in a transgene and transchromosome, such as a mouse with a human heavy chain transgene and a human light chain transartificial chromosome. Such a mouse is referred to herein as a "KM mouse" and is described in detail in Ishida et al., International Publication No. WO02 / 43478.

[0147] Furthermore, alternative genetically modified animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-FLC antibodies described herein.For example, alternative genetically modified systems called Xenomouse (Abgenix, Inc.) can be used, and such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

[0148] In addition, alternative artificial chromosome transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-FLC antibodies described herein. For example, mice carrying both human heavy chain transgenic artificial chromosomes and human light chain transgenic artificial chromosomes, referred to as "TC mice", can be used. Such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. In addition, cattle carrying human heavy chain transgenic and light chain transgenic chromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to produce the anti-FLC antibodies described herein.

[0149] Additional mouse systems for producing human antibodies, e.g., human anti-FLC antibodies, that have been described in the art include (i) the VELOCIMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy and light chain variable regions are replaced by homologous recombination with human heavy and light chain variable regions operably linked to endogenous mouse constant regions such that a chimeric antibody (human V / mouse C) is produced in the mouse, which is then subsequently converted to a fully human antibody using standard recombinant DNA techniques, and (ii) the MEMO® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains unrearranged human heavy chain variable regions rather than a single rearranged human common light chain variable region. Such mice, and their use to produce antibodies, are described, for example, in WO2009 / 15777, US2010 / 0069614, WO2011 / 072204, WO2011 / 097603, WO2011 / 163311, WO2011 / 163314, WO2012 / 148873, US2012 / 0070861, and US2012 / 0073004.

[0150] The human monoclonal anti-FLC antibodies described herein can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are well established in the art. See, e.g., U.S. Pat. Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al., U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al., U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al., and U.S. Pat. Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0151] The human monoclonal anti-FLC antibodies described herein can also be prepared using SCID mice reconstituted with human immune cells so that human antibody responses can be produced by immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0152] The nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise introduce nucleic acid genes into suitable cells, thus producing antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. Sep-Oct 2010;2(5):466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing antibodies, which is a Chinese hamster ovary (CVHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing antibodies. In certain embodiments, described herein are methods of making an antibody, comprising culturing a cell containing nucleic acid encoding the antibody under in vitro conditions sufficient to allow production and secretion of the antibody.

[0153] In certain embodiments, described herein is a master cell bank comprising (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank comprises (a) a CHO cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container capable of withstanding freezing by liquid nitrogen.

[0154] Also described herein is a method of making the antibodies described herein. Such a method includes incubating a cell or cell line containing a nucleic acid encoding the antibody in a cell culture medium under conditions sufficient to allow expression and secretion of the antibody, and further collecting the antibody from the cell culture medium. The collecting step can further include one or more purification steps to remove live cells, cell debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the further purification step(s) include centrifugation, ultracentrifugation, purification with Protein A, Protein G, Protein A / G, or Protein L, and / or ion exchange chromatography. EXAMPLES

[0155] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0156] Example 1: Antibody Production Epitopes embedded within the native LC structure were selected that contribute to the same structural fold in both the kappa (STYSLSSTLT (SEQ ID NO: 1)) and lambda (NKYAASSYLSL (SEQ ID NO: 2)) light chain proteins. Epitopes were selected on the constant domain of the light chain because the sequences are present in all AL patients and therefore bind with less variability between patients compared to antibodies designed against the variable domain. Both antigens comprise the inner chain of the two-layered beta-sandwich motif of the immunoglobulin domain and are more than 85% embedded in the natively folded dimer and monomer (Figure 1A). The antigens are not involved in the formation of disulfide bonds and have significant antigenicity as assessed by EMBOSS explorer (Kolaskar et al., 1990). Because these antigens are embedded in the native structure, antibodies raised against these antigens should not bind to the natively folded light chain.

[0157] Appropriate peptide sequences (GKGGSTYSLSSTLTGGKG (SEQ ID NO: 3) and GKGGNKYAASSYLSLGGKG (SEQ ID NO: 4)) were then designed to generate antibodies that could potentially target only the pathologically misfolded state of the AL protein, leaving the natively folded normal immunoglobulin light chains alone. These antibodies are referred to as anti-κ and anti-λ antibodies.

[0158] The FL-LC protein expression system was used to test the specificity of the anti-κ antibody for the denatured and native forms of κ FL-LC. An indirect ELISA assay showed that the anti-κ antibody was specific for the unfolded form of κ FL-LC protein (Figure 2A). However, a commercially available pan-specific antibody was unable to detect any difference between the folded and unfolded LC protein (Figure 2B). The anti-κ antibody was also confirmed to be specific using denaturing SDS-PAGE Western blot with the anti-κ antibody (Figure 3A) and the commercial antibody (Figure 3B).

[0159] The ability of anti-κ antibodies to bind misfolded κ light chain aggregates in vitro was then tested using a thioflavin T (ThT) fluorescence assay. The pH dependence of κ light chain aggregates was measured. An increased tendency to aggregate was shown between pH 2.5 and pH 5 (Figure 4). The aggregates produced were then used to perform ELISA with both anti-κ and a commercial pan-specific antibody. A correlation was observed between the amount of aggregates produced (ThT) and binding of the anti-κ antibody, but not the commercial antibody (Figure 5). These results were confirmed by performing immunogold electron microscopy (IGEM) on the aggregates. Briefly, 200 mesh copper grids were coated with aggregates at either pH 4.5 or pH 6, blocked, and probed with anti-κ antibodies. An anti-rabbit secondary antibody that binds gold was used to visualize 6 nm electron opaque gold particles (black dots by EM). Binding of anti-κ antibodies to aggregates was observed at both pHs (Figure 5A-C).

[0160] Example 2: Removal of AL aggregates in vitro The ability of anti-κ antibodies to promote immune clearance of AL aggregates in vitro was tested by a phagocytosis assay. The assay was performed using THP-1 monocytic cells and light chain aggregates labeled with a pH-sensitive rhodamine (pHrodo) dye that fluoresces at low pH. Aggregates were incubated with (Figure 6B) or without (Figure 6A) anti-κ antibodies, and monocytic cells were added. The presence of pHrodo fluorescence within the cells indicates uptake of the labeled aggregates and their internalization in endolysosomes. pHrodo uptake was assessed by standard flow cytometry methods gating on fluorescence intensity. A three-fold increase in uptake into monocytic cells was observed in the presence of anti-κ antibodies, indicating effective clearance of immune complexes (Figures 6A-B).

[0161] The specificity of the anti-κ antibody was also verified by immunohistochemistry using human cardiac tissue, showing that the anti-κ antibody does not bind to lambda light chain amyloid (Fig. 7A-J) or ATTR amyloid, another protein that forms cardiac amyloid (Fig. 8A-E).

[0162] Example 3: Immunoreactivity of anti-κ antibodies in vivo To evaluate the immunoreactivity of anti-κ antibodies against AL species in vivo, an indirect ELISA assay was performed using plasma from patients with AL-CM. Anti-κ antibodies successfully discriminated between patients with AL-CM and those who had been previously treated with antimyeloma chemotherapy or had non-AL cardiac amyloidosis (e.g., ATTR-CM), regardless of the patient's AL isotype (Figure 9).

[0163] Example 4: Production of anti-κ monoclonal antibodies In this example, the design and generation of an anti-κ monoclonal antibody is described. To generate antibodies against misfolded immunoglobulin light chains, we selected two internal β chains as antigens, one containing residues 171-180 of SEQ ID NO:5 in the constant domain of the kappa light chain (STYSLSSTLT; SEQ ID NO:1), and the other containing residues 174-184 of SEQ ID NO:7 in the constant domain of the lambda light chain (NKYAASSYLSL; SEQ ID NO:2).

[0164] Additional residues were added to these epitopes to generate antigenic peptides (acetyl-CGKGGSTYSLSSTLTGGKG-amide; SEQ ID NO:3, and acetyl-CGGNKYAASSYLSLGG-amide; SEQ ID NO:9). These peptides were then covalently attached to the surface of keyhole limpet hemocyanin via the side chain of the N-terminal Cys residue using maleimide chemistry. The peptide complexes were used as immunogens to generate antibodies that could potentially target only the pathologically misfolded state of the light chain protein, leaving the natively folded normal immunoglobulin light chains untouched. Monoclonal antibodies were generated by mouse hybridoma technology.

[0165] Among several monoclonal antibodies produced by hybridoma technology using mouse kappa antigen peptide (SEQ ID NO: 1), one hybridoma clone was identified that produced an antibody (herein referred to as LX-96) with optimal properties of specific binding to misfolded forms. This hybridoma cell contains DNA sequences encoding the antibody heavy (FIG. 10C) and light (FIG. 10D) chains, and has the annotated amino acid sequence of FIGS. 10A-10B. Analysis of the constant domain suggests that the antibody is of the IgG1 / kappa subclass, and sequence analysis of the variable domain reveals unique CDR sequences (Tables 3 and 4). These six CDR sequences endow LX-96 with the property of avoiding the native structure and binding to misfolded and aggregated forms of kappa light chains.

[0166] [Table 3-1]

[0167] [Table 3-2]

[0168] [Table 4-1]

[0169] [Table 4-2]

[0170] Example 5: Characterization of anti-κ monoclonal antibodies To verify the binding specificity of LX-96 only for non-native misfolded / unfolded kappa light chains, we performed immunochemical assays using AL disease-associated kappa light chain mutants (AL09 or AL12) expressed and purified from T7 SHuffle cells (New England Biolabs). Using indirect ELISA, we show that LX-96 has strong binding affinity for both misfolded / unfolded kappa light chain mutants, but not for natively folded kappa light chains, produced by unfolding AL09 or AL12 with 6 M guanidine hydrochloride, followed by reduction with dithiothreitol and alkylation with iodoacetamide (Figure 11A and 11C). This binding activity is in contrast to a commercially available anti-kappa light chain antibody (Agilent DAKO), which cannot distinguish between native and misfolded / unfolded AL09 (Figure 11B). To obtain a measure of the relative binding affinity of LX-96 for misfolded versus native kappa light chains, a competitive ELISA was performed. Misfolded AL09 was coated on a 96-well plate and a mixture of LX-96 and competitor was added during primary antibody binding. Misfolded AL09 exhibited an inhibition constant (IC) of 30 nM for binding to LX-96. 50 ) (Figure 12), demonstrating strong binding affinity. In contrast, the natively folded kappa light chain showed no measurable competition for binding, indicating that LX-96 specifically binds to the misfolded light chain but not to the natively folded light chain.

[0171] Example 6: Binding Affinity (K) Measurements Using Biolayer Interferometry D Quantification of We quantified the monovalent binding affinity of LX-96 using biolayer interferometry (BLI). LX-96 was applied to an anti-mouse IgG Fc capture (AMC) biosensor and infiltrated with solutions of various concentrations of misfolded AL09 (0-200 nM) or native AL09 (0-3000 nM). From the sensorgrams, we observed a rapid binding process between misfolded AL09 and LX-96, followed by a slow dissociation process (Figure 13A). This suggests a very tight binding interaction between the antibody and the target. Using Octet Systems Software, we fitted the binding and dissociation sensorgrams and determined the binding affinity of LX-96 for misfolded AL09 to be 1.119 ± 0.01 nM (Figure 13A), whereas no measurable binding was detected for native AL09 at the concentrations assayed (Figure 13B). We repeated the assay for a different disease-causing kappa light chain variant (AL12) and found that the misfolded form was D We determined that LX-96 specifically binds to the misfolded forms of the different kappa light chain variants with similar single-digit nanomolar affinities, with a binding affinity of 7.5±0.1 nM (FIG. 13C) and no significant binding to the native form (FIG. 13D).

[0172] To further verify that LX-96 specifically binds to misfolded / unfolded kappa light chains, native Western blots were performed using AL09 in native, misfolded / unfolded, and amyloid states (Figure 14A-B). Misfolded / unfolded AL09 was generated by unfolding AL09 with 6 M guanidine hydrochloride, followed by reduction with dithiothreitol and alkylation with iodoacetamide, and amyloid state AL09 was generated by incubating AL09 at pH 7, 57°C, and 500 rpm for 5 days. Thioflavin T (ThT) fluorescence was used to confirm the presence of amyloid. Western blots were prepared with a primary antibody concentration of 5 μg / mL for LX-96 and DAKO kappa rabbit polyclonal antibody (commercial control). Secondary antibodies (goat anti-mouse 680LT and goat anti-rabbit 680LT from Licor) were diluted 1:10,000 according to the manufacturer's instructions. Western blot analysis shows that the commercially available kappa light chain antibodies recognize all forms of AL09 (native, misfolded / unfolded, and amyloid forms), whereas LX-96 recognizes only the misfolded / unfolded and amyloid forms, but not native AL09. Under conditions where amyloid is formed, native forms of AL09 are still present in small amounts as detected by the commercial antibody control. However, these species are also not recognized by LX-96, further demonstrating amyloid specificity.

[0173] Example 7: LX-96 specifically recognizes kappa-AL amyloid fibrils in human tissues We evaluated LX-96 reactivity by immunohistochemical analysis of cardiac, liver, and pancreatic tissues from the confirmed amyloidosis cases. In the cases of biopsy-confirmed kappa-AL amyloidosis, we verified the presence of kappa light chains in the tissues using a nonspecific anti-kappa antibody control and confirmed on-target staining of LX-96 to kappa-AL amyloid deposits in both cases of cardiac kappa-AL amyloidosis (FIGS. 15D and 15E) and hepatic kappa-AL amyloidosis (FIG. 15B).

[0174] We then evaluated the cross-reactivity of LX-96 in three pathological control tissues. In these cases, amyloid distribution in the tissues was confirmed by staining using the dye Congo Red or by using commercial antibodies (Figures 16A-I). In the case of ATTR cardiac amyloidosis, we observed positive Congo Red staining of amyloid (Figure 16A), and commercial anti-lambda and anti-kappa light chain antibodies bound non-specifically to non-amyloid (Figures 16B and C). On the other hand, for cardiac tissue, LX-96 showed no staining (Figure 16D). To evaluate cross-reactivity to lambda AL cardiac amyloid deposits, we first verified co-staining of tissues with Congo Red and anti-lambda antibodies (Figures 16E-F). While a commercially available anti-kappa antibody showed non-specific, non-amyloid binding to this tissue (FIG. 16G), LX-96 showed no cross-reactivity (FIG. 16H). Furthermore, no cross-reactivity was observed in the case of biopsy-confirmed IAPP (islet amyloid polypeptide) amyloidosis (FIG. 16I). In summary, this immunohistochemical analysis provides evidence that LX-96 has the ability to specifically bind to kappa light chain amyloid deposits ex vivo in various tissues and has no off-target interactions with native proteins or other non-specific amyloid forms.

[0175] Example 8: Cell-free inhibition of AL09 aggregation by LX-96 binding. Next, we evaluated the binding effect of LX-96 on AL09 aggregates in vitro under conditions that allow amyloid formation. A solution of FITC-labeled AL09 (indicated weight %: 2%) at a final concentration of 15 μM was treated with low pH (4.0) and stirred at 600 rpm to induce aggregation for 14 hours. The aggregates were centrifuged at high speed to pellet the insoluble aggregated material, and the soluble fraction of AL09 was indirectly measured by FITC fluorescence. We introduced substoichiometric concentrations (0-4 μM) of LX-96 to AL09 at the beginning of the aggregation process and measured the soluble fraction of AL09 at the end. LX-96 was able to inhibit AL09 aggregation at substoichiometric concentrations, with an estimated IC 50 The values ​​were less than 1 μM (FIG. 17).

[0176] Example 9: LX-96 induces antibody-mediated phagocytic uptake of kappa light chain amyloid in mouse monocytes Finally, we sought to examine the ability of LX-96 to promote immune clearance of AL09 aggregates in vitro. Thus, we performed a phagocytosis assay using RAW264.7 (ATCC) mouse monocytic cells and AL09 amyloid aggregates labeled with a pH-sensitive rhodamine (pHrodo) dye, which becomes more fluorescent due to a decrease in pH upon phagocytosis into lysosomes. AL09 amyloid aggregates produced by the method described above (57°C, 500 rpm, 5 days) were labeled and either preincubated with an isotype control, LX-96, or without antibody and incubated at 37°C, CO for 3 hours. 25% to give a final AL09 concentration of 100 μg / mL and antibody concentration of 20 μg / mL. Cells were imaged using bright field and pHrodo fluorescence channels to visualize phagocytic uptake of labeled AL09 amyloid aggregates. Visual assessment showed that LX-96 significantly increased the number of RAW cells internalizing AL09 aggregates compared to isotype or no antibody controls, as demonstrated by increased numbers of pHrodo positive cells (Figure 18B, Figure 18D, and Figure 18F). Quantifying uptake by mean pHrodo fluorescence per cell confirmed that LX-96A significantly increased phagocytic uptake of L09 compared to both isotype control (p=0.0236) and no antibody control (p=0.0102), indicating significantly increased clearance of immune complexes (Figure 18G).

[0177] Example 10: LX-96-dependent phagocytic elimination in a mouse amyloidoma model Next, we used NU / NU mice to evaluate the ability of LX-96 to induce immune responses in amyloidoma mouse models. We first tracked LX-96 distribution using a colocalization assay (Figure 19). 100 μg of AL09 amyloid was mixed with Matrigel and injected subcutaneously into the flank of mice to form amyloidomas. Fluorescently labeled LX-96 was injected at a single dose of 10 mg / kg via tail vein injection, and mice were imaged over several hours using an IVIS® Spectrum In Vivo Imaging System. We observed colocalization of LX-96 with amyloidomas within 60 minutes of injection, suggesting that LX-96 can reach extracellular amyloid deposits through the bloodstream. We then used a phagocytic uptake assay to evaluate whether LX-96 could enhance immune responses in mice. As described above, mice were implanted with 100 μg of pHrodo-labeled AL09 amyloid and administered LX-96 (or isotype antibody control) at a single dose of 10 mg / kg via tail vein injection. PHrodo fluorescence was measured 3 hours later. Injection of isotype antibody did not alter PHrodo (an indicator of phagocytosis) signal, whereas mice injected with LX-96 showed a large increase in phagocytosis signal (Figure 20A). We repeated the assay using two other antibody delivery modes, namely co-mixing (co-implantation) with amyloidoma prior to implantation and direct subcutaneous injection (subQ) into the amyloidoma, and observed a similar enhancement of approximately 2-fold increase in phagocytosis with all three delivery methods (Figure 20B). We then measured the rate of amyloid reduction in the presence or absence of LX-96 by implanting two equivalently sized fluorescently labeled amyloidomas in the same animals (Figure 21A). In this assay, one amyloidoma was left untreated and the adjacent implant was premixed with 10 mg / kg LX-96. Amyloidoma reduction was measured and quantified by fluorescent imaging over a 3-day period (FIG. 21B).Although both amyloidomas were reduced in size, the rate of reduction was significantly increased (3-fold) in the presence of LX-96, achieving 80% of amyloid reduction within 1 day compared to 3 days in the absence of antibody (Figure 21C). Furthermore, we confirmed the mechanism of action of LX-96 by resecting amyloidomas (premixed with either LX-96 or isotype control) and staining the resected tissues by immunohistochemistry using macrophage or neutrophil markers (Figure 22). The staining results suggest that LX-96 enhances the phagocytic clearance of amyloidomas by enhancing the recruitment of macrophages and neutrophils at the site. Altogether, these in vivo findings suggest that LX-96 can enhance the phagocytic clearance of deposited amyloid in an animal model.

[0178] The foregoing description of specific embodiments fully discloses the general nature of the present disclosure so that others may easily modify and / or adapt various applications of such specific embodiments by applying knowledge within the skill of the art without undue experimentation and without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation, and thus the terms or terms used herein should be interpreted in light of such teachings and guidance by those skilled in the art.

[0179] Other aspects of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the following claims.

[0180] All publications, patents, and patent applications disclosed in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody that specifically binds to a human immunoglobulin free light chain (FLC), wherein the anti-FLC antibody cross-competes with a reference antibody for binding to a kappa light chain, said reference antibody binding to an epitope on the immunoglobulin kappa light chain set forth in STYSLSSTLT (SEQ ID NO: 1), said reference antibody comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 14 and the light chain variable region amino acid sequence of SEQ ID NO:

10.

2. An antibody that specifically binds to human immunoglobulin free light chain (FLC), wherein the anti-FLC antibody cross-competes with a reference antibody for binding to a lambda light chain, the reference antibody binding to an epitope on the immunoglobulin lambda light chain represented by NKYAASSYLSL (sequence number 2).

3. An antibody described in any one of claims 1 or 2, which binds to aggregates of immunoglobulin light chain peptides.

4. An antibody described in any one of claims 1 to 3, which is a monoclonal antibody.

5. The antibody of any one of claims 1 to 4, comprising an antigen-binding portion of an antibody selected from the group consisting of Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, and bispecific antibody fragments. (a) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11; (b) a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12; (c) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; (d) VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15; (e) a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (f) a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; 6. The antibody of claim 1 , comprising:

7. The antibody described in claim 6, comprising a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

14.

8. The antibody described in claim 7, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

14.

9. The antibody described in claim 6, comprising a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

10.

10. The antibody described in claim 6, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

10.

11. The antibody of any one of claims 1 to 10, which binds to AL09 amyloid with a K D between 1 picomolar and 10 nanomolar.

12. A polynucleotide or multiple polynucleotides encoding an antibody described in any one of claims 1 to 11.

13. A host cell comprising a polynucleotide or a plurality of polynucleotides according to claim 12.

14. A pharmaceutical composition comprising an antibody described in any one of claims 1 to 11 and a pharma- ceutically acceptable excipient.

15. A method for producing an antibody that specifically binds to FLC, comprising culturing a host cell described in claim 13 under suitable conditions and isolating the antibody from the host cell or its supernatant.

16. A method for diagnosing AL-mediated amyloidosis in vitro, comprising contacting a biological sample obtained from a subject with an antibody described in any one of claims 1 to 11.

17. Use of an antibody according to any one of claims 1 to 11 in the manufacture of said antibody for use in a method for treating a subject having one or more amyloid deposits.

18. The use described in claim 17, wherein the subject has systemic amyloidosis.

19. The use described in claim 17, wherein the subject has AL amyloidosis.

20. Use of an antibody according to any one of claims 1 to 11 in the manufacture of said antibody for use in a method for treating a plasma cell disorder in a subject in need of such treatment.

21. The use of claim 20, wherein the plasma cell disorder comprises plasma cell dysplasia.

22. The use of claim 21, wherein the plasma cell dysplasia is selected from monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasma cell leukemia (PCL), and any combination thereof.

23. Use of an antibody according to any one of claims 1 to 11 in the manufacture of said antibody for use in a method for treating systemic amyloidosis in a subject in need of such treatment.

24. The use described in claim 23, wherein the systemic amyloidosis includes AL amyloidosis.

25. A method for producing an antibody directed against a kappa light chain constant region, comprising the step of contacting an antibody-producing cell or its conditioned medium, a polyclonal antibody mixture, or a phage display library with a polypeptide comprising the sequence of SEQ ID NO:

1.

26. The method of claim 25, wherein the polypeptide is less than 50 amino acids in length.

27. The method of claim 25, wherein the polypeptide consists of sequence number 1.

28. The method described in claim 25, further comprising a step of selecting an antibody that binds to sequence number 1.

29. A method for producing an antibody directed against a lambda light chain constant region, comprising the step of contacting an antibody-producing cell or its conditioned medium, a polyclonal antibody mixture, or a phage display library with a polypeptide comprising the sequence of SEQ ID NO:

2.

30. The method of claim 29, wherein the polypeptide is less than 50 amino acids in length.

31. The method of claim 29, wherein the polypeptide consists of sequence number 2.

32. The method of claim 29, further comprising a step of selecting an antibody that binds to sequence number 2.

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