High sensitivity biotinylated peptide binding ELISA assays

Abstract

Immune globulin light chain 'AL' amyloidosis is the most common systemic amyloidosis form, and accounts for about 70% of definite cases of developed countries. Since there are available therapeutic methods, such as therapeutic methods based on the antibody C11-1F4, and therapeutic methods being developed for AL amyloidosis, there is a need for a more accurate method for quality control of the c11-1F4 antibody or antigen-binding fragment thereof. Immunoassays, methods, and kits for testing 11-1F4 antibody specificity are provided. The sensitivity of the immunoassays, methods, and kits is more than 10 times that of conventional ELISA protocols for evaluating the binding of c11-1F4 to LEN peptides.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 340,766, filed on May 11, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to electronically submitted sequence listings

[0004] This application contains a sequence listing, which has been submitted electronically in XML format and the entire contents of which are incorporated herein by reference. The XML copy created on May 1, 2023 is named 0657_WO_SL.xml and is 3,360 bytes in size. Background Art

[0005] Immunoglobulin light chain (AL) amyloidosis is the most common form of systemic amyloidosis, accounting for approximately 70% of diagnosed cases in developed countries. Because there are available treatments, such as those based on the antibody C11-1F4, and treatments being developed for AL amyloidosis, there is a need for more precise methods for quality control of the c11-1F4 antibody or antigen-binding fragment thereof. Summary of the invention

[0006] In an embodiment, an immunoassay system may include a solid substrate coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO:1 and a biotinylated control peptide comprising the amino acid sequence of SEQ ID NO:2.

[0007] In an embodiment, the LEN peptide may consist of the amino acid sequence of SEQ ID NO:1.

[0008] In an embodiment, the control peptide may consist of the amino acid sequence of SEQ ID NO:2.

[0009] In embodiments, the solid substrate can be coated with a biotinylated LEN peptide at a concentration between about 0.1 μg / mL and 1 μg / mL. The solid substrate can be coated with a biotinylated LEN peptide at a concentration of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.65, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 1 μg / mL. The solid substrate can be coated with a biotinylated LEN peptide at a concentration of about 0.1 μg / mL.

[0010] In embodiments, the solid substrate can be coated with a biotinylated control peptide at a concentration between about 0.1 μg / mL and 1 μg / mL. The solid substrate can be coated with the biotinylated control peptide at a concentration of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.65, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 1 μg / mL. The solid substrate can be coated with a control peptide at a concentration of about 0.1 μg / mL.

[0011] In embodiments, the solid support can be a bead, a plate, a matrix, a polymer, a test tube, a thin plate, a culture dish or a test strip. The plate can be a multi-well plate. The multi-well plate can be composed of a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate.

[0012] In embodiments, the solid substrate may be a polystyrene, polypropylene, cyclic olefin, or glass solid substrate.

[0013] In an embodiment, the kit may include an immunoassay system as claimed in any one of claims 1 to 13 and a second antibody that specifically binds to an 11-1F4 antibody or an antigen-binding fragment thereof. The antibody may be conjugated to a detectable label. The detectable label may be a fluorescent label, a luminescent label, a bioluminescent label, a radioactive label, a chemiluminescent label, a colorimetric label, a fluorescent label, an enzyme label or a combination thereof. The label may be horseradish peroxidase (HRP). The 11-1F4 antibody may be a chimeric 11-1F4 antibody (c11-1F4 antibody) or an antigen-binding fragment thereof. The 11-1F4 antibody may be a humanized 11-1F4 antibody or an antigen-binding fragment thereof.

[0014] In an embodiment, a method for testing the binding specificity of the 11-1F4 antibody may include: contacting the 11-1F4 antibody or its antigen-binding fragment with the immunoassay system described herein; washing the solid support; adding a secondary antibody that specifically binds to the 11-1F4 antibody or its antigen-binding fragment; washing the solid support; and performing a detection step.

[0015] In an embodiment, washing may include washing at least 3 times with a wash buffer. The wash buffer may include PBS and 0.05% Tween-20 (polysorbate 20).

[0016] In an embodiment, the solid support may be coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1. The solid support may be coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1 and comprises incubating at the solid support with a solution comprising 0.1 μm / mL of the biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1 for about 1 hour at 37° C. The solution may comprise PBS (phosphate buffered saline).

[0017] In an embodiment, the solid support may be coated with a biotinylated control peptide comprising the amino acid sequence of SEQ ID NO: 2. The solid support may be coated with a biotinylated control peptide comprising the amino acid sequence of SEQ ID NO: 2 and comprises incubating at the solid support with a solution comprising 0.1 μm / mL of the biotinylated control peptide comprising the amino acid sequence of SEQ ID NO: 2 for about 1 hour at about 37° C. The solution may comprise PBS (phosphate buffered saline).

[0018] In an embodiment, the method may further comprise a blocking step prior to step (a), the blocking step comprising incubating the plate with a blocking buffer comprising PBS and 1% BSA at about 37°C for about 60 minutes.

[0019] In embodiments, the method may further include adding a control antibody to the solid support in the absence of the 11-1F4 antibody.

[0020] In embodiments, the control antibody may be a human IgG1-k antibody.

[0021] In an embodiment, the secondary antibody that specifically binds to the 11-1F4 antibody or antigen-binding fragment thereof can be conjugated to a detectable label.

[0022] In an embodiment, the detectable label can be a fluorescent label, a luminescent label, a bioluminescent label, a radioactive label, a chemiluminescent label, a colorimetric label, a fluorescent label, an enzyme label, or a combination thereof. The label can be horseradish peroxidase (HRP).

[0023] In an embodiment, a method for testing the binding specificity of an 11-1F4 antibody may include providing a solid support; coating at least a portion of the solid support with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1, comprising incubating at the solid support with a solution comprising 0.1 μm / mL of the biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1 for about 1 hour at about 37° C.; coating at least a portion of the solid support with a biotinylated control comprising the amino acid sequence of SEQ ID NO: 2, comprising incubating at the solid support with a solution comprising 0.1 μm / mL of the biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 3 for about 1 hour at about 37° C.; NO:2 is incubated with a solution of a biotinylated control peptide having an amino acid sequence of about 1 hour; the solid support is washed, optionally at least three times; the solid support is contacted with a blocking buffer comprising PBS (phosphate buffered saline) and 1% BSA (bovine serum albumin), and incubated at about 37°C for about 1 hour; a solution comprising the 11-1F4 antibody or its antigen-binding fragment is contacted with the solid support, and incubated at about 37°C for about 1 hour; the solid support is washed, optionally at least three times; a secondary antibody that specifically binds to the 11-1F4 antibody or its antigen-binding fragment is added, and incubated at about 37°C for about 1 hour; the solid support is washed, optionally at least three times; and a detection step is performed.

[0024] In an embodiment, the solution that may include the 11-1F4 antibody or antigen-binding fragment thereof includes an assay buffer containing PBS (phosphate buffered saline), 1% BSA (bovine serum albumin), and 0.05% Tween-20 (polysorbate 20).

[0025] In an embodiment, the second antibody can be conjugated with a detectable label. The detectable label can be a fluorescent label, a luminescent label, a bioluminescent label, a radioactive label, a chemiluminescent label, a colorimetric label, a fluorescent label, an enzyme label, or a combination thereof. The label can be horseradish peroxidase (HRP).

[0026] In an embodiment, the solution comprising the 11-1F4 antibody or antigen-binding fragment thereof may comprise a series of serial dilutions of the solution 11-1F4 antibody or antigen-binding fragment thereof.

[0027] In an embodiment, the 11-1F4 antibody may be a chimeric 11-1F4 antibody (c11-1F4 antibody) or an antigen-binding fragment thereof.

[0028] In an embodiment, the 11-1F4 antibody can be a humanized 11-1F4 antibody or an antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To further understand the nature, objects and advantages of the present disclosure, reference should be made to the following detailed description, read in conjunction with the following drawings.

[0030] Figure 1 Combined data from three independent assays are depicted. EC 50 =2.6±0.2 ng / mL (0.018±0.001 nM). Biotinylated and control peptide coating concentration: 0.1 μg / ml.

[0031] Figure 2 Depicted is a schematic diagram of an 8x12 plate showing antibody concentrations (rows A to H), antibodies used (c11-1F4 in columns 1 to 6 and IgG1-K in columns 7 to 9), and controls (columns 10 to 12) for an exemplary biotinylated LEN peptide plate (1 μg / mL).

[0032] Figure 3 A schematic diagram of an 8x12 plate is depicted showing antibody concentrations (rows A to H), antibodies used (c11-1F4 is columns 1 to 6 and IgG1-K is columns 7 to 9), and controls (columns 10 to 12) for an exemplary biotinylated control peptide plate (1 μg / mL). DETAILED DESCRIPTION

[0033] It should be understood that the present disclosure is not limited to the specific embodiments of the present disclosure described below, as changes may be made to the specific embodiments and these changes still fall within the scope of the appended claims. It should also be understood that the terminology used is only for the purpose of describing specific embodiments and is not intended to be limiting. Instead, the scope of the present disclosure will be determined by the appended claims.

[0034] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] definition

[0036] "Antibodies" (Ab) and "immunoglobulins" (Ig) are glycoproteins with the same structural features. While antibodies exhibit binding specificity for a specific antigen, immunoglobulins include antibodies and other antibody-like molecules that lack antigen specificity. The term "antibody" specifically encompasses monoclonal antibodies, including antibody fragment clones.

[0037] "Native antibodies and immunoglobulins" are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. Chothia et al. J. Mol. Biol. 186:651(1985); Novotny and Haber Proc. Natl. Acad. Sci. USA 82:4592(1985).

[0038] As used herein, "variable" refers generally to certain parts of the variable domain that differ greatly in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called frameworks (FRs). The variable domains of natural heavy and light chains each contain four FR regions, which mainly adopt a β-folded configuration and are connected by three CDRs, which form a loop that is connected and in some cases forms part of the β-folded structure. The CDRs in each chain are closely together through the FR region and contribute to the formation of the antigen binding site of the antibody together with the CDRs from the other chain. Johnson and Wu "Kabat Database and its applications: 30 years after the first variability plot." Nucleic Acids Res .(2000)28(1):214–218. The constant domains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cellular cytotoxicity.

[0039] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0040] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. In the two-chain Fv species, this region is composed of a dimer of a heavy chain variable domain and a light chain variable domain tightly non-covalently associated. In the single-chain Fv species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and the heavy chain can be associated in a "dimer" structure similar to the two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. In general, these six CDRs give the antibody antigen binding specificity. However, even a single variable domain (or half an Fv containing only three CDRs specific to an antigen) has the ability to recognize and bind to an antigen, but the affinity is lower than the complete binding site.

[0041] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical conjugates of antibody fragments are also known.

[0042] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0043] Depending on the amino acid sequence of their heavy chain constant domain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. "Therapeutic Antibody Engineering" (1st edition) Strohl & Strohl Woodhead Publishing (2012)

[0044] "Antibody fragments" comprise a portion of an intact antibody, typically the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; single-chain antibody molecules, including single-chain Fv (scFv) molecules; and multispecific antibodies formed from antibody fragments. "Human Monoclonal Antibodies: Methods and Protocols" (2nd ed.) Steinitz (Ed.) Humana Press (2019).

[0045] As used herein, the term "monoclonal antibody" refers to an antibody (or antibody fragment) obtained from a substantially homogeneous antibody population, for example, the individual antibodies constituting the population are substantially identical and are derived from a single clone. Monoclonal antibodies are highly specific, being directed against a single antigenic site (epitope). In addition, unlike polyclonal antibody preparations that typically include different antibodies directed against multiple epitopes, each monoclonal antibody binds to a single epitope on an antigen. Monoclonal antibodies are also easier to produce than polyclonal antibodies because monoclonal antibodies are produced in hybridoma cell cultures. The modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies described herein can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared by recombinant DNA methods. See, for example, U.S. Patent No. 4,816,567. "Monoclonal antibodies" also include clones of antibody fragments containing antigen recognition and binding sites isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0046] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) and fragments of such antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as long as they exhibit the desired biological activity. U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)); "Antibody Engineering" Volume 2 (2nd Edition) Kontermann & Dübel. Springer Press (2010).

[0047] A "human antibody" (also referred to as a "fully human antibody") is an antibody that includes a human framework region and all CDRs from human immunoglobulins. In one example, the framework and CDRs are derived from the same starting human heavy chain and / or light chain amino acid sequence. However, a framework from one human antibody can be engineered to include CDRs from a different human antibody.

[0048] The "humanized" form of a non-human (e.g., mouse) antibody is an immunoglobulin, an immunoglobulin chain, or a fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing a minimal sequence derived from a non-human immunoglobulin. A humanized antibody may be, for example, a human immunoglobulin (acceptor antibody), wherein residues from the acceptor CDR are replaced by residues from a CDR or synthetic sequence (donor antibody) from a non-human species (such as a mouse, rat, or rabbit) having the desired specificity, affinity, and ability. Alternatively, antibodies identified or produced in species other than humans, such as mouse antibodies, can be "humanized" by replacing one or more amino acids present in the mouse antibody sequence with amino acids present in the corresponding sequence position in the human antibody sequence. Because there are several sequence differences between, for example, mouse antibody sequences and human antibody sequences, "fully humanized" antibodies are produced by replacing human positions at each difference site. In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced ("back mutation") by corresponding non-human residues. In addition, humanized antibodies may include residues that are neither present in the recipient antibody nor in the CDR or framework sequences that are imported. These modifications are made in order to further improve and optimize antibody performance. In an embodiment, all CDRs can be derived from donor immunoglobulins in humanized immunoglobulins. Constant regions do not need to be present, but if present, they should be substantially identical to human immunoglobulin constant regions, for example, at least about 85%-90%, such as about 95% or more identical. Therefore, all parts of humanized immunoglobulins (possibly except CDR) are substantially identical to the corresponding parts of natural human immunoglobulin sequences. Humanization or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by genetic engineering. See, for example, U.S. Patent No. 5,585,089.

[0049] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the structure required for antigen binding. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0050] The term "diabodies" refers to small antibody fragments with two antigen binding sites, which fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the two domains are forced to pair with the complementary domains of another chain and generate two antigen binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc Natl Acad Sci USA, 90:6444-6448 (1993).

[0051] An "isolated" antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteins or non-proteinaceous solutes. The antibody may be purified, for example: (1) to greater than 95% by weight, as determined by the Lowry method (or optionally, to more than 99% by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably a silver stain. An isolated antibody may include, for example, an antibody in situ in a recombinant cell, because at least one component of the antibody's natural environment is not present. The isolated antibody is prepared by at least one purification step.

[0052] As used herein, "epitope" refers generally to an antigen or portion of an antigen to which an antibody binds. As described herein, for example, the 11-1F4 antibody binds to a portion of human light chain amyloid fibrils in an animal (e.g., a mammal, such as a human). An epitope with immunogenic activity is a portion of human light chain amyloid fibrils that elicits an antibody response in an animal, including binding and effector functions. Immunospecificity, i.e., the degree to which an antibody binds to a particular epitope, can be determined by methods known in the art, for example, by immunoassays as described herein. An antigenic epitope is not necessarily immunogenic.

[0053] 11-1F4 antibody-peptide epitope binding assay

[0054] This system and method described herein provides a 10-fold increase in sensitivity for the 11-1F4 antibody-peptide epitope binding assay compared to similar assays reported in the literature.During monoclonal antibody drug development and for antibody characterization, reliable and highly sensitive binding assays are needed.

[0055] High sensitivity assays as described herein provide reliable quality control assays for any humanization and / or any other modified forms of the 11-1F4 antibody and the antibody. Compared with previously described assays, the increase in sensitivity ensures that the difference in the affinity of the antibody to its epitope can be more easily observed. Compared with previously described in conjunction with ELISA (enzyme-linked immunosorbent assay), the increase in sensitivity also has the advantage of using less reagents in the assay.

[0056] This peptide ELISA assay uses biotinylated peptides and streptavidin-coated plates. This configuration increases the assay sensitivity 10-fold (compared to other peptide ELISA assays).

[0057] The use of biotinylated peptides and streptavidin-coated plates resulted in consistent distribution and conformation of the peptide coating, leading to a 10-fold increase in assay sensitivity. The higher assay sensitivity of this assay means a lower likelihood of nonspecific signal interference, providing a better quality control method. This innovation also translates into less antibody required for each individual test, providing an economic advantage.

[0058] The solid support described herein can be coated with one or both of a LEN peptide comprising the amino acid sequence of DIVMTQSPDSLAVSLGERA TIN (SEQ ID NO: 1) and / or a control peptide (CT) comprising the amino acid sequence of AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA (SEQ ID NO: 2). The LEN peptide is a peptide of the atomic LEN protein, such as a 22 amino acid N-terminal fragment (Solomon, A. and McLaughlin, C., J. Biol. Chem., 244: 3303-404, 1969). The LEN peptide and the control peptide can be biotinylated, such as covalently attached to biotin. Methods for peptide biotinylation are known in the art.

[0059] Anti-AL antibody

[0060] 11-1F4 (also known as CAEL-101) and related variants (Solomon, A. et al., Am. J. Pathol., 137:855–62, 1990; Solomon, A. et al., Clin. Cancer Res., 9:3831s-3838s, 2003; Wall, J. et al., Proc. Natl. Acad. Sci. USA., 115:E10839-E10848, 2018; U.S. Patent Nos. 8,105,594; 10,046,050; and 10,213,506; U.S. Patent Application Publication No. 2020 / 0181246) represent an IgG1k monoclonal antibody that directly binds to a conformational epitope present on human light chain amyloid fibrils, regardless of the κ or λ isotype. It has been demonstrated that 11-1F4 causes neutrophil chemotaxis and activation, causing Fcγ receptor-mediated opsonization and proteolysis of deposited amyloid fibrils, leading to the reversal of AL amyloid tumors in mouse models. Although not bound by theory, the 11-1F4 antibody can bind to a conformational epitope on human light chain amyloid fibrils. The 11-1F4 antibody can selectively bind to an epitope (linear epitope) comprising the amino acid sequence DIVMTQSPDSLAVSLGERATIN (SEQ ID NO: 1). Chimeric, human and humanized (partial or complete) can be used in the context of this assay.

[0061] Immunoassay systems (e.g., enzyme-linked immunosorbent assay (ELISA)) and methods described herein can be used to determine the specificity of the 11-1F4 antibody or its antigen-binding fragment. The binding affinity of the antibody can be determined, for example, by Scatchard analysis (Frankel, M. and Gerhard, W., Mol. Immunol., 16: 101-6, 1979). Binding affinity can be measured, for example, by antigen / antibody dissociation rate. High binding affinity can be measured by competitive radioimmunoassay. Binding affinity can be measured by ELISA such as immunoassay described herein.

[0062] Certain assay methods require reagent fixation. Fixation requires separation of anti-human light chain amyloid fibril antibodies from any remaining free human light chain amyloid fibrils in solution. This is conventionally accomplished by insolubilizing anti-human light chain amyloid fibril antibodies prior to the assay procedure, such as by adsorption to a water-insoluble matrix or surface (U.S. Patent No. 3,720,760), by covalent coupling (e.g., cross-linking using glutaraldehyde), or by insolubilizing anti-human light chain amyloid fibril antibodies, such as by immunoprecipitation.

[0063] Secondary Antibodies

[0064] The detectably labeled secondary antibody used with the immunoassay systems and methods described herein can be any antibody or antigen-binding fragment thereof that specifically binds to the 11-1F4 antibody or antigen-binding fragment thereof.

[0065] The label used with the secondary antibody can be any label with detectable function that does not interfere with the binding of human light chain amyloid fibrils and anti-human light chain amyloid fibrils. Many labels are known for use in immunoassays, examples of which include directly detectable moieties, such as fluorescent dyes, chemiluminescent and radioactive labels, and moieties that must react or derivatize to be detected, such as enzymes. Examples of such labels include radioisotopes, 32 P. 14 C. 125 I. 3 H and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase (e.g., firefly luciferase and bacterial luciferase) (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (e.g., glucose oxidase), galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases (such as uricase and xanthine oxidase), enzymes (such as HRP, lactoperoxidase or microperoxidase) that combine to oxidize dye precursors with hydrogen peroxide, biotin / avidin, spin labeling, phage labeling, stable free radicals or combinations thereof. For example, the secondary antibody can be HRP-goat anti-human Fcy.

[0066] Conventional methods can be used to covalently bind these labels to proteins or polypeptides. For example, coupling agents such as dialdehydes, carbodiimides, bismaleimides, bisimidoesters, bis-nitrobenzidine, etc. can be used to label antibodies with the above-mentioned fluorescent, chemiluminescent and enzyme labels (U.S. Patent Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., Nature. 1962; 194: 495-496; David et al., Biochemistry. 1974; 13: 1014-1021; Pain et al., J Immunol Methods, 1981; 40: 219-230; and Nygren, J Histochem Cytochem. 1982; 30 (5): 407-412). Labels that can be used for the materials and methods described herein include enzymes such as horseradish peroxidase and alkaline phosphatase.

[0067] Conjugation of such labels, including enzymes, to antibodies is standard procedure for those of ordinary skill in the immunoassay arts (O'Sullivan et al., "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. JJ Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, NY, 1981), pp. 147-166).

[0068] Solid support

[0069] The immunoassay systems and methods described herein may include a solid support. For example, the solid support may be a bead, a plate, a matrix, a polymer, a test tube, a thin plate, a culture dish, or a test strip. Combinations of solid supports may be used in the immunoassay systems and methods described herein. The plate may be a multi-well plate. The multi-well plate may be a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate. The solid substrate may be made of any acceptable material, such as polystyrene, polypropylene, cycloolefins, or glass.

[0070] The solid support can be coated with a biotinylated LEN peptide comprising the amino acid sequence DIVMTQSPDSLAVSLGERATIN (SEQ ID NO: 1) at a concentration between about 0.01 μg / mL and 1 μg / mL. For example, the concentration can be about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.65, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 μg / mL. Preferably, the concentration is about 0.1 μg / mL.

[0071] The solid support can be coated with a biotinylated control peptide comprising the amino acid sequence AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA (SEQ ID NO: 2) at a concentration between about 0.01 μg / mL and 1 μg / mL. For example, the concentration can be about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.65, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 μg / mL. Preferably, the concentration is about 0.1 μg / mL.

[0072] Buffer

[0073] Buffers can be used in the immunoassay systems and methods described herein to form solutions with the 11-1F4 antibody, wash solution, blocking solution, or a combination thereof. Exemplary buffers include, but are not limited to: a coating buffer comprising phosphate buffered saline (PBS); a coating buffer comprising PBS and 0.05% (polysorbate 20); a blocking buffer containing PBS and 1% BSA (bovine serum albumin); and a blocking buffer containing PBS, 1% BSA, and 0.05% (Polysorbate 20) assay buffer. Buffers are known in the art, and commercially available buffers can be used.

[0074] condition

[0075] Typically, the reagents are incubated at about 37°C for about 60 minutes.

[0076] In some embodiments, the incubation step may be continued for about 30 minutes to 90 minutes. For example, the incubation step may be continued for about 60 minutes. For example, the incubation step may be continued for about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 minutes. The incubation step may last about 45 to 75 minutes, 55 to 65 minutes, 50 to 70 minutes, or 40 to 80 minutes.

[0077] The reacting step can be performed at about 37° C. For example, the reacting step can be performed at a temperature between about 35° C. and 45° C. The reacting step can be performed at a temperature of about 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C. The reacting step can be performed at a temperature between about 37° C. and 40° C., 36° C. and 41° C., or 36° C. and 40° C.

[0078] Reagent test kit

[0079] The kit may include one or more containers containing one or more ingredients of the pharmaceutical composition including a buffer, a solid support, optionally a multiwell plate, a LEN peptide (biotinylated) and a control peptide (biotinylated). The multiwell plate may be coated with the LEN peptide (biotinylated) and the control peptide (biotinylated). Optionally, accompanying such one or more containers may be a statement in tabular form issued by a governmental agency regulating the manufacture, use or sale of a drug or biological product, the statement reflecting approval by the agency for manufacture, use or sale for human administration.

[0080] The test kit can also include a control antibody that does not react with human light chain amyloid fibrils. In another specific embodiment, the test kit contains a tool for detecting the combination of antibodies and human light chain amyloid fibrils (for example, antibodies can be conjugated with detectable substrates such as fluorescent compounds, enzyme substrates, radioactive compounds or luminescent compounds, or the second antibody identifying the first antibody can be conjugated with detectable substrates). The test kit can include recombinantly produced or chemically synthesized human light chain amyloid fibrils. The human light chain amyloid fibrils provided in the test kit can also be attached to a solid support. In a more specific embodiment, the detection tool of the above-mentioned test kit includes the solid support to which human light chain amyloid fibrils are attached. Such test kits can also include the anti-human antibodies of unattached reporter factor labels. In this embodiment, the combination of antibodies and human light chain amyloid fibrils can be detected by the combination of the antibodies of the reporter factor labels.

[0081] A diagnostic kit for screening biological samples of antigens containing polypeptides as described herein. The diagnostic kit may comprise a substantially separated antibody specifically immunoreactive with human light chain amyloid fibrils, and a tool for detecting the binding of human light chain amyloid fibrils to the antibody. The antibody may be attached to a solid support. The antibody may be a monoclonal antibody. The detection tool of the kit may comprise a second labeled monoclonal antibody. Alternatively, or in addition, the detection tool may comprise a labeled competing antigen.

[0082] In one diagnostic configuration, a biological sample is reacted with a solid phase reagent having surface-bound human light chain amyloid fibrils. After the human light chain amyloid fibrils bind to the specific antibody, unbound serum components are removed by washing, a reporter-labeled anti-human antibody is added, unbound anti-human antibody is removed by washing, and the reagent reacts with the reporter-labeled anti-human antibody so that the reporter binds to the reagent in proportion to the amount of anti-human light chain amyloid fibril antibody bound to the solid support. Typically, the reporter is an enzyme that is detected by incubating the solid phase in the presence of a suitable fluorescent, luminescent, or colorimetric substrate.

[0083] The solid surface reagents in the above assays are prepared by known techniques for attaching protein materials to solid support materials such as polymer beads, impregnated rods, 96-well plates or filter materials. These attachment methods generally include non-specific adsorption of proteins to the support or covalent attachment of proteins, usually through free amine groups to chemically reactive groups on the solid support, such as activated carboxyl, hydroxyl or aldehyde groups. Alternatively, streptavidin-coated plates can be used in conjunction with biotinylated antigens.

[0084] Thus, described herein are assay systems or kits for practicing such diagnostic methods. The kits generally include a support having surface-bound recombinant human light chain amyloid fibrils, and a reporter-labeled anti-human antibody for detecting surface-bound anti-human light chain amyloid fibril antibodies.

[0085] Example 1

[0086] ELISA LEN-c11-1F4 assay

[0087] This example describes the use of the immunoassay systems and methods described herein to titrate c11-1F4 antibodies against biotinylated peptides (LEN, control) on streptavidin-coated plates. Antibody concentrations ranged from 5 μg / mL to 5×10 -7 μg / mL, diluted 10 times. IgG1-κ antibody was used as a negative control.

[0088] Buffer

[0089] The following buffers were used: Coating buffer: PBS (phosphate buffered saline); Washing buffer: PBS + 0.05% (polysorbate 20); blocking buffer: PBS + 1% BSA (bovine serum albumin); and assay buffer: PBS + 1% BSA + 0.05%

[0091] Plate coating

[0092] Add 50 μL of 0.1 μg / mL biotinylated LEN peptide (DIVMTQSPDSLAVSL GERATIN) (SEQ ID NO: 1) (New England Peptide) in PBS and 50 μL of 0.1 μg / mL biotinylated control peptide (AVSEHQLLHDKGKSIQDLRRRF FLHHLIAEIHTA) (SEQ ID NO: 2) in PBS to each well of the first 12×8 well microplate (StreptaWell Microplate, Transparent, Roche) to make plate #1.

[0093] 50 μL of 0.1 μg / mL biotinylated control peptide (AVSEHQLLHDKGKSIQ DLRRRFFLHHLIAEIHTA) (SEQ ID NO: 2) in PBS was added to each well of a second 12×8 well microplate (StreptaWell Microplate, Transparent, Roche) to make plate #2.

[0094] The plates were incubated at about 37°C for about 1 hour.

[0095] Closed

[0096] After incubation at 37° C. for about 1 hour, the coated plates were washed three times in a MicroTek plate washer. About 200 μL of blocking buffer was added to each well, and the plates were incubated at about 37° C. for about 1 hour.

[0097] Antibody Binding

[0098] Prepare a 5 μg / mL c11-1F4 antibody solution in assay buffer (c11-1F4 anti-light chain amyloid antibody (TPP-3841) 4.54 mg / mL from GeneArt, lot 2753245). Aliquot approximately 140 μL of the 5 μg / mL c11-1F4 antibody solution into the first 6 wells of the first row of an 8×12 well prep plate (i.e., Figure 2 6 of row A of the plate schematically shown in FIG.

[0099] Prepare a 5 μg / mL human IgG1-K-ULNL solution (human IgG1-K-ULNL, SouthernBiotech 0151K-01) in assay buffer. Divide approximately 140 μL of the 5 μg / mL human IgG1-K-ULNL solution into the next three wells in the first row of the preparation plate (i.e., Figure 2 The last three columns ( Figure 2 The remaining wells in row A, columns 10-12 of ) received 126 μL of assay buffer. Figure 2 Serial dilutions are performed on rows B through H of the preparation plate, starting with 14 μL from row A and diluting into 126 μL of buffer in the next row (row B), then repeating for subsequent rows (i.e., 14 μL from row B is diluted into 126 μL of buffer in row C, and so on). From each well of the preparation plate, transfer 50 μL to the corresponding well of each of two assay plates: Plate #1 and Plate #2 (see, e.g., Figure 2 ). The assay plate was incubated at about 37°C for about 1 hour.

[0100] Detection

[0101] Dilute HRP-goat anti-human Fcy 1:1000 in assay buffer. Wash the assay plates (plate #1 and plate #2) three times in a MicroTek plate washer. Divide 50 μL of labeled secondary antibody into each well of the assay plate. Then incubate the assay plate at about 37°C for about 1 hour.

[0102] Development and reading

[0103] The assay plate was washed three times in a MicroTek plate washer. 50 μL of TMB substrate was aliquoted into each well of the assay plate and the assay plate was incubated at room temperature (about 25° C.) for about 4 minutes. 50 μL of stop solution was aliquoted into each well of the assay plate to stop development. The absorbance was read at 450 nm.

[0104] The methods described herein (eg, ELISA LEN peptide / c11-1F4 antibody) showed unexpected improvements when compared to other ELISA protocols.

[0105] Using an ELISA protocol, O'Nuallain et al. (Biochemistry. 2007; 46(45): 13049-13058) tested the binding of the mouse c11-1F4 antibody to plate-immobilized phage and LEN peptides, as well as the inhibition of antibody binding to immobilized Len(1–22) by these molecules, and report the EC values ​​in Table 1 50 =0.24±0.1nM.

[0106] Using an ELISA protocol, Wall et al. (Proc Natl Acad Sci US A. 2018; 115(46): E10839-E10848) tested the binding of the chimeric c11-1F4 antibody and found that the estimated EC 50 (the midpoint of the binding transition) was ∼0.4 nM, which was indistinguishable from the binding of 11-1F4 to the κ4 N-terminal peptide LEN(1–22), which is derived from the Bence Jones protein LEN used as an immunogen to generate m11-1F4.

[0107] Unexpectedly, the ELISA protocol described herein is more than 10 times more sensitive than the ELISA protocol routinely used to evaluate the binding of chimeric c11-1F4 to the LEN peptide (EC 50 =0.018±0.001; see Figure 1 ).

[0108] All references cited in this specification are incorporated herein by reference as if each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0109] It should be understood that each of the above elements, or two or more together, may also find useful applications in other types of methods than those described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can easily adapt it to various applications by applying existing knowledge without omitting the essential features that clearly constitute the general or specific aspects of the present disclosure set forth in the appended claims from the viewpoint of the prior art. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is limited only by the following claims.

Claims

1. An immunoassay system comprising a solid substrate coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1 and a biotinylated control peptide comprising the amino acid sequence of SEQ ID NO:

2.

2. The immunoassay system of claim 1, wherein the LEN peptide consists of the amino acid sequence of SEQ ID NO:

1.

3. The immunoassay system according to claim 1 or 2, wherein the control peptide consists of the amino acid sequence of SEQ ID NO:

2.

4. The immunoassay system of any one of claims 1-3, wherein the solid substrate is coated with the biotinylated LEN peptide at a concentration between about 0.1 μg / mL and 1 μg / mL.

5. The immunoassay system of any one of claims 1-4, wherein the solid substrate is coated with the biotinylated LEN peptide at a concentration of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.65, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.01 μg / mL.

6. The immunoassay system of any one of claims 1-5, wherein the solid substrate is coated with the biotinylated LEN peptide at a concentration of about 0.1 μg / mL.

7. The immunoassay system of any one of claims 1-6, wherein the solid substrate is coated with the biotinylated control peptide at a concentration between about 0.1 μg / mL and 1 μg / mL.

8. The immunoassay system of any one of claims 1-7, wherein the solid substrate is coated with the biotinylated control peptide at a concentration of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.65, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.01 μg / mL.

9. The immunoassay system of any one of claims 1-8, wherein the solid substrate is coated with the control peptide at a concentration of about 0.1 μg / mL.

10. The immunoassay system of any one of claims 1-9, wherein the solid support is a bead, a plate, a matrix, a polymer, a test tube, a sheet, a culture dish, or a test strip.

11. The immunoassay system of claim 10, wherein the plate is a multi-well plate.

12. The immunoassay system of claim 11, wherein the multi-well plate consists of a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate.

13. The immunoassay system of any one of claims 1 to 12, wherein the solid substrate is a polystyrene, polypropylene, cyclic olefin or glass solid substrate.

14. A kit comprising the immunoassay system according to any one of claims 1 to 13 and a secondary antibody that specifically binds to the 11-1F4 antibody or an antigen-binding fragment thereof.

15. The kit of claim 14, wherein the antibody is conjugated to a detectable label.

16. The kit of claim 15, wherein the detectable label is a fluorescent label, a luminescent label, a bioluminescent label, a radioactive label, a chemiluminescent label, a colorimetric label, a fluorescent label, an enzyme label, or a combination thereof.

17. The kit of claim 15 or 16, wherein the label is horseradish peroxidase (HRP).

18. The kit of any one of claims 14 to 17, wherein the 11-1F4 antibody is a chimeric 11-1F4 antibody (c11-1F4 antibody) or an antigen-binding fragment thereof.

19. The kit of any one of claims 14-17, wherein the 11-1F4 antibody is a humanized 11-1F4 antibody or an antigen-binding fragment thereof.

20. A method for testing the binding specificity of 11-1F4 antibody, comprising: (a) contacting the 11-1F4 antibody or an antigen-binding fragment thereof with the immunoassay system according to any one of claims 1 to 13; (b) washing the solid support; (c) adding a secondary antibody that specifically binds to the 11-1F4 antibody or the antigen-binding fragment thereof; (d) washing the solid support; and (e) performing a detection step.

21. The method of claim 20, wherein the washing comprises washing at least 3 times with wash buffer.

22. The method of claim 21, wherein the wash buffer comprises PBS and 0.05% Tween-20 (polysorbate 20).

23. The method of any one of claims 20-22, wherein the solid support is coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO:

1.

24. The method of any one of claims 20-22, wherein the solid support is coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1, and the method comprises incubating at the solid support for about 1 hour at about 37°C with a solution comprising 0.1 μm / mL of the biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO:

1.

25. The method of claim 24, wherein the solution comprises PBS (phosphate buffered saline).

26. The method of any one of claims 20-25, wherein the solid support is coated with a biotinylated control peptide comprising the amino acid sequence of SEQ ID NO:

2.

27. The method of any one of claims 20-26, wherein the solid support is coated with a biotinylated control peptide comprising the amino acid sequence of SEQ ID NO: 2, and the method comprises incubating at the solid support for about 1 hour at about 37°C with a solution comprising 0.1 μm / mL of the biotinylated control peptide comprising the amino acid sequence of SEQ ID NO:

2.

28. The method of claim 27, wherein the solution comprises PBS (phosphate buffered saline).

29. The method of claim 20, wherein the method further comprises a blocking step prior to step (a), the blocking step comprising incubating the plate with a blocking buffer comprising PBS and 1% BSA at about 37°C for about 60 minutes.

30. The method of claim 20, wherein the method further comprises adding a control antibody to the solid support in the absence of the 11-1F4 antibody.

31. The method of claim 30, wherein the control antibody is a human IgG1-k antibody.

32. The method of any one of claims 20-31, wherein the second antibody that specifically binds to the 11-1F4 antibody or antigen-binding fragment thereof is conjugated to a detectable label.

33. The method of claim 32, wherein the detectable label is a fluorescent label, a luminescent label, a bioluminescent label, a radioactive label, a chemiluminescent label, a colorimetric label, a fluorescent label, an enzyme label, or a combination thereof.

34. The method of claim 32 or 33, wherein the label is horseradish peroxidase (HRP).

35. A method for testing the binding specificity of 11-1F4 antibody, comprising (a) providing a solid support; (b) coating at least a portion of the solid support with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1, comprising incubating at about 37°C at the solid support with a solution comprising 0.1 μm / mL of the biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1 for about 1 hour; (c) coating at least a portion of the solid support with a biotinylated control comprising the amino acid sequence of SEQ ID NO: 2, comprising incubating at the solid support at about 37° C. with a solution comprising 0.1 μm / mL of a biotinylated control peptide comprising the amino acid sequence of SEQ ID NO: 2 for about 1 hour; (d) washing the solid support, optionally at least three times; (e) contacting the solid support with a blocking buffer comprising PBS (phosphate buffered saline) and 1% BSA (bovine serum albumin), and incubating at about 37° C. for about 1 hour; (f) contacting a solution comprising the 11-1F4 antibody or an antigen-binding fragment thereof with the solid support and incubating at about 37° C. for about 1 hour; (g) washing the solid support, optionally at least three times; (h) adding a secondary antibody that specifically binds to the 11-1F4 antibody or antigen-binding fragment thereof, and incubating at about 37° C. for about 1 hour; (i) washing the solid support, optionally at least three times; as well as (j) performing a detection step.

36. The method of claim 35, wherein the solution comprising the 11-1F4 antibody or antigen-binding fragment thereof comprises an assay buffer containing PBS (phosphate buffered saline), 1% BSA (bovine serum albumin) and 0.05% Tween-20 (polysorbate 20).

37. The method of claim 35 or 36, wherein the secondary antibody is conjugated to a detectable label.

38. The method of claim 37, wherein the detectable label is a fluorescent label, a luminescent label, a bioluminescent label, a radioactive label, a chemiluminescent label, a colorimetric label, a fluorescent label, an enzyme label, or a combination thereof.

39. The method of claim 37 or 38, wherein the label is horseradish peroxidase (HRP).

40. The method of any one of claims 35-39, wherein the solution comprising the 11-1F4 antibody or antigen-binding fragment thereof comprises a series of serial dilutions of the solution 11-1F4 antibody or antigen-binding fragment thereof.

41. The method of any one of claims 35-40, wherein the 11-1F4 antibody is a chimeric 11-1F4 antibody (c11-1F4 antibody) or an antigen-binding fragment thereof.

42. The method of any one of claims 35-40, wherein the 11-1F4 antibody is a humanized 11-1F4 antibody or an antigen-binding fragment thereof.

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