Highly sensitive biotinylated peptide binding ELISA assay

Abstract

Immunoglobulin light chain "AL" amyloidosis is the most common form of systemic amyloidosis, accounting for approximately 70% of diagnosed cases in developed countries. For example, with treatments based on the antibody C11-1F4 available and in development for AL amyloidosis, there is a need for more accurate methods for quality control of c11-1F4 antibodies or antigen-binding fragments thereof. Provided are immunoassays, methods, and kits for testing the specificity of the 11-1F4 antibody. The immunoassays, methods, and kits are more than 10 times more sensitive than the ELISA protocol routinely used to assess binding of c11-1F4 to the LEN peptide.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 340,766, filed May 11, 2022, which is incorporated by reference in its entirety.

[0002] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically in XML format, 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 technology]

[0003] Immunoglobulin light chain (AL) amyloidosis is the most common form of systemic amyloidosis, accounting for approximately 70% of cases diagnosed in developed countries. For example, since there are treatments available based on the antibody C11-1F4 and are in development for AL amyloidosis, there is a need for more accurate methods for the quality control of the c11-1F4 antibody or its antigen-binding fragments. Summary of the Invention

[0004] In one embodiment, an immunoassay system can 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.

[0005] In one embodiment, the LEN peptide may consist of the amino acid sequence of SEQ ID NO:1.

[0006] In one embodiment, the control peptide may consist of the amino acid sequence of SEQ ID NO:2.

[0007] In one embodiment, the solid substrate can be coated with biotinylated LEN peptide at a concentration of about 0.1 μg / mL to 1 μg / mL. The solid substrate can be coated with 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 μg / mL. The solid substrate can be coated with biotinylated LEN peptide at a concentration of about 0.1 μg / mL.

[0008] In one embodiment, the solid substrate may be coated with a biotinylated control peptide at a concentration of about 0.1 μg / mL to 1 μg / mL. The solid substrate may be coated with a 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 μg / mL. The solid substrate may be coated with a control peptide at a concentration of about 0.1 μg / mL.

[0009] In one embodiment, the solid support can be a bead, a plate, a matrix, a polymer, a test tube, a sheet, a culture dish, or a test strip. The plate can be a multi-well plate. The multi-well plate can 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.

[0010] In one embodiment, the solid substrate can be a polystyrene, polypropylene, cycloolefin, or glass solid substrate.

[0011] In one embodiment, the kit may comprise 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. 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 fluorogenic label, an enzymatic 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.

[0012] In one embodiment, a method for testing the binding specificity of the 11-1F4 antibody may include contacting the 11-1F4 antibody or an antigen-binding fragment thereof with an immunoassay system described herein; washing the solid support; adding a secondary antibody that specifically binds to the 11-1F4 antibody or an antigen-binding fragment thereof; washing the solid support; and performing a detection step.

[0013] In one embodiment, the washing may include at least three washes with a washing buffer, which may include PBS and 0.05% TWEEN®-20 (polysorbate 20).

[0014] In one embodiment, a solid support may be coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1. A solid support may be coated with a biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1, which comprises incubating the solid support with a solution comprising 0.1 μg / mL of biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO: 1 for about 1 hour at about 37° C. The solution may comprise PBS (phosphate buffered saline).

[0015] In one 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, which comprises incubating the solid support with a solution comprising 0.1 μg / 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).

[0016] In one embodiment, the method may further comprise a blocking step prior to step (a), comprising incubating the plate with a blocking buffer comprising PBS and 1% BSA at about 37° C. for about 60 minutes.

[0017] In one embodiment, the method may further comprise adding a control antibody to the solid support in the absence of the 11-1F4 antibody.

[0018] In one embodiment, the control antibody may be a human IgG1-k antibody.

[0019] In one embodiment, a secondary antibody that specifically binds to the 11-1F4 antibody or antigen-binding fragment thereof can be conjugated to a detectable label.

[0020] In one 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 fluorogenic label, an enzyme label, or a combination thereof. The label can be horseradish peroxidase (HRP).

[0021] In one embodiment, a method for testing the binding specificity of the 11-1F4 antibody includes 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, wherein the coating with the biotinylated LEN peptide comprises incubating the solid support with a solution comprising 0.1 μg / mL of the biotinylated LEN peptide comprising the amino acid sequence of SEQ ID NO:1 at about 37° C. for about 1 hour; coating at least a portion of the solid support with a biotinylated control comprising the amino acid sequence of SEQ ID NO:2, wherein the solid support is incubating with a solution comprising 0.1 μg / mL of the biotinylated control peptide comprising the amino acid sequence of SEQ ID NO:2 at about 37° C. for about 1 hour. 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; contacting the solid support with a solution comprising the 11-1F4 antibody or antigen-binding fragment thereof and incubating at about 37° C. for about 1 hour; optionally washing the solid support at least three times; 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; optionally washing the solid support at least three times;

[0022] In one embodiment, a solution containing the 11-1F4 antibody or antigen-binding fragment thereof may comprise an assay buffer containing PBS (phosphate buffered saline), 1% BSA (bovine serum albumin), and 0.05% TWEEN®-20 (polysorbate 20).

[0023] In one embodiment, the secondary antibody can be conjugated to 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 fluorogenic label, an enzyme label, or a combination thereof.The label can be horseradish peroxidase (HRP).

[0024] In one embodiment, the solution comprising the 11-1F4 antibody or antigen-binding fragment thereof may comprise a series of serial dilutions of the 11-1F4 antibody or antigen-binding fragment thereof solution.

[0025] In one embodiment, the 11-1F4 antibody can be a chimeric 11-1F4 antibody (c11-1F4 antibody) or an antigen-binding fragment thereof.

[0026] In one embodiment, the 11-1F4 antibody can be a humanized 11-1F4 antibody or an antigen-binding fragment thereof.

[0027] For a fuller understanding of the nature, objects, and advantages of the present disclosure, reference should be made to the following detailed description, which should be read in conjunction with the following drawings. [Brief description of the drawings]

[0028] [Figure 1] Combined data from three independent assays are shown. EC50=2.6±0.2 ng / mL (0.018±0.001 nM). Coating concentration of biotinylated and control peptide: 0.1 μg / ml.

[0029] [Diagram 2] FIG. 13 shows a schematic map of an 8×12 plate for an exemplary biotinylated-LEN peptide plate (1 μg / mL), showing antibody concentrations (rows A-H), antibodies used (c11-1F4 in columns 1-6, IgG1-K in columns 7-9), and controls (columns 10-12).

[0030] [Diagram 3] FIG. 13 shows a schematic map of an 8×12 plate showing antibody concentrations (rows A-H), antibodies used (c11-1F4 columns 1-6, IgG1-K columns 7-9), and controls (columns 10-12) for an exemplary biotinylated control peptide plate (1 μg / mL). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] It is to be understood that the disclosure is not limited to the specific embodiments disclosed below, since the specific embodiments may vary and still be within the scope of the appended claims. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments, and is not intended to be limiting. Instead, the scope of the disclosure is established by the appended claims.

[0032] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. 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 disclosure belongs.

[0033] definition "Antibodies" (Ab) and "immunoglobulins" (Ig) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. The term "antibody" specifically encompasses monoclonal antibodies, including antibody fragment clones.

[0034] "Native antibodies and immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons consisting 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, but the number of disulfide bonds varies among 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 several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the variable domain of the light chain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form the 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).

[0035] "Variable" as used herein broadly refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies, and are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called frameworks (FRs). Natural heavy and light chain variable domains each contain four FR regions, which adopt a predominantly β-sheet configuration and are connected by three CDRs, which form loops that connect (and in some cases form part of) the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions, and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. Johnson & 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 involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0036] 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, a designation reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0037] An "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight non-covalent association. In a single-chain Fv species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that in the two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0038] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy 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 ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0039] 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.

[0040] Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond 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. st Ed.)Strohl&Strohl Woodhead Publishing(2012)

[0041] An "antibody fragment" comprises 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. See "Human Monoclonal Antibodies: Methods and Protocols" (2003), "Antibody Fragments: Antibodies for Human Monoclonal Antibodies: Methods and Protocols" (2003), "Antibody Fragments: Antibody Fragments ... nd Ed.) Steinitz (Ed.) Humana Press (2019).

[0042] The term "monoclonal antibody" as used herein refers to an antibody (or antibody fragment) obtained from a population of substantially homogenous antibodies, e.g., the individual antibodies constituting the population are essentially identical and originate from a single clone. Monoclonal antibodies are highly specific and directed against a single antigenic site (epitope). Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against multiple epitopes, each monoclonal antibody binds to a single epitope on the antigen. Monoclonal antibodies are also more convenient to produce than polyclonal antibodies because they are produced in hybridoma cell cultures. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogenous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies described herein may be produced by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be produced by recombinant DNA methods. See, e.g., U.S. Pat. No. 4,816,567. "Monoclonal antibodies" also include antigen-recognition and binding site-containing antibody fragment clones (Fv clones) 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).

[0043] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies so 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 (2 nd Ed.) Kontermann & Durbel. Springer Press (2010).

[0044] A "human antibody" (also called a "fully human antibody") is an antibody that contains human framework regions and all of the CDRs from a human immunoglobulin. In one example, the framework and CDRs are derived from the same original human heavy and / or light chain amino acid sequence. However, a framework from one human antibody can be engineered to contain CDRs from a different human antibody.

[0045] Humanized forms of non-human (e.g., murine) antibodies are immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. A humanized antibody can be, for example, a human immunoglobulin (recipient antibody) in which residues from the CDRs of the recipient are replaced by residues from the CDRs of a non-human species (such as mouse, rat, or rabbit) or synthetic sequences (donor antibody) having the desired specificity, affinity, and capacity. Alternatively, an antibody identified or produced in a species other than human, e.g., a murine antibody, can be "humanized" by replacing one or more amino acids present in the murine antibody sequence with amino acids present in a human antibody sequence at the corresponding sequence positions. For example, since there are several sequence differences between murine and human antibody sequences, a "fully humanized" antibody can be obtained by substituting human positions at all sites of difference. In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues ("backmutations"). Furthermore, a humanized antibody may contain residues found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In one embodiment, all of the CDRs in a humanized immunoglobulin may be derived from the donor immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by genetic engineering. See, e.g., U.S. Patent No. 5,585,089.

[0046] "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. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0047] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which comprise 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, these domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0048] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antibody can be purified, for example, (1) to greater than 95% by weight (or optimally greater than 99% by weight) of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody can include within its scope an antibody in situ, for example, in a recombinant cell, since at least one component of the antibody's natural environment will not be present. An isolated antibody is prepared by at least one purification step.

[0049] "Epitope" as used herein broadly refers to an antigen or a portion of an antigen to which an antibody binds. As described herein, the 11-1F4 antibody binds to a portion of a human light chain amyloid fibril, e.g., in an animal, e.g., a mammal, e.g., a human. An epitope with immunogenic activity is a portion of a human light chain amyloid fibril that elicits an antibody response in an animal that includes both binding and effector functions. Immune specificity, i.e., the degree to which an antibody binds to a particular epitope, can be determined by methods known in the art, e.g., by immunoassays described herein. An antigenic epitope does not necessarily have to be immunogenic.

[0050] 11-1F4 antibody-peptide epitope binding assay The system and method described herein provides an 11-1F4 antibody-peptide epitope binding assay with a 10-fold increase in sensitivity compared to similar assays reported in the literature. Reliable and sensitive binding assays are required during the monoclonal antibody drug development process and for antibody characterization.

[0051] The highly sensitive assay described herein provides a reliable quality control assay for the 11-1F4 antibody, as well as any humanized and / or other modified forms of this antibody. The increased sensitivity compared to previously described assays ensures that differences in the affinity of antibodies to epitopes can be more easily observed. The increased sensitivity also has the advantage that fewer reagents are used in the assay compared to previously described binding ELISA (enzyme-linked immunoassay).

[0052] This peptide ELISA assay uses biotinylated peptides and streptavidin-coated plates, a setup that increased the sensitivity of the assay by 10-fold (compared to other peptide ELISA assays).

[0053] The use of biotinylated peptides and streptavidin-coated plates results in a uniform distribution and conformation of the peptide coating, thereby increasing assay sensitivity by 10-fold. The higher assay sensitivity of the assay means that there is less chance of non-specific signal interference, providing a better method of quality control. This innovation also provides economic benefits, as it translates into lower amounts of antibodies being required per individual test.

[0054] The solid support described herein may be coated with one or both of the LEN peptide, which comprises the amino acid sequence DIVMTQSPDSLAVSLGERATIN (SEQ ID NO: 1), and / or the control peptide (CT), which comprises the amino acid sequence AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA (SEQ ID NO: 2). The LEN peptide is a peptide derived from the LEN protein, e.g., a 22 amino acid N-terminal fragment (Solomon, A. & McLaughlin, C., J. Biol. Chem., 244:3303-404, 1969). The LEN peptide and the control peptide may be biotinylated, e.g., covalently linked to biotin. Methods for biotinylating peptides are known in the art.

[0055] Anti-AL antibody 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), represents an IgG1k monoclonal antibody that directly binds to a conformational epitope present on human light chain amyloid fibrils, regardless of kappa or lambda isotype. 11-1F4 has been shown to induce chemotaxis and activation of neutrophils, trigger Fcy receptor-mediated opsonization and proteolysis of deposited amyloid fibrils, and result in the reversal of AL amyloidoma in mouse models. Without being bound by theory, the 11-1F4 antibody may bind to a conformational epitope on human light chain amyloid fibrils. The 11-1F4 antibody may selectively bind to an epitope containing the amino acid sequence DIVMTQSPDSLAVSLGERATIN (SEQ ID NO: 1) (linear epitope). Chimeric, human, and humanized (either partial or complete) versions can be used in the context of this assay.

[0056] Immunoassay systems (e.g., enzyme-linked immunosorbent assay (ELISA)) and methods described herein may be used to determine the specificity of the 11-1F4 antibody or antigen-binding fragment thereof. The binding affinity of the antibody can be determined, for example, by Scatchard analysis (Frankel, M. & Gerard, W., Mol. Immunol., 16:101-6, 1979). Binding affinity may be measured, for example, by antigen / antibody dissociation rate. High binding affinity may be measured by competitive radioimmunoassay. Binding affinity may be measured by ELISA, such as the immunoassays described herein.

[0057] Certain assay methods require immobilization of the reagent. Immobilization involves separating the anti-human light chain amyloid fibril antibody from any human light chain amyloid fibril that remains free in solution. This is traditionally accomplished by insolubilizing the anti-human light chain amyloid fibril antibody prior to the assay procedure, such as by adsorption to a water-insoluble matrix or surface (U.S. Pat. No. 3,720,760), covalent coupling (e.g., using glutaraldehyde cross-linking), or by insolubilizing the anti-human light chain amyloid fibril antibody, for example, by immunoprecipitation.

[0058] secondary antibody A detectably labeled secondary antibody for use in 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.

[0059] The label used with the secondary antibody may be any label having detectable functionality that does not interfere with the binding of human light chain amyloid fibrils to the anti-human light chain amyloid fibril antibody. Numerous labels for use in immunoassays are known, including moieties that can be directly detected, such as fluorescent dyes, chemiluminescent, and radioactive labels, as well as moieties that must be reacted or derivatized in order 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, luceriferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide such as heterocyclic oxidases coupled with HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, or combinations thereof. For example, the secondary antibody may be HRP-goat anti-human Fcy.

[0060] Conventional methods are available for covalently binding these labels to proteins or polypeptides. For example, antibodies may be tagged with the above-mentioned fluorescent, chemiluminescent, and enzyme labels using coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidines, etc. (U.S. Patents 3,940,475 (fluorometry) 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 useful in the materials and methods described herein include enzymes such as horseradish peroxidase and alkaline phosphatase.

[0061] Conjugation of such labels, including enzymes, to antibodies is a standard procedure for those skilled in the art of immunoassay technology (O'Sullivan et al., "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay", in Methods in Enzymology, ed. J. J. Langone and H. Vun Vunakis, Vol. 73 (Academic Press, New York, NY, 1981), pp. 147-166).

[0062] solid support 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 sheet, a culture dish, or a test strip. A combination 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 constructed of any acceptable material, for example, polystyrene, polypropylene, cycloolefin, or glass.

[0063] The solid support may be coated with a biotinylated LEN peptide comprising the amino acid sequence DIVMTQSPDSLAVSLGERATIN (SEQ ID NO: 1) at a concentration of about 0.01 μg / mL to 1 μg / mL. For example, the concentration may 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.

[0064] The solid support may be coated with a biotinylated control peptide comprising the amino acid sequence AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA (SEQ ID NO: 2) at a concentration of about 0.01 μg / mL to 1 μg / mL. For example, the concentration may 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.

[0065] buffer solution Buffers may be used in the immunoassay systems and methods described herein to form solutions with the 11-1F4 antibody, wash solutions, block solutions, or combinations thereof. Exemplary buffers include, but are not limited to, a coating buffer comprising phosphate buffered saline (PBS); a wash buffer comprising PBS and 0.05% TWEEN®-20® (polysorbate 20); a blocking buffer comprising PBS and 1% BSA (bovine serum albumin); and an assay buffer comprising PBS, 1% BSA, and 0.05% TWEEN®-20® (polysorbate 20). Buffers are known in the art, and commercially available buffers may be used.

[0066] conditions Generally, the reagents are incubated at about 37° C. for about 60 minutes.

[0067] For example, the step may be about 30 minutes to 90 minutes. The incubation step may be about 60 minutes. For example, the incubation step may be 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 be for about 45-75 minutes, 55-65 minutes, 50-70 minutes, or 40-80 minutes.

[0068] The reacting step may be carried out at about 37° C. For example, the reacting step may be carried out at a temperature of about 35° C. to 45° C. The reacting step may be carried out 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 may be carried out at a temperature of about 37° C. to 40° C., 36° C. to 41° C., or 36° C. to 40° C.

[0069] kit The kit may include one or more containers filled with one or more of the components of the pharmaceutical composition including a buffer, a solid support, optionally a multi-well plate, the LEN peptide (biotinylated), and a control peptide (biotinylated). The multi-well plate may be coated with the LEN peptide (biotinylated), and the control peptide (biotinylated). Optionally, such container(s) may be affixed with a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting approval by the agency of the manufacture, use, or sale for administration to humans.

[0070] The kit may further include a control antibody that does not react with human light chain amyloid fibrils. In another specific embodiment, the kit contains a means for detecting binding of an antibody to human light chain amyloid fibrils (e.g., the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody may be conjugated to a detectable substrate). The kit may include recombinantly produced or chemically synthesized human light chain amyloid fibrils. The human light chain amyloid fibrils provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection means of the above kit includes a solid support to which human light chain amyloid fibrils are attached. Such a kit may also include a reporter-labeled anti-human antibody that is not attached. In this embodiment, the binding of the antibody to human light chain amyloid fibrils can be detected by the binding of the reporter-labeled antibody described above.

[0071] A diagnostic kit for use in screening biological samples containing antigens of the polypeptides described herein. The diagnostic kit may include a substantially isolated antibody specifically immunoreactive with human light chain amyloid fibrils and a means for detecting 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 means of the kit may include a secondary, labeled monoclonal antibody. Alternatively, or additionally, the detection means may include a labeled competing antigen.

[0072] In one diagnostic configuration, a biological sample is reacted with a solid-phase reagent having human light chain amyloid fibrils bound to its surface. After the human light chain amyloid fibrils bind to the specific antibody, unbound serum components are washed away, a reporter-labeled anti-human antibody is added, unbound anti-human antibody is washed away, and the reagent is reacted with the reporter-labeled anti-human antibody to bind the reporter to the reagent. This binding is proportional to the amount of anti-human light chain amyloid fibril antibody bound on 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.

[0073] The solid surface reagent in the above assay is prepared by known techniques for attaching protein materials to solid support materials, such as polymer beads, dipsticks, 96-well plates, or filter materials.These attachment methods generally include non-specific adsorption of protein to support, or covalent binding of protein to chemically reactive groups, such as activated carboxyl, hydroxyl, or aldehyde groups, on solid support, typically via free amine groups.Alternatively, streptavidin-coated plates can be used in conjunction with biotinylated antigen(s).

[0074] Thus, described herein are assay systems or kits for carrying out this diagnostic method, which generally include a support having surface-bound recombinant human light chain amyloid fibrils, and a reporter-labeled anti-human antibody for detecting the surface-bound anti-human light chain amyloid fibril antibodies. EXAMPLES

[0075] Example 1 ELISA LEN-c11-1F4 assay This example describes the use of the immunoassay system and methods described herein for titration of c11-1F4 antibody against a biotinylated peptide (LEN, control) on streptavidin-coated plates. Antibody concentrations ranged from 5 μg / mL to 5 × 10 -7The concentrations ranged from 1 μg / mL and were diluted 10-fold. An IgG1-kappa antibody was used as a negative control.

[0076] buffer solution The following buffers were used: Coating Buffer: PBS (Phosphate Buffered Saline); Washing Buffer: PBS + 0.05% TWEEN®-20® (Polysorbate 20); Blocking Buffer: PBS + 1% BSA (Bovine Serum Albumin); and Assay Buffer: PBS + 1% BSA + 0.05% TWEEN®-20®.

[0077] Plate Coating 50 μL of a 0.1 μg / mL solution of biotinylated LEN peptide (DIVMTQSPDSLAVSLGERATIN) (SEQ ID NO: 1) (New England Peptide) in PBS and 50 μL of a 0.1 μg / mL solution of biotinylated control peptide (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA) (SEQ ID NO: 2) in PBS were added to each well of a first 12×8 well microplate (StreptaWell Microplate, Transparent, Roche) to create Plate #1.

[0078] 50 μL of a 0.1 μg / mL solution of biotinylated control peptide (AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA) (SEQ ID NO: 2) in PBS was added to each well of a second 12×8 well microplate (StreptaWell Microplate, Transparent, Roche) to create Plate #2.

[0079] The plates were incubated at about 37° C. for about 1 hour.

[0080] blocking 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 37° C. for about 1 hour.

[0081] antibody binding A solution of c11-1F4 antibody at 5 μg / mL was prepared in assay buffer (c11-1F4 anti-light chain amyloid antibody (TPP-3841) 4.54 mg / mL, lot #2753245, GeneArt). Approximately 140 μL of the 5 μg / mL c11-1F4 antibody solution was dispensed into the first 6 wells of the first row of an 8×12 well preparatory plate (i.e., row A, columns 1-6 of the plate shown diagrammatically in FIG. 2).

[0082] A solution of 5 μg / mL human IgG1-K-ULNL was prepared in assay buffer (human IgG1-K-ULNL, Southern Biotech 0151K-01). Approximately 140 μL of the 5 μg / mL human IgG1-K-ULNL solution was dispensed into the next three wells of the first row of the preparation plate (i.e., row A, columns 7-9 of the plate shown diagrammatically in FIG. 2). The remaining wells of the last three columns (row A, columns 10-12 in FIG. 2) received 126 μL of assay buffer. A serial dilution into the remaining rows (rows B to H in FIG. 2) was performed, starting with taking 14 μL from row A and diluting it into 126 μL of buffer in the next row (row B), then repeating for the subsequent rows (i.e., 14 μL from row B into 126 μL of buffer in row C, etc.). 50 μL from each well of the preparation plate was transferred to a corresponding well in each of two assay plates, Plate #1 and Plate #2 (see, e.g., FIG. 2). The assay plates were incubated at about 37° C. for about 1 hour.

[0083] detection HRP-Goat anti-human Fcy was diluted 1:1000 in assay buffer. The assay plates (plates #1 and #2) were washed three times with a MicroTek plate washer. 50 μL of labeled secondary antibody was dispensed into each well of the assay plate. The assay plate was then incubated at about 37° C. for about 1 hour.

[0084] Color development and reading The assay plate was washed three times in a MicroTek plate washer. 50 μL of TMB substrate was dispensed into each well of the assay plate and the assay plate was incubated at room temperature (approximately 25° C.) for approximately 4 minutes. Color development was stopped by dispensing 50 μL of stop solution into each well of the assay plate. Absorbance was read at 450 nm.

[0085] When compared to other ELISA protocols, the methods described herein (eg, ELISA LEN peptide / c11-1F4 antibody) show unexpected improvements.

[0086] Using an ELISA protocol, O'Nuallain et al. (Biochemistry. 2007;46(45):13049-13058) tested the binding of mouse c11-1F4 antibody to plate-immobilized phage and LEN peptides, as well as the inhibitory effect of these molecules on antibody binding to immobilized Len(1-22), and reported EC 50 =0.24±0.1 nM.

[0087] Using an ELISA protocol, Wall et al. (Proc Natl Acad Sci USA. 2018;115(46):E10839-E10848) tested the binding of the chimeric c11-1F4 antibody and found that EC 50 We found that the binding transition midpoint was estimated to be approximately 0.4 nM, but could not be distinguished from the binding of 11-1F4 to the κ4 N-terminal peptide LEN(1–22), derived from the Bence Jones protein LEN, which served as the immunogen to generate m11-1F4.

[0088] Unexpectedly, the ELISA protocol described herein is more than 10-fold more sensitive (EC 50 =0.018±0.001; see Figure 1).

[0089] All references cited herein are incorporated by reference as if each was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date of the present application and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by reason of prior invention.

[0090] It will be understood that each of the above elements, or two or more together, may find useful application in other types of methods different from those described above. Without further analysis, the foregoing sufficiently reveals the gist of the present disclosure so that others, applying their current knowledge, can readily adapt the disclosure to various applications without omitting features that properly constitute essential characteristics of the general or specific aspects of the present disclosure as set forth in the appended claims. The foregoing embodiments are presented by way of example only, and the scope of the present disclosure is limited only by the appended claims.

Claims

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

2.

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

2.

3. The immunoassay system according to claim 1, wherein the solid substrate is coated with the biotinylated LEN peptide at a concentration of about 0.1 μg / mL to 1 μg / mL, and / or the solid substrate is coated with the biotinylated control peptide at a concentration of about 0.1 μg / mL to 1 μg / mL.

4. The immunoassay system according to claim 1, wherein the solid substrate is a bead, a plate, a matrix, a polymer, a test tube, a sheet, a culture dish, or a test strip.

5. The immunoassay system according to claim 4, wherein the plate is a multiwell plate, a two-well plate, a four-well plate, a six-well plate, a twelve-well plate, a twenty-four-well plate, a forty-eight-well plate, a ninety-six-well plate, or a three-eight-well plate.

6. A kit comprising an immunoassay system according to any one of claims 1 to 5, and a secondary antibody that specifically binds to an 11-1F4 antibody or its antigen-binding fragment.

7. The kit according to claim 6, wherein the secondary antibody is conjugated with a detectable label.

8. The kit according to claim 6, wherein the 11-1F4 antibody is a chimeric 11-1F4 antibody (c11-1F4 antibody), a humanized 11-1F4 antibody, or an antigen-binding fragment thereof.

9. A method for testing the binding specificity of the 11-1F4 antibody, (a) Contacting the 11-1F4 antibody or its antigen-binding fragment with the immunoassay system according to any one of claims 1 to 5; (b) Cleaning the solid support; (c) Adding a secondary antibody that specifically binds to the 11-1F4 antibody or its antigen-binding fragment; (d) Cleaning the solid support; and (e) Perform the detection process The method, including the method described above.

10. The method according to claim 9, wherein the washing comprises at least three washes with a washing buffer.

11. The method according to claim 9, further comprising: coating the solid support with a biotinylated LEN peptide containing the amino acid sequence of SEQ ID NO: 1; incubating the solid support with a solution containing 0.1 μg / mL of biotinylated LEN peptide containing the amino acid sequence of SEQ ID NO: 1 at approximately 37°C for approximately 1 hour; and / or incubating the solid support with a solution containing 0.1 μg / mL of biotinylated control peptide containing the amino acid sequence of SEQ ID NO: 2 at approximately 37°C for approximately 1 hour.

12. The method according to claim 9, further comprising a blocking step of incubating the plate with a blocking buffer containing PBS and 1% BSA at about 37°C for about 60 minutes prior to step (a).

13. The method according to claim 9, further comprising adding a control antibody to the solid support in the absence of the 11-1F4 antibody, wherein the control antibody is optionally a human IgG1-k antibody.

14. The method according to claim 9, wherein the secondary antibody that specifically binds to the 11-1F4 antibody or its antigen-binding fragment is conjugated with a detectable label.

15. A method for testing the binding specificity of the 11-1F4 antibody, (a) Prepare a solid support; (b) Coating at least a portion of the solid support with a biotinylated LEN peptide containing the amino acid sequence of SEQ ID NO: 1, comprising incubating the solid support with a solution containing 0.1 μg / mL of the biotinylated LEN peptide containing the amino acid sequence of SEQ ID NO: 1 at approximately 37°C for approximately 1 hour; (c) Coating at least a portion of the solid support with a biotinylation control comprising the amino acid sequence of SEQ ID NO: 2, wherein the solid support is incubated with a solution comprising 0.1 μg / mL of a biotinylation control peptide comprising the amino acid sequence of SEQ ID NO: 2 at approximately 37°C for approximately 1 hour; (d) Wash the solid support at least three times, if optional; (e) Contact the solid support with a blocking buffer containing PBS (phosphate-buffered saline) and 1% BSA (bovine serum albumin), and incubate at approximately 37°C for approximately 1 hour; (f) Contact the solid support with a solution containing the 11-1F4 antibody or its antigen-binding fragment and incubate at approximately 37°C for approximately 1 hour; (g) Wash the solid support at least three times, if optional; (h) Add a secondary antibody that specifically binds to the 11-1F4 antibody or its antigen-binding fragment, and incubate at approximately 37°C for approximately 1 hour; (i) Washing the solid support at least three times, as optional; and (j) Perform the detection process The method, including the method described above.

16. The method according to claim 15, wherein the solution containing the 11-1F4 antibody or its antigen-binding fragment comprises an assay buffer containing PBS (phosphate-buffered saline), 1% BSA (bovine serum albumin), and 0.05% TWEEN®-20 (polysorbate 20).

17. The method according to claim 15, wherein the secondary antibody is conjugated with a detectable label.

18. The method according to claim 17, wherein the detectable label is a fluorescent label, an luminescent label, a bioluminescent label, a radioactive label, a chemiluminescent label, a colorimetric label, a fluorescence-generating label, an enzyme label, or a combination thereof.

19. The method according to claim 15, wherein the solution containing the 11-1F4 antibody or its antigen-binding fragment comprises a series of sequential dilutions of the solution of the 11-1F4 antibody or its antigen-binding fragment.

20. The method according to claim 15, wherein the 11-1F4 antibody is a chimeric 11-1F4 antibody (c11-1F4 antibody), a humanized 11-1F4 antibody, or an antigen-binding fragment thereof.

Images