Methods for detecting and quantifying membrane-associated proteins on extracellular vesicles

Abstract

To provide assays for the detection and / or quantification of membrane-associated proteins, e.g., circulating CD20 (cCD20), incorporating an extracellular vesicle-based calibrator comprising the membrane-associated tumor antigen as well as the use of such assays in the detection and treatment of hyperproliferative disorders.SOLUTION: An assay for detecting a membrane-associated protein in a sample, comprises: a) a capture antibody that binds to an extracellular vesicle comprising the membrane-associated protein in the sample, thereby generating a capture antibody-extracellular vesicle complex; and b) a detection antibody that binds to the capture antibody-extracellular vesicle complex to form a detectable bound complex, where a signal from the detectable bound complex is calibrated against one or more known values detected from the extracellular vesicle comprising the protein.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Patent Application No. 62 / 815,863, filed on March 8, 2019, the content of which is incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure provides assays for the detection and / or quantification of membrane - bound proteins, such as circulating CD20 (cCD20), incorporating extracellular vesicle - based calibrators containing membrane - bound proteins, and the use of such assays in the detection and treatment of proliferative disorders.

Background Art

[0003] B - lymphocyte antigen CD20 (also known as human B - lymphocyte restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein having a molecular weight of approximately 35 kD. CD20 can be detected on the surface of pre - and mature B - lymphocytes. CD20 regulates early stages in the activation of B - cell cycle initiation, cell differentiation, and cell proliferation. CD20 is also known to function as a calcium ion channel.

[0004] CD20 is a membrane protein having four transmembrane domains that form a large extracellular loop on the cell surface and both N- and C-termini located within the cytoplasm. Since both its C- and N-termini are located in the cytoplasm, it has been thought that CD20 is less likely to be shed or cleaved from the cell surface upon antibody binding. CD20 can form dimers or oligomers when translocating to lipid rafts. Considering the expression of CD20 in B cell lymphomas, this antigen may serve as a candidate for "targeting" such lymphomas, similar to other membrane-bound tumor antigens. For example, antibodies specific for the CD20 surface antigen of B cells that bind to the extracellular loop have been administered to patients to promote the destruction and depletion of neoplastic B cells. In addition, a chemical substance or radiolabel having the potential to destroy tumors can be conjugated to an anti-CD20 antibody so that the agent is specifically "delivered" to neoplastic B cells. In view of the above, CD20 antibodies are playing an increasingly important role in the treatment of patients with lymphoproliferative diseases, including patients with chronic lymphocytic leukemia, non-Hodgkin lymphoma, or Hodgkin disease.

[0005] Membrane-bound proteins can circulate, along with other proteins, in cell membrane fragments or large membrane complexes. For example, circulating CD20 protein may exist in association with membrane-bound particles in circulation as a full-length protein. Thus, when present in circulation, drug targets of these membrane-bound proteins, such as CD20, can bind to therapeutic antibodies and sequester them, and thus act as a drug sink for anti-CD20 antibodies intended to target tumors. Such sequestration can reduce the therapeutic efficiency of the therapeutic antibody. Membrane-bound proteins, such as circulating CD20, can also act as biomarkers for lymphoproliferative diseases such as chronic lymphocytic leukemia, non-Hodgkin lymphoma, or Hodgkin disease, and as markers associated with the likelihood of response to treatment according to the presence of specific tumor antigens. Considering the important role of membrane-bound proteins, such as circulating CD20, in the detection and treatment of hyperproliferative disorders, there is still a need in the art for assays to determine the amount of membrane-associated tumor antigens, such as circulating CD20, present in an individual.

SUMMARY OF THE INVENTION

[0006] The subject matter of the present disclosure relates to assays and methods for the detection and / or quantification of membrane proteins, such as circulating CD20 (cCD20), incorporating an extracellular vesicle-based calibrator comprising a membrane-bound protein, and the use of such assays in the detection and treatment of proliferative disorders.

[0007] In some embodiments, the present disclosure provides an assay for detecting a membrane-bound protein in a sample, the assay comprising: a) a capture antibody that binds to extracellular vesicles comprising a membrane-bound protein in the sample to generate a capture antibody-extracellular vesicle complex; and b) a detection antibody that binds to the capture antibody-extracellular vesicle complex to form a detectable binding complex, wherein the signal from the detectable binding complex is calibrated against one or more known values detected from extracellular vesicles comprising the protein.

[0008] In some embodiments, the assay of the present disclosure further comprises an extracellular vesicle calibrator.

[0009] In some embodiments, the assay of the present disclosure comprises a capture antibody that does not compete for binding with the detection antibody. In some embodiments, the capture antibody binds to an epitope different from the detection antibody. In some embodiments, the capture antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, and combinations thereof. In some embodiments, the detection antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, and combinations thereof.

[0010] In some embodiments, the present disclosure is directed to assays in which membrane-bound proteins in a sample are utilized. In some embodiments, the sample is selected from the group consisting of plasma samples, serum samples, tissue culture supernatant samples, and combinations thereof. In some embodiments, the membrane-bound protein is selected from the group consisting of human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof.

[0011] In some embodiments, the present disclosure is directed to a method for quantifying the concentration of a circulating protein in a sample, the method comprising: a) determining the level of a target protein in extracellular vesicles in the sample; and b) comparing the level of the protein in the extracellular vesicles in the sample to a calibration curve generated using extracellular vesicles containing the target protein.

[0012] In some embodiments, the present disclosure is directed to a method for quantifying the concentration of a circulating protein in a sample, the method comprising: a) generating a calibration curve using extracellular vesicles containing the protein; and b) comparing the level of the target protein in the extracellular vesicles in the sample to the calibration curve to determine the amount of the protein in the extracellular vesicles in the sample.

[0013] In some embodiments, the present disclosure is directed to a method for determining whether a patient having B cell lymphoma is likely to respond to anti-CD20 therapy, the method comprising: a) obtaining a sample from the patient; b) determining the amount of circulating CD20 in extracellular vesicles in the sample; c) comparing the level of CD20 in the extracellular vesicles in the sample to a calibration curve generated using extracellular vesicles containing CD20; and d) determining whether the patient is likely to respond to CD20 therapy based on the amount of circulating CD20 in the extracellular vesicles determined in the sample. In some embodiments, the anti-CD20 therapy comprises administration of an anti-CD20 antibody.

[0014] In some embodiments, the present disclosure is directed to a method for determining the affinity of a non-target protein antibody, such as an anti-CD20 antibody, the method comprising subjecting the antibody to surface plasmon resonance (SPR) analysis, the SPR analysis comprising the use of a target protein as a ligand, such as extracellular vesicles expressing CD20, and the antibody as an analyte, such as an anti-CD20 antibody. In some embodiments, the SPR analysis is used as described herein to distinguish between two or more anti-target antibodies. In some embodiments, the SPR analysis enables ranking of anti-target antibodies. In some embodiments, the selection of a particular anti-target antibody is performed by ranking two or more anti-target antibodies via SPR analysis and selecting the anti-target antibody ranked highest, or by selecting an anti-target antibody exhibiting a desired affinity.

[0015] In some embodiments, the present disclosure is directed to a method for determining the activation of T cells obtained from a patient, the method comprising: a) incubating extracellular vesicles expressing CD20 with T cells and a CD20 T cell-dependent bispecific antibody; and b) determining the activation of the T cells.

[0016] In some embodiments, the present disclosure is directed to a method of treating a tumor in a subject in need of treatment, the method comprising: a) obtaining a sample from the subject; b) generating a calibration curve using extracellular vesicles containing a tumor antigen; c) comparing the level of the tumor antigen in the extracellular vesicles in the sample to the calibration curve to determine the amount of the target tumor antigen in the extracellular vesicles in the sample; d) determining, based on the level of the tumor antigen in the extracellular vesicles in the sample, whether the subject is likely to exhibit a response to antibody therapy; and e) applying a treatment in response to the determination in d).

[0017] In some embodiments, the method of the present disclosure further comprises detecting the presence of extracellular vesicles using an extracellular marker, the extracellular marker being selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

[0018] In some embodiments, the present disclosure is directed to a method in which the concentration of a membrane-bound protein and the calibration curve are determined using an immunoassay, ELISA, and / or Western blot. In some embodiments, the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof. In some embodiments, the tumor antigen is selected from the group consisting of a human CD20 antigen, a mouse CD20 antigen, a rat CD20 antigen, a rabbit CD20 antigen, a cynomolgus monkey CD20 antigen, a human CD3 antigen, a mouse CD3, a rat CD3 antigen, a rabbit CD3 antigen, and a cynomolgus monkey CD3 antigen, a human FcRH5 antigen, a human Ly6G6 antigen, a human HER2 antigen, a human EGFR antigen, a human HER3 antigen, a human HER4 antigen, a human PSMA antigen, and combinations thereof.

[0019] In some embodiments, the present disclosure is directed to a method that utilizes an anti-CD20 antibody, the anti-CD20 antibody being selected from the group consisting of rituximab, ofatumumab, obinutuzumab, CD20 T cell-dependent bispecific antibodies, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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Mode for Carrying Out the Invention

[0021] The present disclosure provides assays for the detection and / or quantification of membrane-bound proteins, such as circulating CD20, incorporating extracellular vesicle-based calibrators comprising membrane-bound proteins, and the use of such assays in the detection and treatment of proliferative disorders. In some embodiments, the assays of the present disclosure comprise determining the level of CD20 present in extracellular vesicles in a sample and comparing the level of CD20 in the sample to a calibration curve generated using extracellular vesicles comprising CD20 to quantify the concentration of a membrane-bound protein, such as an extracellular vesicle-bound protein, such as circulating CD20. In some embodiments, an immunoassay using one or more antibodies, such as ELISA or Western blot, is used to determine the concentration of the membrane-bound protein in the sample or in connection with the preparation of the calibration curve.

[0022] Without limitation and for clarity, the detailed description of the subject matter disclosed herein is divided into the following subsections: I. Definitions; II. Immunoassays; III. Antibodies; IV. Kits; and V. Exemplary embodiments.

[0023] I. Definitions Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used in this specification, the following terms have the meanings set forth below unless otherwise specified.

[0024] As used in this specification, the term "about" or "approximately" can mean an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations of a given value per implementation. Where a particular value is recited in the present application and claims, unless otherwise stated, the term "about" can mean an acceptable error range for the particular value, e.g., ±10% of the value modified by the term "about".

[0025] For the purposes of this specification, the term "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework that is derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0026] The term "affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0027] The term "affinity matured" antibody refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs) and has an improved affinity for an antigen of the antibody as compared to the parent antibody that does not have such modifications.

[0028] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0029] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0030] An "antibody that binds" to a target antigen, e.g., a CD20 protein, is one that binds the antigen with sufficient affinity such that the antibody is useful as an assay reagent, e.g., a capture or detection antibody. Typically, such an antibody does not significantly cross-react with other polypeptides. With respect to binding of a polypeptide to a target molecule, the terms "specific binding" to, or "specifically binds" to, or "specific for" a particular polypeptide or epitope on a particular polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a target molecule compared to the binding of a control molecule, which is typically a molecule having a similar structure that does not have binding activity.

[0031] The term "anti-tumor antigen antibody" refers to an antibody that has sufficient affinity to bind to a tumor antigen, such as CD20, such that the antibody is useful as an agent in targeting tumor antigens, for example, as an agent in the assays described herein. In some embodiments, the extent to which the anti-tumor antigen antibody binds to unrelated proteins is, for example, less than about 10% of the binding of the antibody to the targeted tumor antigen as measured by a radioimmunoassay (RIA). In some embodiments, the antibody that binds to the targeted tumor antigen has a dissociation constant (K d ) of ≦1M, ≦100 mM, ≦10 mM, ≦1 mM, ≦100 μM, ≦10 μM, ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM or ≦0.001 nM. In some embodiments, the K D of the antibody that binds to CD20, as disclosed herein, is 10 -3 M or less, or 10 -8 M or less, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 M. In some embodiments, the K D of the antibody that binds to the targeted tumor antigen, as disclosed herein, can be from 10 -10 M to 10 -13 M. In some embodiments, the anti-tumor antigen antibody binds to an epitope of the targeted tumor antigen that is conserved among different species of the targeted tumor antigen.

[0032] A "reference antibody" and an "antibody that competes for binding" refer to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are described in "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0033] A "B cell" is a lymphocyte that matures within the bone marrow and includes naive B cells, memory B cells, or effector B cells (plasma cells). The B cells herein may be normal or non-malignant B cells.

[0034] A "binding domain" means a part of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab’2, scFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and VH and / or VL domains of antibodies), receptors, ligands, aptamers, and other molecules having identified binding partners.

[0035] As used herein, a "capture antibody" refers to an antibody that specifically binds to a target molecule, e.g., the form of CD20, in a sample. Under certain conditions, the capture antibody forms a complex with the target molecule, whereby the antibody-target molecule complex can be separated from the rest of the sample. In some embodiments, such separation may include washing away substances or materials in the sample that did not bind to the capture antibody. In some embodiments, the capture antibody can bind to the surface of a solid support such as, but not limited to, a plate or beads, e.g., paramagnetic beads.

[0036] As used herein, the term "CD20" refers to the CD20 antigen, an approximately 35 kDa glycoprotein found on the surface of greater than 90% of B cells derived from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until the differentiation of plasma cells. CD20 is present on both normal and malignant B cells. Aliases for CD20 in the literature include "B lymphocyte restricted antigen" or "Bp35". The CD20 antigen is described, for example, in Clark et al. PNAS (USA) 82:1766 (1985).

[0037] As used herein, the term "CD20 nucleic acid" refers to nucleic acids including DNA and mRNA that encode at least a portion of the CD20 protein and / or complementary nucleic acids.

[0038] "Detecting a tumor antigen" means evaluating whether a sample contains a tumor antigen. Usually, a tumor antigen protein, such as the CD20 protein, is detected, but detecting a tumor antigen nucleic acid, such as a CD20 nucleic acid, is also encompassed by this expression herein.

[0039] As used herein, the term "tumor antigen nucleic acid" refers to nucleic acids including DNA and mRNA that encode at least a portion of the tumor antigen protein and / or complementary nucleic acids.

[0040] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0041] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five main classes of antibodies, namely, IgA, IgD, IgE, IgG, and IgM, and some 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, or μ, respectively.

[0042] Throughout this specification and the claims, it will be understood that the word "comprise", or variations such as "comprises" or "comprising", means the inclusion of the stated integer or group of integers, but does not mean the exclusion of any other integer or group of integers.

[0043] The term "correlates" or "correlating" refers to the comparison, in any manner, of the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of a first analysis or protocol may be used when performing a second protocol, and / or the results of a first analysis or protocol may be used to determine whether a second analysis or protocol should be performed. With respect to embodiments of gene expression analysis or protocols, the results of a gene expression analysis or protocol can be used to determine whether a particular treatment regimen should be performed.

[0044] The term "detecting" as used herein is used to include both qualitative and quantitative measurements of a target molecule, such as CD20 or a processed form thereof. In some embodiments, detecting includes simply identifying the presence of a target molecule in a sample and determining whether the target molecule is present in the sample at a detectable level.

[0045] As used herein, "detection antibody" refers to an antibody that specifically binds to a target molecule in a sample or sample-capture antibody combination material. Under certain conditions, the detection antibody forms a complex with the target molecule or target molecule-capture antibody complex. The detection antibody can be detected directly using a label that can be amplified, or indirectly, for example, using another antibody that is labeled and binds to the detection antibody. In the case of a direct label, the detection antibody is typically conjugated to a detectable moiety by several means including, but not limited to, for example, biotin or ruthenium.

[0046] As used herein, the term "detection means" refers to the part or technique used to detect the presence of an antibody detectable by a signal report read later in an assay. Typically, the detection means uses a detection reagent that amplifies an immobilized label, such as a label captured on a microtiter plate, for example, avidin, streptavidin-HRP, or streptavidin-β-D-galactopyranose.

[0047] The "effective amount" of an agent refers to the amount necessary to cause a physiological change in the cell or tissue to which the agent is administered.

[0048] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies by antibody isotype. Examples of antibody effector functions include the following. C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0049] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0050] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain generally consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL). FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0051] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain containing the Fc region as defined herein.

[0052] "Heteromultimer", "heteromultimer complex", or "heteromultimeric protein" refers to a molecule comprising at least a first hinge-containing polypeptide and a second hinge-containing polypeptide, wherein the second hinge-containing polypeptide has an amino acid sequence different from that of the first hinge-containing polypeptide by at least one amino acid residue. The heteromultimer can comprise a "heterodimer" formed by the first and second hinge-containing polypeptides, or can form a higher-order tertiary structure in which polypeptides are present in addition to the first and second hinge-containing polypeptides. The polypeptides of the heteromultimer can interact with each other by non-peptide, covalent bonds (e.g., disulfide bonds) and / or non-covalent interactions (e.g., hydrogen bonds, ionic bonds, van der Waals forces, and / or hydrophobic interactions).

[0053] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably herein and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", and these cells include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not have the exact same nucleic acid content as the parental cell and may contain mutations. Progeny of mutants that have the same function or biological activity as those originally screened or selected in the transformed cell are included herein.

[0054] "Human antibody" refers to an antibody produced by a human or human cell, or having an amino acid sequence corresponding to an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody-encoding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0055] The "human consensus framework" is a framework that represents the amino acid residues that most commonly occur in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup such as those in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Vols. 1-3. In some embodiments, for VL, the subgroup is subgroup kappa I as in Kabat et al. (supra). In some embodiments, for VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0056] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., HVRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to the antibody that has been humanized.

[0057] As used herein, the terms "hypervariable region" or "HVR" refer to each of the regions of an antibody variable domain that are hypervariable (Complementarity Determining Regions or "HVRs"), and / or form structurally defined loops (hypervariable loops), and / or contain antigen contact residues (antigen contacts). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (supra). Generally, an antibody contains six HVRs, i.e., three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987)); (b) HVRs occurring at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262:732 - 745 (1996)); and, (d) combinations of (a), (b), and / or (c) that include the HVR amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1), 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3) includes. As used interchangeably herein, "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0058] "Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecule(s) including, but not limited to, a cytotoxic agent.

[0059] "Isolated" nucleic acid refers to a nucleic acid molecule separated from the components of its natural environment. An isolated nucleic acid includes nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0060] As used interchangeably herein, "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0061] "Isolated nucleic acid encoding an antibody" (including reference to a specific antibody, e.g., an anti-CD20 antibody) refers to one or more nucleic acid molecules encoding an antibody heavy chain and light chain (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors and such nucleic acid molecule(s) present at one or more locations within a host cell.

[0062] As used herein, the term "label" or "detectable label" refers to any chemical group or moiety that can bind to a substance to be detected or quantified, such as an antibody. The label is a detectable label suitable for the sensitive detection or quantification of a substance. Non-limiting examples of detectable labels include luminescent labels, such as fluorescent, phosphorescent, chemiluminescent, bioluminescent, and electrochemiluminescent labels, radioactive labels, enzymes, particles, magnetic substances, and electroactive species, among others, but are not limited thereto. Alternatively, a detectable label can signal its presence by participating in a specific binding reaction. Non-limiting examples of such labels include haptens, antibodies, biotin, streptavidin, His-tag, nitrilotriacetic acid, glutathione S-transferase, and glutathione, among others.

[0063] As used herein, the term "membrane-bound protein" refers to any membrane-bound target including an antigen, peptide, and protein. The membrane-bound protein can include integral membrane proteins and / or surface membrane proteins. Non-limiting examples of membrane-bound proteins include human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, and cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof, among others, but are not limited thereto.

[0064] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a collection of substantially homogeneous antibodies, i.e., the individual antibodies comprising the collection are identical and / or bind to the same epitope, except for possible variant antibodies, such as those containing naturally occurring mutations or mutations occurring during the production of a monoclonal antibody preparation. Such variants are usually present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain various antibodies to various determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the subject matter disclosed herein can be made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0065] As used herein, the term "accompanying document" refers to the instructions customarily included in a commercial package that contain information regarding the use of the components of the package.

[0066] "Pathogenic" cells are those that cause disease or an abnormality and can be present within or surrounding a diseased tissue or cell.

[0067] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity and, if necessary, introducing gaps, without considering conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for alignment of sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this specification, the % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the work of Genentech, Inc., and the source code has been filed with the U.S. Copyright Office (Washington D.C., 20559) together with user documentation and is registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or may be compiled from the source code. The ALIGN-2 program should be compiled when used on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by and do not vary with the ALIGN-2 program.

[0068] Under circumstances where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or can be expressed as a given amino acid sequence A having, or containing, a specific % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y where X is the number of amino acid residues scored as exact matches in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0069] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that are naturally or artificially modified, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), and other modifications known in the art are included in this definition. The terms "polypeptide" and "protein" as used herein specifically include antibodies.

[0070] As used herein, "sample" refers to a small part of a larger quantity of material. In some embodiments, the sample includes, but is not limited to, cells in culture, cell supernatant, cell lysate, serum, plasma, biological fluids (e.g., blood, plasma, serum, feces, urine, lymph, ascites, lavage fluid, saliva, and cerebrospinal fluid), and tissue samples. The source of the sample can be a solid tissue (e.g., fresh, frozen, and / or preserved organs, tissue samples, biopsy materials, or aspirates), blood or any blood component, a body fluid (e.g., urine, lymph, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid), or cells derived from an individual including circulating cells.

[0071] As used herein, "treatment" refers to a clinical intervention aimed at modifying the natural course of a treated individual or cell and can be performed before or during a clinical pathologic process. Desirable effects of treatment include prevention of the development or recurrence of a disease or its condition or symptoms, reduction of the state or symptoms of a disease, decrease in the direct or indirect pathologic consequences of a disease, decrease in the rate of disease progression, improvement or alleviation of a disease state, and remission or improvement of prognosis. In some embodiments, the methods and compositions of the present disclosure are useful in attempts to delay the onset of a disease or disorder.

[0072] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (VH and VL, respectively) generally have similar structures, and each domain includes four conserved framework regions (FRs) and three hypervariable regions (HVRs). (e.g., Kindt et al. Kuby Immunology, 6 th(See, e.g., ed., W.H.Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Further, an antibody that binds to a particular antigen may be isolated using the VH or VL domain of the antibody that binds to the antigen and screening a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0073] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors can induce the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0074] II. Immunoassays The present disclosure provides assays for the detection and / or quantification of membrane-bound proteins, such as circulating CD20, incorporating extracellular vesicle-based calibrators comprising membrane-bound proteins, and the use of such assays in the detection and treatment of proliferative disorders. In some embodiments, the assays of the present disclosure include determining the level of CD20 present in extracellular vesicles in a sample and comparing the level of CD20 in the sample to a calibration curve generated using extracellular vesicles comprising CD20 to quantify the concentration of a membrane-bound protein, such as an extracellular vesicle-bound protein, such as circulating CD20. In some embodiments, immunoassays using one or more antibodies, such as ELISA or Western blot, are used to determine the concentration of a membrane-bound tumor antigen in a sample or in connection with the preparation of a calibration curve.

[0075] In some embodiments, the present disclosure provides an immunoassay method for the detection and quantification of membrane-bound proteins. For example, the immunoassay methods of the present disclosure can incorporate strategies known in the art, including but not limited to sandwich assays, enzyme-linked immunosorbent assay (ELISA) assays, digital forms of ELISA, electrochemical assay (ECL) assays, and magnetic immunoassay methods.

[0076] In some embodiments, the present disclosure provides an extracellular vesicle (EV)-based calibrator. The EV calibrator can be a membrane-bound protein calibrator. For example, the EV calibrator can be similar to native CD20 in validation. Since ocrelizumab binds to the epitope of CD20 that has a tertiary structure (in the form of a loop) due to four transmembrane spans, it is important to generate a membrane-bound protein calibrator.

[0077] In some embodiments, the present disclosure provides a method for generating an EV calibrator. Exemplary methods include subculturing the seed line every 3 to 4 days, diluting the seed line in the production medium into the production culture, transfecting the production culture (e.g., DNA / jetPEI complex), collecting the EV calibrator from the transfected production culture, and purifying the EV calibrator. The purified EV calibrator can be characterized by western blot.

[0078] In some embodiments, the present disclosure provides a method for a continuous assay. The continuous assay can be a three-day continuous assay. The three-day continuous assay can be used when an improvement in sensitivity is desired. For example, on the first day, the plate can be coated with a capture antibody at 4°C. On the second day, the sample can be added to the plate and incubated overnight at 4°C. On the third day, a detection antibody (e.g., conjugated to biotin) and a reagent (e.g., HRP). In a non-limiting embodiment, Ocre (capture antibody), Ofa (detection antibody), and HRP 100 ng / mL (signal) can be used for the three-day continuous assay.

[0079] In some embodiments, the present disclosure provides a method for a cross-linking assay. The cross-linking assay can be a two-day cross-linking assay. The two-day cross-linking assay can be used when a shortening of the time to result is desired. For example, on the first day, the sample can be incubated with a master mix containing a biotin-conjugated ocrelizumab antibody and a dig-conjugated ofatumumab antibody. On the second day, the sample can be transferred to a streptavidin plate, and then an HRP-conjugated anti-DIG antibody can be added and incubated for detection. In a non-limiting embodiment, the plate can be read at 450 nm as the detection absorbance and 630 nm as the reference absorbance. The sample concentration can be determined by inputting the data into a 5-parameter logistic curve fitting using a curve weighting of 1 / y 2 and can be determined by inputting the data into a 5-parameter logistic curve fitting using a curve weighting of 1 / y.

[0080] In some embodiments, the method of the present disclosure includes contacting a sample obtained from a subject with a capture antibody such as those described herein under conditions that permit binding of the capture anti-FGF21 antibody in the sample to the CD20 protein. For example, without limitation, the sample can be incubated with a capture antibody that binds to an epitope present on CD20 to generate a sample-capture antibody combination material. The conditions for incubation of the sample and the capture antibody can be selected to maximize the sensitivity of the assay and / or minimize dissociation and to ensure that the CD20 protein present in the sample binds to the capture antibody.

[0081] In some embodiments, the capture antibody used in the immunoassay method disclosed herein can be used at a concentration of about 0.1 μg / ml to about 5.0 μg / ml. For example, without limitation, the capture antibody can be used at a concentration of about 0.1 μg / ml to about 0.5 μg / ml, about 0.1 μg / ml to about 1.0 μg / ml, about 0.1 μg / ml to about 1.5 μg / ml, about 0.1 μg / ml to about 2.0 μg / ml, about 0.1 μg / ml to about 2.5 μg / ml, about 0.1 μg / ml to about 3.0 μg / ml, about 0.1 μg / ml to about 3.5 μg / ml, about 0.1 μg / ml to about 4.0 μg / ml, about 0.1 μg / ml to about 4.5 μg / ml, about 0.5 μg / ml to about 5.0 μg / ml, about 1.0 μg / ml to about 5.0 μg / ml, about 1.5 μg / ml to about 5.0 μg / ml, about 2.0 μg / ml to about 5.0 μg / ml, about 2.5 μg / ml to about 5.0 μg / ml, about 3.0 μg / ml to about 5.0 μg / ml, about 3.5 μg / ml to about 5.0 μg / ml, about 4.0 μg / ml to about 5.0 μg / ml, about 4.5 μg / ml to about 5.0 μg / ml, about 0.5 μg / ml to about 2.0 μg / ml, or about 0.5 μg / ml to about 1.0 μg / ml, such as about 0.5 μg / ml.

[0082] In some embodiments, the capture antibody can be diluted in a coating buffer. Non-limiting examples of coating buffers include PBS, carbonate buffer, bicarbonate buffer, or combinations thereof. In some embodiments, the coating buffer is sodium bicarbonate. In some embodiments, the coating buffer is PBS. In some embodiments, the coating buffer can be used at a concentration of about 10 mM to about 1 M. For example, without limitation, the coating buffer can be about 10 mM to about 100 mM, about 10 mM to about 200 mM, about 10 mM to about 300 mM, about 10 mM to about 400 mM, about 10 mM to about 500 mM, about 10 mM to about 600 mM, about 10 mM to about 700 mM, about 10 mM to about 800 mM, about 10 mM to about 900 mM, about 100 mM to about 1 M, about 200 mM to about 1 M, about 300 mM to about 1 M, about 400 mM to about 1 M, about 500 mM to about 1 M, about 600 mM to about 1 M, about 700 mM to about 1 M, about 800 mM to about 1 M, or about 900 mM to about 1 M.

[0083] The capture antibody, as used herein, can be immobilized on a solid phase. For example, without limitation, the solid phase can be any inert support or carrier useful in immunoassay, including supports in the form of, for example, surfaces, particles, porous matrices, and beads. Non-limiting examples of commonly used supports include small sheets, SEPHADEX®, gels, polyvinyl chloride, plastic beads, and assay plates or test tubes made from polyethylene, polypropylene, and polystyrene, such as 96-well microtiter plates, and particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. In some embodiments, the solid phase used for immobilization can be beads. For example, without limitation, the capture antibodies disclosed herein are immobilized on paramagnetic beads. In some embodiments, the immobilized capture antibody is coated on a microtiter plate that can be used to analyze multiple samples at once.

[0084] In some embodiments, the paramagnetic beads that bind to the capture antibody are from about 0.1×10 7 beads / ml to about 10.0×10 7 beads / ml, for example, from about 0.1×10 7 beads / ml to about 0.5×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 1.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 2.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 3.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 4.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 5.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 6.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 7.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 8.0×10 7 beads / ml, from about 0.1×10 7 beads / ml to about 9.0×10 7 beads / ml, from about 0.5×10 7 beads / ml to about 10.0×10 7 beads / ml, from about 1.0×10 7 beads / ml to about 10.0×10 7 beads / ml, from about 2.0×10 7 beads / ml to about 10.0×10 7 beads / ml, from about 3.0×10 7 beads / ml to about 10.0×10 7 beads / ml, from about 4.0×10 7 beads / ml to about 10.0×10 7 beads / ml, from about 5.0×10 7 beads / ml to about 10.0×10 7 beads / ml, from about 6.0×10 7 beads / ml to about 10.0×107 beads / ml, about 7.0×10 7 beads / ml to about 10.0×10 7 beads / ml, about 8.0×10 7 beads / ml to about 10.0×10 7 beads / ml, about 9.0×10 7 beads / ml to about 10.0×10 7 beads / ml, about 0.5×10 7 beads / ml to about 1.0×10 7 beads / ml, about 0.5×10 7 beads / ml to about 2.0×10 7 beads / ml or about 0.5×10 7 beads / ml to about 3.0×10 7 beads / ml can be used. In some embodiments, the paramagnetic beads are about 0.5×10 7 beads / ml to about 2.0×10 7 beads / ml can be used. In some embodiments, the paramagnetic beads are about 1.0×10 7 beads / ml, for example about 1.22×10 7 beads / ml concentration, or about 0.5×10 7 beads / ml, for example about 0.59×10 7 beads / ml concentration can be used.

[0085] The immunoassay method disclosed herein can further include contacting the sample-capture antibody combination material with a detection antibody. In some embodiments, the detection antibody binds to an epitope present on CD20. In some embodiments, the detection antibody binds to an epitope present on the sample-capture antibody combination material but does not bind to the capture antibody in the absence of CD20. In some embodiments, the detection antibody bound to the sample-capture antibody combination is subsequently measured or quantified for the detection antibody using a detection means, such as one or more detection reagents, to determine the amount of CD20 protein to which the detection antibody is bound.

[0086] In some embodiments, the detection antibody can be used at a concentration of about 0.1 μg / ml to about 5.0 μg / ml. For example, but not limited to, the detection antibody can be used at a concentration of about 0.1 μg / ml to about 0.5 μg / ml, about 0.1 μg / ml to about 1.0 μg / ml, about 0.1 μg / ml to about 1.5 μg / ml, about 0.1 μg / ml to about 2.0 μg / ml, about 0.1 μg / ml to about 2.5 μg / ml, about 0.1 μg / ml to about 3.0 μg / ml, about 0.1 μg / ml to about 3.5 μg / ml, about 0.1 μg / ml to about 4.0 μg / ml, about 0.1 μg / ml to about 4.5 μg / ml, about 0.5 μg / ml to about 5.0 μg / ml, about 1.0 μg / ml to about 5.0 μg / ml, about 1.5 μg / ml to about 5.0 μg / ml, about 2.0 μg / ml to about 5.0 μg / ml, about 2.5 μg / ml to about 5.0 μg / ml, about 3.0 μg / ml to about 5.0 μg / ml, about 3.5 μg / ml to about 5.0 μg / ml, about 4.0 μg / ml to about 5.0 μg / ml, about 4.5 μg / ml to about 5.0 μg / ml, about 1.0 μg / ml to about 3.0 μg / ml, about 0.5 μg / ml to about 3.0 μg / ml, or about 0.5 μg / ml to about 2.0 μg / ml. In some embodiments, for an immunoassay to detect total CD20 protein, a detection antibody at a concentration of about 0.1 μg / ml to about 1.0 μg / ml, such as about 0.4 μg / ml or about 0.8 μg / ml, can be used. In some embodiments, for an immunoassay to detect active CD20 protein, a detection antibody at a concentration of about 1.0 μg / ml to about 3.0 μg / ml, such as about 1.1 μg / ml or about 2.1 μg / ml, can be used.

[0087] In some embodiments, the anti-CD20 antibody for use in the methods of the present disclosure can be labeled. Labels include, but are not limited to, labels or moieties that are directly detected, such as fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, radioactive labels, and moieties that are indirectly detected via an enzyme reaction or intermolecular interaction, such as an enzyme or a ligand. Non-limiting examples of 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. Patent No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase coupled with uricase and xanthine oxidase, etc., heterocyclic oxidases), biotin / avidin, spin labels, bacteriophage labels, and stable free radicals, etc. In some embodiments, the detection antibody is labeled with biotin, e.g., the detection antibody is conjugated to biotin.

[0088] In some embodiments, the detection reagent for the biotinylated detection antibody is avidin, streptavidin-HRP, or streptavidin-β-D-galactopyranose (SBG). In some embodiments, the readout of the detection reagent is fluorescence quantification or colorimetric quantification. For example, but not limited to, tetramethylbenzidine and hydrogen peroxide can be used as the readout. In some embodiments, when the detection reagent is streptavidin-HRP, the readout can be colorimetrically quantified by using tetramethylbenzidine and hydrogen peroxide. Alternatively, in some embodiments, resorufin β-D-galactopyranoside can be used as the readout. For example, but not limited to, when the detection reagent is SBG, the readout can be fluorescence-quantified by using resorufin β-D-galactopyranoside.

[0089] In some embodiments, a detection reagent, such as SBG, can be used at a concentration of about 50 to about 500 pM. For example, without limitation, the detection reagent can be used at a concentration of about 50 to about 100 pM, about 50 to about 150 pM, about 50 to about 200 pM, about 50 to about 250 pM, about 50 to about 300 pM, about 50 to about 350 pM, about 50 to about 400 pM, about 50 to about 450 pM, about 100 to about 500 pM, about 150 to about 500 pM, about 200 to about 500 pM, about 250 to about 500 pM, about 300 to about 500 pM, about 350 to about 500 pM, about 400 to about 500 pM, about 450 to about 500 pM, about 100 to about 400 pM, or about 200 to about 400 pM. In some embodiments, the detection reagent can be used at a concentration of about 100 pM to about 400 pM. For example, SBG can be used at a concentration of about 110 pM, about 155 pM, or about 310 pM. In some embodiments, SBG is used at a concentration of about 310 pM. In some embodiments, a detection reagent, such as HRP, can be used at a dilution of about 1 / 10 to about 1 / 1000. For example, without limitation, the detection reagent can be used at a dilution of about 1 / 10 to about 1 / 100, about 1 / 10 to about 1 / 500, about 1 / 100 to about 1 / 1000, or about 1 / 500 to about 1 / 1000. In some embodiments, the detection reagent can be used at a dilution of about 1 / 100 to about 1 / 1000. For example, HRP can be used at a dilution of about 1 / 100 or about 1 / 500.

[0090] In some embodiments, the method of the present disclosure can include blocking the capture antibody with a blocking buffer. In some embodiments, the blocking buffer is PBS, bovine serum albumin (BSA), and / or a biocide, such as ProClin TMIt can include [(Sigma-Aldrich, Saint Louis, MO)]. In some embodiments, the method can include a plurality of washing steps. In some embodiments, the solution used for washing is generally, but not limited to, a buffer (e.g., a "washing buffer") such as a PBS buffer containing a detergent, such as Tween20. For example, but not limited to, the capture antibody can be washed after blocking, and / or the sample can be separated from the capture antibody, e.g., by washing, to remove unbound materials.

[0091] In some embodiments, the immunoassay method disclosed herein has a detection sensitivity of about 2 pg / ml to about 20 pg / ml, e.g., the sensitivity within a well. For example, but not limited to, the immunoassay disclosed herein has a sensitivity of about 2 pg / ml to about 3 pg / ml, about 2 pg / ml to about 4 pg / ml, about 2 pg / ml to about 5 pg / ml, about 2 pg / ml to about 6 pg / ml, about 2 pg / ml to about 7 pg / ml, about 2 pg / ml to about 8 pg / ml, about 2 pg / ml to about 10 pg / ml, about 2 pg / ml to about 11 pg / ml, about 2 pg / ml to about 12 pg / ml, about 2 pg / ml to about 13 pg / ml, about 2 pg / ml to about 14 pg / ml, about 2 pg / ml to about 15 pg / ml, about 2 pg / ml to about 16 pg / ml, about 2 pg / ml to about 17 pg / ml, about 2 pg / ml to about 18 pg / ml, about 2 pg / ml to about 19 pg / ml, about 3 pg / ml to about 15 pg / ml, about 3 pg / ml to about 10 pg / ml, or about 3 pg / ml to about 5 pg / ml. In some embodiments, the immunoassay disclosed herein has a sensitivity of about 2 pg / ml or more, 1 pg / ml or more, or 0.5 pg / ml or more. In some embodiments, the immunoassay disclosed herein has a detection sensitivity of about 0.2 pg / ml to about 2.0 pg / ml, e.g., about 0.2 pg / ml to about 0.5 pg / ml, about 0.2 pg / ml to about 1.0 pg / ml, or about 0.2 pg / ml to about 1.5 pg / ml, e.g., the sensitivity within a well. For example, but not limited to, the immunoassay disclosed herein, e.g., Simoa HD-1 Analyzer TMThe single molecule immunoassay using it has a sensitivity of about 0.2 pg / ml to about 0.5 pg / ml, for example, the sensitivity within a well.

[0092] The sample to be analyzed by the immunoassay method of the present disclosure can be a clinical sample, cells in culture, cell supernatant, cell lysate, serum sample, plasma sample, other biological fluid (e.g., lymph) sample, or tissue sample. In some embodiments, the source of the sample can be a solid tissue (e.g., from fresh, frozen, and / or preserved organs, tissue samples, serum, plasma, biopsy material, or aspirate), or cells from a subject. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a plasma sample. In some embodiments, a sample, e.g., a blood or plasma sample, is obtained from a subject and can be treated with one or more protease, esterase, DDP-IV, and / or phosphatase inhibitors. For example, without limitation, the sample can be treated with a cocktail of protease and phosphatase inhibitors, such as MS-SAFE (Sigma-Aldrich, Saint Louis, MO). In some embodiments, the sample is treated with an anticoagulant or collected in a tube containing an anticoagulant, e.g., K2-EDTA. In some embodiments, the sample can be collected using a P800 Blood Collection System (BD Biosciences, San Jose, CA).

[0093] In some embodiments, the present disclosure provides a method for measuring the affinity of a therapeutic agent using surface plasmon resonance analysis (SPR). For example, the binding interaction between a target protein expressed on extracellular vesicles, e.g., CD20, and a non-target protein antibody, e.g., an anti-CD20 antibody, can be evaluated by SPR analysis, where extracellular vesicles expressing the target, e.g., CD20, can be used as a ligand, and the anti-target antibody, e.g., the anti-CD20 antibody, can be used as an analyte. By SPR analysis, the dissociation equilibrium constant (K D ), the dissociation rate constant (k d ), and the association rate constant (k aThe value of [[ID=]] can be calculated. In some embodiments, the therapeutic agent can include rituximab, ocrelizumab, ofatumumab, obinutuzumab, CD20 T cell-dependent bispecific antibodies, and combinations thereof. In some embodiments, SPR analysis is used as described herein to distinguish between two or more anti-target antibodies. In some embodiments, SPR analysis enables ranking of anti-target antibodies. In some embodiments, the selection of a particular anti-target antibody is performed by ranking two or more anti-target antibodies via SPR analysis and selecting the anti-target antibody ranked highest, or by selecting an anti-target antibody that exhibits a desired affinity.

[0094] III. Antibodies The present disclosure further provides antibodies that bind to CD20. The antibodies of the present disclosure are useful for the detection and quantification of CD20 protein levels in a sample. In some embodiments, the antibodies of the present disclosure can be used in immunoassay methods for detecting and quantifying the CD20 proteins disclosed herein. For example, but not limited to, the antibodies of the present disclosure can be used to detect the level of circulating CD20 protein in a sample.

[0095] In some embodiments, the antibodies of the present disclosure can be humanized. In some embodiments, the antibodies of the present disclosure include an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In some embodiments, the antibodies of the present disclosure can be monoclonal antibodies including chimeric antibodies, humanized antibodies, or human antibodies. In some embodiments, the antibodies of the present disclosure can be antibody fragments, such as Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragments. In some embodiments, the antibody is an IgG. In some embodiments, the antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody is a full-length antibody, such as an intact IgG1 antibody as defined herein, or other antibody classes or isotypes. In some embodiments, the antibodies disclosed herein can be labeled, for example, conjugated to biotin. In some embodiments, the antibodies of the present disclosure can incorporate any one or a combination of the features as described in Items 1-7 detailed below.

[0096] A. Exemplary Antibodies In some embodiments, the antibodies of the present disclosure, such as anti-CD20 antibodies, have a dissociation constant (K D ) of ≦1M, ≦100 mM, ≦10 mM, ≦1 mM, ≦100 μM, ≦10 μM, ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM or ≦0.001 nM. In some embodiments, the antibodies of the present disclosure have a K -3 of about 10 -8 or less, or 10 -8 M or less, for example, 10 -13 M to 10 -9 M, for example, 10 -13 M to 10 D . In some embodiments, the antibodies disclosed herein have a K -10 of about 10 -13 M to 10 D . For example, without limitation, the capture antibody or detection antibody of the present disclosure binds to its target antigen with a K -10 of about 10 -13K of M D is coupled with.

[0097] In some embodiments, K D can be measured by a radiolabeled antigen binding assay (RIA). In some embodiments, the RIA can be performed with the Fab version of the antibody of interest and its antigen. For example, without limitation, the solution binding affinity of the Fab for the antigen is determined by equilibrating the Fab with a minimum concentration of 125 I)-labeled antigen in the presence of a series of titrations of unlabeled antigen and then capturing the antigen bound to an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked for 2-5 hours at room temperature (approximately 23 °C) with 2% (w / v) bovine serum albumin in PBS. In non-adsorptive plates (Nunc #269620), 100 pM or 26 pM of 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to the capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard) is added and the plate is counted on a TOPCOUNT for 10 minutes TMCount using a gamma counter (Packard). The concentration of each Fab that results in less than 20% of the maximum binding is selected for use in a competitive binding assay.

[0098] In some embodiments, K D can be measured using a BIACORE® surface plasmon resonance assay. For example, but not limited to, an assay using a BIACORE®-2000, BIACORE®-3000, BIACORE X100, or BIACORE T200 processing unit (Biacore, Inc., Piscataway, NJ) is performed at 25° C. using an immobilized antigen CM5 chip at ~10 response units (RU). In some embodiments, a carboxymethylated dextran biosensor chip (CM5, Biacore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. After diluting the antigen to 5 μg / mL (~0.2 μM) with 10 mM sodium acetate (pH 4.8), it is injected at a flow rate of 5 μL / min to achieve approximately 10 response units (RU) of the binding protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at 25° C. at a flow rate of approximately 25 μl / min into PBS containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant (PBST). The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple 1:1 Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (K d ) can be calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on-rate by the above surface plasmon resonance assay is 10 6 M -1s -1 If it exceeds, the on-rate can be measured with a spectrophotometer equipped with stop-flow (Aviv Instruments) or an 8000 series SLM-AMINCO spectrophotometer equipped with a stirred cuvette TM by using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab type) (pH 7.2) in PBS at 25°C in the presence of antigen of increasing concentration.

[0099] 1. Antibody fragment In some embodiments, the antigen-antibody of the present disclosure is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below. For an overview of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For an overview of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); WO 93 / 16185; US Pat. Nos. 5,571,894 and 5,587,458. See also US Pat. No. 5,869,046 for a description of Fab and F(ab’)2 fragments that contain salvage receptor binding epitope residues and have an extended half-life in vivo.

[0100] In some embodiments, the antibodies of the present disclosure can be diabodies. A diabody is an antibody fragment that contains two antigen-binding sites that can be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Also, tribodies and tetrabodies, which are additional antibody fragments within the scope of the antibodies of the present disclosure, are described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0101] In some embodiments, the antibodies of the present disclosure can be single-domain antibodies. A single-domain antibody is an antibody fragment that contains all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516).

[0102] Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage) as described herein.

[0103] 2. Chimeric and Humanized Antibodies In some embodiments, the antibodies of the present disclosure are chimeric antibodies. Some chimeric antibodies are disclosed, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, the chimeric antibodies of the present disclosure include non-human variable regions (e.g., variable regions derived from non-human primates such as mice, rats, hamsters, rabbits, or monkeys), and human constant regions. In further examples, the chimeric antibody can be a "class switch" antibody whose class or subclass has changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0104] In some embodiments, the chimeric antibodies of the present disclosure can be humanized antibodies. Typically, non-human antibodies are humanized to retain the specificity and affinity of the parent non-human antibody while reducing immunogenicity to humans. Generally, a humanized antibody includes one or more variable domains, wherein the HVRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody also optionally includes at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) so as to, for example, restore or improve antibody specificity or affinity.

[0105] For humanized antibodies and methods for their production, for example, there is a review in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing the specific determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall’Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (further describing the "guided selection" approach of FR shuffling).

[0106] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0107] 3. Human Antibodies In some embodiments, the antibodies of the present disclosure can be human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0108] Human antibodies can be prepared by administering an immunogen to a transgenic animal modified to produce intact human antibodies or fully intact antibodies having human variable regions that respond to antigen administration. Such animals typically contain all or part of the human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or are present extrachromosomally, or are randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also, for example, see U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE TM technique; U.S. Patent No. 5,770,429, which describes the HUMAB® technique; U.S. Patent No. 7,041,870, which describes the K-M MOUSE® technique; and U.S. Patent Application Publication No. 2007 / 0061900, which describes the VELOCIMOUSE® technique. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0109] In addition, human antibodies can be produced by methods using hybridomas. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Also, human antibodies generated via human B cell hybridoma techniques are described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies derived from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma techniques (triooma techniques) are also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0110] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0111] 4. Antibodies from Libraries The antibodies of the present disclosure can be isolated by screening a combinatorial library for antibodies having one or more desired activities. For example, methods for generating phage display libraries and screening such libraries for antibodies having desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0112] In some phage display methods, the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then screened against antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunization sources provide high-affinity antibodies against the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) as described by Griffiths et al., EMBO J, 12:725-734 (1993) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization. In some embodiments, unrearranged V gene segments from stem cells can be cloned and highly variable HVR regions encoded using PCR primers containing random sequences, and a naive library can be synthetically generated by achieving rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0113] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0114] 5. Multispecific Antibodies In some embodiments, the antibodies of the present disclosure can be multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different epitopes. In some embodiments, one of the binding specificities is for an epitope present on CD20 and the other is for any other antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0115] Techniques for making multispecific antibodies are not limited, but include recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)) and "knobs-into-holes" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create antibody Fc heterodimer molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), using "diabody" technology to make bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and can also be made by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0116] Also included herein are engineered antibodies having three or more functional antigen-binding sites that include an "octopus antibody" (see, e.g., US Patent Application Publication No. 2006 / 0025576).

[0117] 6. Antibody Variants The subject matter disclosed herein further provides amino acid sequence variants of the disclosed antibodies. For example, it may be desirable to improve the binding affinity and / or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by incorporating appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, but are not limited to, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the present antibody. Deletions, insertions, and substitutions can be arbitrarily combined to arrive at the final construct, provided that the final antibody modification, i.e., the modified antibody, possesses the desired properties, such as antigen binding.

[0118] a) Substitution, Insertion, and Deletion Variants Antibody variants can have one or more amino acid substitutions, insertions, and / or deletions. Target sites for such mutations include, but are not limited to, the HVRs and FRs. Non-limiting examples of conservative substitutions are shown under the heading "Preferred Substitutions" in Table 1. Non-limiting examples of more substantial changes are shown under the heading "Exemplary Substitutions" in Table 1 and are further described below with respect to amino acid side-chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for the desired activity, such as retained / improved antigen binding, decreased immunogenicity, or improved complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0119] JPEG2025090689000002.jpg169170

[0120] Amino acids can be grouped according to common side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0121] In some embodiments, non-conservative substitutions will involve exchanging a member of one of these classes for another.

[0122] In some embodiments, a substitution variant involves substitution of one or more hypervariable region residues of a parent antibody, such as a humanized antibody or a human antibody. Generally, the resulting variant(s) selected for further testing have an improvement in certain biological properties, such as, but not limited to, increased affinity, reduced immunogenicity, and / or substantially retained specific biological properties of the parent antibody compared to the parent antibody. Non-limiting examples of substitution variants can be, for example, affinity matured antibodies that can be conveniently generated using affinity maturation techniques based on phage display, such as the techniques described herein. In short, one or more HVR residues are mutated, the variant antibody is displayed on phage, and screened for a particular biological activity (e.g., binding affinity).

[0123] In some embodiments, for example, to improve antibody affinity, modifications (e.g., substitutions) can be made to the HVRs. Such modifications can be made to HVR “hotspots,” i.e., residues encoded by codons that mutate frequently during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within residues that contact the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and rescreening from a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity can be introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify antibody variants having the desired affinity. Another method for introducing diversity involves an HVR-directed approach in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.

[0124] In some embodiments, substitutions, insertions, and / or deletions can occur within one or more HVRs so long as such modifications do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications may be, for example, outside of antigen contact residues within the HVRs. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0125] A useful method for identifying residues or regions of an antibody that can be targets for mutagenesis is what is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at positions of amino acids that show functional sensitivity to the first substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they have the desired properties.

[0126] Examples of amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from 1 residue to polypeptides containing more than 100 residues, as well as in-sequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody having an N-terminal methionyl residue. Other insertion-type variants of the antibody molecule include N-terminal or C-terminal fusions of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy: ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0127] b) Glycosylation variants In some embodiments, the antibodies of the present disclosure can be modified to increase or decrease the degree to which the antibody is glycosylated. For example, but not limited to, the addition or deletion of glycosylation sites to the antibody can be conveniently achieved by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0128] If the antibody of the present disclosure includes an Fc region, the carbohydrate bound thereto, if present, can be modified. Natural antibodies produced by mammalian cells typically include branched biantennary oligosaccharides commonly attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNac), galactose, and sialic acid, and fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides of the antibodies of the present disclosure can be modified to produce antibody variants with improved specific properties.

[0129] In some embodiments, antibody variants are provided that have a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such antibodies can be from about 1% to about 80%, from about 1% to about 65%, from about 5% to about 65%, or from about 20% to about 40%, and values therebetween.

[0130] In some embodiments, the amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 within the Fc region (Eu numbering of Fc region residues), although Asn297 can be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations within the antibody. Such fucosylation variants can have improved ADCC function. See, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US Patent Application Publication No. 2003 / 0115614; US Patent Application Publication No. 2002 / 0164328; US Patent Application Publication No. 2004 / 0093621; US Patent Application Publication No. 2004 / 0132140; US Patent Application Publication No. 2004 / 0110704; US Patent Application Publication No. 2004 / 0110282; US Patent Application Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; US Patent Application Publication No. 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0131] Defucosylated antibodies can be produced in any cell line deficient in protein fucosylation. Non-limiting examples of cell lines include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108, Presta, L; and International Publication No. 2004 / 056312, Adams et al., particularly Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 085107).

[0132] For example, there is further provided an antibody variant having a bisecting oligosaccharide in which the bisecting oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Non-limiting examples of such antibody variants are described, for example, in International Publication No. 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue of the oligosaccharide bound to the Fc region are also provided. Such antibody variants can have improved CDC function. Such antibody variants are described, for example, in International Publication No. 1997 / 30087 (Patel et al.), International Publication No. 1998 / 58964 (Raju, S.), and International Publication No. 1999 / 22764 (Raju, S.).

[0133] c) Fc region variant In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variants may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0134] In some embodiments, the present disclosure provides antibody variants having some, but not all, effector functions. Such limited effector functions can make the antibody variant a desirable candidate for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / abolition of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity), but retains FcRn binding ability. NK cells, which are the major cells mediating ADCC, express only FcγRIII, and monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI for flow cytometry (Cell Technology, Inc. Mountain View, CA)) TMNon-radioactive cytotoxicity assays; see, e.g., the CYTOTOX 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, e.g., in an animal model as disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can be performed to confirm that an antibody lacks CDC activity due to its inability to bind C1q. See, e.g., C1q and C3c binding ELISAs in International Publication Nos. WO 2006 / 029879 and WO 2005 / 100402. A CDC assay can be performed to evaluate complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)). In some embodiments, e.g., as described in U.S. Patent No. 6,194,551, International Publication No. WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000), modifications can be made in the Fc region to effect altered (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC).

[0135] Examples of antibodies with reduced effector function include antibodies having one or more substitutions among residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Patent No. 6,737,056). Examples of such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0136] Certain antibody variants with improved or decreased binding to FcR are described. See, e.g., U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0137] In some embodiments, the antibody variants of the disclosure include an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0138] In some embodiments, modifications occurring in the Fc region of the antibodies disclosed herein, such as bispecific antibodies, can generate variant antibodies having an increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), as described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies include an Fc region having one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include variants having a substitution at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a variant having a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0139] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and International Publication No. 94 / 29351, which relate to other examples of Fc region variants.

[0140] d) Cysteine-modified antibody variants In some embodiments, it may be desirable to generate a cysteine engineered antibody, e.g., a “thioMAb,” in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the replaced residues are present at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0141] e) Antibody derivatives In some embodiments, the antibodies of the present disclosure can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during production due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if two or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a treatment under defined conditions, etc.

[0142] In some embodiments, conjugates of antibodies and non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protective moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). In some embodiments, the radiation can be of any wavelength, including, but not limited to, wavelengths that heat the non-proteinaceous moiety to a temperature at which normal cells are not damaged but cells in proximity to the antibody-non-proteinaceous moiety are killed.

[0143] B. Method for Antibody Generation Antibodies, such as the capture antibodies and / or detection antibodies disclosed herein, can be made using techniques available in the art or known techniques. For example, but not limited to, antibodies can be generated using recombinant methods and compositions, as described, for example, in U.S. Patent No. 4,816,567. Detailed procedures for generating antibodies are described in the following examples.

[0144] The subject matter of the present disclosure further provides an isolated nucleic acid encoding an antibody disclosed herein. For example, the isolated nucleic acid can encode an amino acid sequence comprising an antibody, such as the VL of the light chain and / or VH of the heavy chain of the antibody.

[0145] In some embodiments, the nucleic acid can be present in one or more vectors, such as an expression vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Certain vectors can replicate themselves in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell upon introduction into the host cell and are thereby replicated with the host genome. Furthermore, certain vectors, expression vectors, can direct the expression of genes to which they are operably linked. Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, the disclosed subject matter is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

[0146] In some embodiments, a nucleic acid encoding an antibody of the present disclosure, and / or one or more vectors containing the nucleic acid, can be introduced into a host cell. In some embodiments, introduction of the nucleic acid into the cell can be carried out by any known method including, but not limited to, transfection, electroporation, microinjection, infection with a nucleic acid sequence-containing virus or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In some embodiments, the host cell can include the following and is, for example, transformed by the following: (1) a vector containing a nucleic acid encoding an amino acid sequence including the VL of the antibody and an amino acid sequence including the VH of the antibody, or (2) a first vector containing a nucleic acid encoding an amino acid sequence including the VL of the antibody and a second vector containing a nucleic acid encoding an amino acid sequence including the VH of the antibody. In some embodiments, the host cell is a eukaryotic cell, for example, a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cells).

[0147] In some embodiments, a method for producing the disclosed anti-CD20 antibody can include culturing a host cell into which a nucleic acid encoding the antibody has been introduced under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell and / or the host cell culture medium. In some embodiments, the antibody is recovered from the host cell by chromatographic techniques.

[0148] For recombinant production of the antibodies of the present disclosure, a nucleic acid encoding the antibody, such as described above, can be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0149] Suitable host cells for cloning or expression of the antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in the soluble fraction and further purified.

[0150] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including strains and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of antibodies having a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006). Suitable host cells for the expression of glycosylated antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified and can be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0151] Host cells suitable for the expression of glycosylated antibodies can be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified and can be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0152] In some embodiments, plant cell cultures can be utilized as host cells. For example, see U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES TM technique for generating antibodies in transgenic plants).

[0153] In some embodiments, vertebrate cells can also be used as hosts. For example, without limitation, mammalian cell lines adapted to grow in suspension may be useful. Non-limiting examples of useful mammalian host cell lines include monkey kidney CV1 cell line transformed by SV40 (COS-7), human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), e.g., TRI cells as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0154] In some embodiments, techniques for making bispecific and / or multispecific antibodies include recombinant co-expression of two immunoglobulin heavy-chain / light-chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knobs-into-holes" engineering (see, e.g., U.S. Patent No. 5,731,168), but are not limited thereto. Bispecific antibodies can be made by manipulating electrostatic steering effects to create antibody Fc heterodimer molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), using "diabody" technology to make bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and can also be made, for example, by preparing trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0155] Bispecific and multispecific molecules of the present disclosure can also be produced using chemical techniques (e.g., Kranz (1981) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" techniques (e.g., U.S. Pat. No. 4,474,893), or recombinant DNA techniques. Bispecific and multispecific molecules of the presently disclosed subject matter can also be prepared by conjugating component binding specificities, e.g., a first epitope and a second epitope binding specificity, as described herein using methods known in the art. For example, and not by way of limitation, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Non-limiting examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins.Mitt.(1985) No.78, 118-132; Brennan(1985) Science 229:81-83), Glennie(1987) J.Immunol.139:2367-2375). When the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region can be modified to contain an odd number of sulfhydryl residues, e.g., one, prior to conjugation.

[0156] In some embodiments, both binding specificities of a bispecific antibody can be encoded in the same vector, expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecules are MAb×MAb, MAb×Fab, Fab×F(ab’)2, or ligand×Fab fusion proteins. In some embodiments, the bispecific antibodies of the present disclosure can be single-chain bispecific antibodies, single-chain bispecific molecules comprising one single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants. The bispecific and multispecific molecules can be single-chain molecules or can comprise at least two single-chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858. Engineered antibodies having three or more functional antigen-binding sites (e.g., epitope-binding sites), including “octopus antibodies,” are also included herein (see, for example, U.S. Patent Application Publication No. 2006 / 0025576).

[0157] In some embodiments, an animal system can be used to generate the antibodies of the present disclosure. One animal system for preparing hybridomas is the mouse system. Hybridoma generation in mice is a very well-established technique. Immunization protocols and techniques for isolation of immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known (see, for example, Harlow and Lane (1988), Antibodies, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York).

[0158] IV. Kit The subject matter disclosed in the present application further provides a kit comprising materials useful for performing the immunoassays disclosed herein. In some embodiments, the kit includes a container containing an antibody disclosed herein, such as an anti-CD20 antibody. Non-limiting examples of suitable containers include bottles, test tubes, vials, and microtiter plates. The container can be formed from a variety of materials such as glass or plastic. In some embodiments, the kit further includes a package insert providing instructions for using an antibody, such as an anti-CD20 antibody, in the disclosed immunoassay method.

[0159] In some embodiments, the kit can include one or more containers containing one or more antibodies. For example, without limitation, the kit can include at least one container containing a capture antibody and at least one container containing a detection antibody.

[0160] In some embodiments, a kit for detecting a tumor antigen protein in a sample includes a first container containing a capture antibody that binds to an epitope present within the amino acid sequence of the target protein, a second container containing a detection antibody that binds to an epitope present within the amino acid sequence of the target protein, and a third container containing a detection reagent. In some embodiments, the capture antibody and the detection antibody bind to different epitopes present within the amino acid sequence of the target protein.

[0161] In some embodiments, the capture and / or detection antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, CD20 T cell-dependent bispecific antibodies, and combinations thereof.

[0162] In some embodiments, the capture antibody and / or the detection antibody can be provided in the kits of the present disclosure at a concentration of about 0.1 μg / ml to about 5.0 μg / ml. For example, the capture antibody and / or the detection antibody can be provided in an ELISA kit at a concentration of about 0.1 μg / ml to about 5.0 μg / ml. In non-limiting embodiments, the capture antibody and / or the detection antibody can be provided in a Quanterix kit at a concentration of about 0.1 μg / ml to about 2.0 μg / ml. In some embodiments, the detection antibody can be labeled, for example, with biotin.

[0163] In some embodiments, the detection reagent provided in the kits of the present disclosure can be avidin, streptavidin-HRP, or streptavidin-β-D-galactopyranoside (SBG). In some embodiments, the kits of the present disclosure can further include tetramethylbenzidine, hydrogen peroxide, and / or resorufin β-D-galactopyranoside. In some embodiments, when the kit includes streptavidin-HRP, the kit can further include tetramethylbenzidine and hydrogen peroxide. In some embodiments, when the kit includes SBG, the kit can further include resorufin β-D-galactopyranoside. In some embodiments, SBG can be provided in the kit at a concentration of about 100 pM to about 400 pM.

[0164] In some embodiments, the capture antibody can be provided, for example, but not limited to, bound to the surface of a solid support such as a plate or beads, such as paramagnetic beads. Alternatively or in addition, the kit can further include a surface of a solid support that can be linked to the capture antibody. In some embodiments, the solid support can be paramagnetic beads, and can be provided at a concentration of about 0.1×10 7 beads / ml to about 10.0×10 7 beads / ml.

[0165] Alternatively or in addition, the kit can include other materials that are desirable from a commercial and user perspective, such as other buffers, diluents, and filters. In some embodiments, the kit can include materials for collecting and / or processing a blood sample.

[0166] The following examples are merely illustrative of the subject matter disclosed herein and should not be construed as limiting in any way.

[0167] IV. Exemplary Embodiments A1. In some non-prior art embodiments, the present disclosure provides an assay for detecting a membrane-bound protein in a sample, comprising: a) a capture antibody that binds to extracellular vesicles containing the membrane-bound protein in the sample to generate a capture antibody-extracellular vesicle complex; and b) a detection antibody that binds to the capture antibody-extracellular vesicle complex to form a detectable binding complex, wherein the signal from the detectable binding complex is calibrated against one or more known values detected from extracellular vesicles containing the protein. A2. In some embodiments of A1, the capture antibody does not compete with the detection antibody for binding. A3. In some embodiments of A1 and A2, the capture antibody binds to an epitope different from that of the detection antibody. A4. In some embodiments of A1-A3, the membrane-bound protein is selected from the group consisting of human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof. A5. In some embodiments of A1-A4, the capture antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, and combinations thereof. A6. In some embodiments of A1-A5, the detection antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, and combinations thereof. In some embodiments of A7.A1 - A6, the assay further comprises an extracellular vesicle calibrator. In some embodiments of A8.A1 - A7, the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof.

[0168] B1. In some non - limiting embodiments, the present disclosure is directed to a method for quantifying the concentration of a circulating protein in a sample, comprising: a) determining the level of a target protein in extracellular vesicles in the sample; and b) comparing the level of the target protein in extracellular vesicles in the sample to a calibration curve generated using extracellular vesicles containing the target protein. B2. In some embodiments of B1, the target protein is selected from the group consisting of a human CD20 antigen, a mouse CD20 antigen, a rat CD20 antigen, a rabbit CD20 antigen, a cynomolgus monkey CD20 antigen, a human CD3 antigen, a mouse CD3, a rat CD3 antigen, a rabbit CD3 antigen, a cynomolgus monkey CD3 antigen, a human FcRH5 antigen, a human Ly6G6 antigen, a human HER2 antigen, a human EGFR antigen, a human HER3 antigen, a human HER4 antigen, a human PSMA antigen, and combinations thereof. B3. In some embodiments of B1 or B2, the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof. B4. In some embodiments of B1 - B3, the concentration of the target protein and the calibration curve are determined using an immunoassay, ELISA, and / or Western blot. B5. In some embodiments of B1 - B4, the method further comprises detecting the presence of an extracellular vesicle marker, and the extracellular marker is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

[0169] C1. In some non-limiting embodiments, the present disclosure is directed to a method for quantifying the concentration of a circulating protein in a sample, the method comprising: a) generating a calibration curve using extracellular vesicles comprising the protein; and b) comparing the level of the protein in the extracellular vesicles in the sample to the calibration curve to determine the amount of the protein in the extracellular vesicles in the sample. C2. In some embodiments of C1, the protein is selected from the group consisting of human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof. C3. In some embodiments of C1 or C2, the sample is selected from the group consisting of plasma samples, serum samples, tissue culture supernatant samples, and combinations thereof. C4. In some embodiments of C1-C3, the concentration of the circulating protein and the calibration curve are determined using immunoassays, ELISA, and / or Western blot. C5. In some embodiments of C1-C4, the method further comprises detecting the presence of an extracellular vesicle marker, the extracellular marker being selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

[0170] D1. In some non-limiting embodiments, the present disclosure is directed to a method for determining whether a patient having B cell lymphoma is likely to respond to anti-CD20 therapy, the method comprising: a) obtaining a sample from the patient; b) determining the amount of circulating CD20 in the extracellular vesicles in the sample; c) comparing the level of CD20 in the extracellular vesicles in the sample to a calibration curve generated using extracellular vesicles comprising CD20; and d) determining whether the patient is likely to respond to CD20 therapy based on the amount of circulating CD20 in the extracellular vesicles determined in the sample. In some embodiments of D2.D1, the anti-CD20 therapy comprises administration of an anti-CD20 antibody. In some embodiments of D3.D1 or D2, the anti-CD20 antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, CD20 T cell-dependent bispecific antibodies, and combinations thereof. In some embodiments of D4.D1-D3, the sample is selected from the group consisting of plasma samples, serum samples, tissue culture supernatant samples, and combinations thereof. In some embodiments of D5.D1-D4, the concentration of the circulating protein and the calibration curve are determined using an immunoassay, ELISA, and / or Western blot. In some embodiments of D6.D1-D5, the method further comprises detecting the presence of an extracellular marker, which is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

[0171] In some non-limiting embodiments, the present disclosure is directed to a method for determining the affinity of an anti-CD20 antibody, comprising subjecting the anti-CD20 antibody to surface plasmon resonance (SPR) analysis, wherein the SPR analysis comprises the use of extracellular vesicles expressing CD20 as a ligand and the anti-CD20 antibody as an analyte. In some embodiments of E2.E1, the anti-CD20 antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, CD20 T cell-dependent bispecific antibodies, and combinations thereof. In some embodiments of E3.E1-E2, the method further comprises detecting the presence of an extracellular marker, which is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

[0172] F1. In some non-limiting embodiments, the present disclosure is directed to a method for determining the activation of T cells obtained from a patient, the method comprising: a) incubating extracellular vesicles expressing CD20 with T cells and a CD20 T cell-dependent bispecific antibody; and b) determining the activation of the T cells. F2. In some embodiments of F1, the method further comprises detecting the presence of an extracellular marker, the extracellular marker being selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

[0173] G1. In some non-limiting embodiments, the present disclosure is directed to a method for treating a tumor in a subject in need of treatment, the method comprising: a) obtaining a sample from the subject; b) generating a calibration curve using extracellular vesicles containing a tumor antigen; c) comparing the level of the tumor antigen in the extracellular vesicles in the sample to the calibration curve to determine the amount of the target tumor antigen in the extracellular vesicles in the sample; d) determining whether the subject is likely to exhibit a response to antibody therapy based on the level of the tumor antigen in the extracellular vesicles in the sample; and e) applying a treatment in response to the determination in d). G2. In some embodiments of G1, the method further comprises detecting the presence of an extracellular marker, the extracellular marker being selected from the group consisting of CD81, CD63, CD9, and combinations thereof. G3. In some embodiments of G1-G2, the antibody is selected from the group consisting of rituximab, ofatumumab, obinutuzumab, and combinations thereof. G4. In some embodiments of G1-G3, the target tumor antigen is selected from the group consisting of human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof. In some embodiments of G5.G1 - G4, the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof. In some embodiments of G6.G1 - G5, the concentration of the circulating tumor antigen and the calibration curve are determined using an immunoassay, ELISA, and / or Western blot.

Example

[0174] Example 1. Preparation of a Tumor Antigen Assay Calibration Curve A. Culturing of Tumor Antigen - Expressing Cells Maintenance of the seed strain: The seed strain was passaged every 3 - 4 days. For 3 - day sub - culturing, the cells were seeded at 0.8×10 6 cells / mL in Expi293 expression medium filled to 32% (e.g., 80 mL in a 250 mL baffled - free shaking flask) or 50% (e.g., 1 L in a 2 L baffled - free shaking flask); agitated at 125 rpm (32% fill) or 160 rpm (50% fill), orbital diameter 25 mm, incubated at 8% CO2, 80% humidity, 37°C; the cells should grow to >4×10 6 cells / mL with a >95% survival rate. For 4 - day sub - culturing, the cells were seeded at 0.4×10 6 cells / mL in Expi293 expression medium filled to 32% (e.g., 80 mL in a 250 mL baffled - free shaking flask) or 50% (e.g., 1 L in a 2 L baffled - free shaking flask); agitated at 125 rpm (32% fill) or 160 rpm (50% fill), orbital diameter 25 mm, incubated at 8% CO2, 80% humidity, 37°C; the cells should grow to >4×10 6 cells / mL with a >95% survival rate.

[0175] Seeding and culturing: The Expi293 seed strain was cultured in Hyclone HyCell TransFX - H medium, 10 mg / mL gentamicin (A466), 10% pluronic F - 68, 20 mM L - glutamine (A0821). Dilutions were made using containers and 125 mL shaking flasks / 50 mL tube spins.

[0176] Count the seed culture for viable cell density and viability, viability (>4×10 6 cells / mL, >95% viability, viability): Calculate the volume of culture required for transfection (V F = 30 mL × number of transfections).

[0177] Prepare the production medium: Supplement Hyclone medium with 0.5 g / L pluronic F-68 (add 5 mL of 10% pluronic F-68 to 1 L of Hyclone medium); 4 mM L-glutamine (add 20 mL of 20 mM L-glutamine (A0821) to 1 L of Hyclone medium); and 0.21 g / L gentamicin (add 21 mL of 10 mg / mL gentamicin (A466) to 1 L of Hyclone medium) (optional).

[0178] Dilute the Expi293 cell line to 2.0×10 6 cells / mL in an appropriate amount of production medium; this is the culture to be transfected. Calculate the seed culture required for dilution using the following formula: V C = X F V F / X C [where: V C = volume of seed culture required (mL) X F = final desired viable cell density for transfection (2.0×10 6 cells / mL) V F = final culture volume required for all transfections (mL) X C = viable cell density of the seed culture (cells / mL)]

[0179] Dilutions of Expi293 production cultures were dispensed at 25.5 mL per flask / tube spin. The flask / tube spins were placed at 37 °C, 8% CO2, 125 rpm (flask orbital diameter 25 mm) or 225 rpm (tube spin orbital diameter 50 mm) and equilibrated (for at least 15 minutes).

[0180] B. Extracellular Vesicle Tumor Antigen Calibrator Purification Protocol A 7-day culture of Expi293 transfected with the pB_EF1_hCD20 construct was harvested and centrifuged at 500 g for 10 minutes. The supernatant was decanted into another 50 ml conical and rotated at 2000 g for 10 minutes. The supernatant was decanted through a 0.22 um vacuum filter and filtered. The filtered medium was concentrated using a 70 ml centrifugal concentrator (Centricon Plus-70, UFC710008): 60 ml of supernatant was loaded, gently mixed supernatant at 3750 rpm at 4 °C for 10 minutes; 3750 rpm at 4 °C for 10 minutes; the filtrate was decanted, additional supernatant was added and rotated additional times until the volume was less than 12 ml. The concentrate was recovered at 750 g (max 1000 g) for 2 minutes at 4 °C. The rotation of the concentrate was kept below 10 minutes to avoid precipitation and aggregation. The concentrated medium was rotated in an ultracentrifuge at 30 krpm and 4 °C for 75 minutes. Tubes were properly balanced within 0.01 g using PBS, including tubes without sample (using H2O for balancing). Max was used for both Accel and Decel. The supernatant was decanted. The pellet must be visible at the bottom of the tube. The pellet was resuspended in 500 uL of PBS and then the ultracentrifuge tube was filled with 12 mL of 1X PBS. The mixture was balanced again with PBS and centrifuged again at 30 krpm (100,000 g) at 4 °C for 75 minutes. The supernatant was drained and the pellet was gently resuspended in 0.5 - 1 ml of PBS.

[0181] C. Assignment of EV Tumor Antigen Calibrator Values by Western Blot Figure 2 shows an exemplary characterization of an EV tumor antigen calibrator prepared as described herein. The values in the figure are assigned by Western blot. Column 1 represents the marker, Column 2 represents 2 μg of EV, Column 3 represents 1 μg of EV, Column 4 represents 0.5 μg of EV, Column 5 represents 0.25 μg of EV, Column 6 represents 250 ng of rhCD20, Column 7 represents 100 ng of rCD20, Column 8 represents 40 ng of rCD20, Column 9 represents 16 ng of rCD20, and Column 10 represents 6.4 ng of rCD20.

[0182] Figure 3 shows the presence of CD20 in plasma samples from healthy and NHL (e.g., DLBCL and FL) donors using anti-CD20 Ab as the capture antibody and anti-CD20 as the detection antibody or anti-tetraspanin antibody as the detection antibody. The presence of CD20 in extracellular vesicles can also be detected using the tetraspanin antibody. For example, capture using CD20 and detection using CD81, CD9, Cd63 demonstrated the co-localization of these markers and the presence of CD20 in the membrane (or extracellular vesicles). Since not all vesicles have all or the same markers, a cocktail was therefore required for detection.

[0183] Figure 4 shows an exemplary ELISA format when detecting CD20 in plasma from healthy and NHL (e.g., DLBCL and FL) using an anti-CD20 antibody. The ELISA data in Figure 4 shows evidence of the co-localization of these markers in neat plasma that has not been ultracentrifuged.

[0184] D. EV tumor antigen standard curve by Quanterix The following materials were used for the Quanterix assay: a) standard curve and sample diluent PBS, 1.5% BSA, 0.05% polysorbate 20, 0.05% Proclin 300, pH 7.4, b) anti-DIG antibody-conjugated beads, c) DIG-conjugated offatumumab as a capture antibody for CD20 antigen, d) biotin-conjugated offatumumab as a detection antibody, e) streptavidin-conjugated beta-galactosidase (SBG) as an enzyme reagent, and f) RGP, a substrate for SBG used as a reporter of signal (see also Figure 10).

[0185] CD20 expressed on extracellular vesicles was diluted in the standard curve diluent starting from a concentration of 500 ng / mL. Serial two-fold dilutions were performed 10 times so that the final concentration was 0.5 ng / mL. Eleven levels + non-specific blank were pipetted into a Quanterix polypropylene low-binding plate. Detection and capture antibodies diluted to 0.5 μg / mL in PBS, 1.5% BSA, 0.05% polysorbate 20, 0.05% Proclin 300, pH 7.4 were prepared and loaded onto the instrument in front of a 96-well plate. The enzyme reagent SA Beta-galactosidase was diluted to a concentration of 150 pM in its own buffer. Raw data from the instrument was downloaded into an Excel cvs file and regressed using 5 pl fit of SoftMax Pro software. Table 2 provides an exemplary EV CD20 standard curve by Quanterix.

[0186] JPEG2025090689000003.jpg94170

[0187] Example 2. Detection of Tumor Antigens with and without a Calibration Curve Plasma collection. 6 mL of whole blood was collected and transferred to a plasma lavender top Vacutainer tube (plasma collection tube, BD #367863). The collection tube was filled completely until the blood flow stopped. After whole blood collection, the plasma lavender top Vacutainer tube was gently and completely inverted 5 times to mix uniformly. The red blood cells did not rupture even when the tube was strongly inverted. Cell lysis can lead to specimen degradation. The specimen was placed on wet ice immediately after blood collection. This process was initiated within 30 minutes after blood collection.

[0188] The sample was frozen immediately after processing. The Vacutainer tube was centrifuged at 1600 × g for 15 minutes at 4°C. Without disturbing the cell buffy coat, plasma was slowly and carefully collected from the top layer of the tube (~3 mL) using a transfer pipette and transferred to two pre-labeled 4.5 mL NUNC tubes. The remaining cell pellet was discarded appropriately. Not all possible plasma was removed. The plasma remained approximately 5 mm away from the buffy coat, avoiding contamination of the plasma by cellular material (mononuclear cells). The plasma was mixed by inverting 5 - 6 times and aliquoted into pre-labeled 2.0 mL Sarstedt tubes. The samples were transferred to a freezer at -70 / -80°C (preferred) or -20°C (alternatively) in an upright position for storage.

[0189] The samples were stored at -70 / -80°C (preferred) or -20°C (alternatively) until analysis.

[0190] Continuous assay. If improved sensitivity is desired, a 3-day continuous assay (Figure 5A) can be used. Day 1: The plate was coated overnight at 4°C with the capture antibody. Day 2: The sample was added and incubated overnight at 4°C. Day 3: The detection antibody (conjugated to biotin) and SA-HRP were added. The following antibodies and signal conditions were used: Ocre 1 ug / ml (capture antibody), Ofa 0.5 ug / ml (detection antibody), and HRP 100 ng / mL (signal). Table 3 provides exemplary data generated by the continuous assay.

[0191] JPEG2025090689000004.jpg185170

[0192] Crosslinking assay. The two-day crosslinking assay (Figure 5B) can be used when it is desirable to shorten the time to results. Day 1: 100 uL of sample was incubated overnight at 4 °C with 100 uL of master mix (Ab-DIG + Ab-biotin). Day 2: 100 uL of the sample containing the master mix was removed and transferred to an SA plate. The signal was detected with anti-DIG-HRP. The following antibody and signal conditions were used: master mix 1 ug / mL (ofa-DIG + anti-DIG-biotin) and HRP 50 ng / mL (signal). Table 4 provides exemplary data generated by the crosslinking assay.

[0193] JPEG2025090689000005.jpg176170

[0194] Signal detection without using a calibration curve: Samples and reagents were diluted with a standard curve diluent without an EV calibrator. Samples were serially diluted 2-fold. The diluted samples were pipetted into a Quanterix polypropylene low-binding plate. Beads conjugated to anti-DIG antibody, ofatumumab-DIG, and ofatumumab-biotin were diluted to a concentration of 0.5 ug / mL in the standard curve diluent. The beads were diluted to a nominal bead concentration of 1.4×10 9 beads / mL. Enzyme (streptavidin β-galactosidase, SBG) was diluted to 150 pM in SBG diluent. A 96-well plate containing beads, detector, enzyme, substrate, and standards / samples was loaded into the instrument. Raw data was downloaded from the instrument to an Excel cvs file. The raw data was processed using an Excel spreadsheet.

[0195] As shown in Table 5, there was no calibration curve generated using recombinant human. Ocrelizumab was used as the capture antibody and ofatumumab was used as the detection antibody. Commercially available recombinant human likely did not induce a signal with the Ocre / Ofa combination due to lacking loop formation by transmembrane helices. Linear peptides or recombinant proteins not bound to the membrane did not induce a signal in the ELISA, suggesting that ocrelizumab or ofatumumab do not bind to CD20 in their conformation.

[0196] JPEG2025090689000006.jpg62170

[0197] Signal detection using the calibration curve: CD20 EV was diluted with a standard curve diluent at an initial concentration of 500 ng / mL. Two-fold serial dilutions were performed 10 times so that the final concentration was 0.5 ng / mL. 11 levels + non-specific blank were pipetted into a Quanterix polypropylene low-binding plate.

[0198] Ofatumumab-DIG and ofatumumab-biotin were diluted to a concentration of 0.5 μg / mL in BA003 + 1.5% BSA. Anti-DIG Ab-beads were diluted to a nominal bead concentration of 1.4×10 9 beads / mL in BA003 + 1.5% BSA. The enzyme (streptavidin β-galactosidase, SBG) was diluted to 150 pM in SBG diluent. A 96-well plate containing beads, detector, enzyme, substrate, and standard / sample was loaded into the instrument. Raw data was exported and analyzed using Softmax Pro.

[0199] Table 6 provides the dose-dependent signal (average enzyme per bead, see also Figure 10), standard deviation, CD observed concentration, coefficient of variation of concentration, and recovery. The following equations were used: Signal: average enzyme per bead (AEB), Coefficient of variation (%C.V.) = (standard deviation / theoretical value) × 100, JPEG2025090689000007.jpg16170, and Difference from the theoretical value (recovery %) = [(average calculated concentration / theoretical concentration) - 1] × 100

[0200] JPEG2025090689000008.jpg83170

[0201] Example 3. Bead - based immunoassay format Another format useful for detecting proteins, such as tumor antigens, includes bead - based immunoassays, such as the Quanterix platform. In such an assay, anti - DIG antibody followed by an offatumumab - DIG coating can be used to capture cCD20 and offatumumab - biotin, and subsequently streptavidin β - galactosidase can be used for detection (Figure 6).

[0202] In an exemplary bead - based immunoassay format, offatumumab - DIG and offatumumab - biotin were diluted to a concentration of 0.5 μg / mL in BA003 + 1.5% BSA. Beads labeled with an antibody against Digoxin (anti - DIG - beads) were diluted to a nominal bead concentration of 1.4×10 9 beads / mL in BA003 + 1.5% BSA. The enzyme (streptavidin β - galactosidase, SBG) was diluted to 150 pM in SBG diluent. A 96 - well plate containing beads, detector, enzyme, substrate, and standards / samples was loaded onto the instrument.

[0203] As shown in FIG. 6, in the first step, the sample, anti-DIG beads, offatumumab-biotin, and offatumumab DIG detector were pipetted into a cuvette to form a sandwich for approximately 67 cadences of incubation (50 minutes). Next, in the second step, the sandwich was labeled with SBG and incubated for 7 cadences (5 minutes). Between steps, the beads were pelleted with a magnet followed by a washing step. The beads were resuspended in resorufin β-D-galactopyranoside (RGP) substrate and transferred to a Simoa Disc for imaging. Table 7 provides the dose-dependent average enzyme per bead (AEB), coefficient of variation (CV), calculated concentration, CV of concentration, recovery, and signal-to-background ratio.

[0204] JPEG2025090689000009.jpg88170

[0205] Example 4. Effect of Detergent on Detectability A set of CD20 controls (5 and 50 ng / mL) was prepared in PBS, 1.5% BSA, 0.15% polysorbate 20, 0.05% Proclin 300, pH 7.4. A second set was prepared in PBS, 1.5% BSA, 0.05% polysorbate 20, 0.05% Proclin 300, pH 7.4. The controls were assayed on a Quanterix instrument. As shown in FIG. 7, in this assay the lower detergent showed an improved signal-to-background (S / B) ratio.

[0206] Example 5. Drug Resistance Assay Drug resistance control was prepared in a buffer matrix to reduce endogenous effects (Figure 8). CD20 TDB and CD20 were each diluted to twice the nominal concentration in PBS, 1.5% BSA, 0.05% polysorbate 20, 0.05% Proclin 300, pH 7.4 and then combined 1:1. The final concentrations were 0, 0.05, 0.5, and 5 μg / mL of TDB and 50 ng / mL of CD20. Controls were assayed and quantified against a standard curve. The drug resistance test showed approximately 50% interference with 50 ng / mL of CD20 EV in the presence of 5 μg / mL of TDB (e.g., anti-CD20-CD3).

[0207] Example 6. Characterization of anti-CD20 TDB against CD20 expressed on extracellular vesicles by Biacore TM Using The binding interaction between CD20 expressed on extracellular vehicles (EV) and anti-CD20 TDB was evaluated by surface plasmon resonance (SPR) technique using a Biacore TM T200 instrument (GE Healthcare; Piscataway, NJ). The values of the dissociation equilibrium constant (K D ), dissociation rate constant (kd), and association rate constant (ka) were calculated using the heterogeneous analyte binding model with Biacore TM T200 evaluation software (Version 3.0; GE Healthcare).

[0208] CD20 EVs were captured on different flow cells (FCs) on the SA sensor chip using an indirect capture method (Figure 9A). Biotinylated anti-CD81 and anti-CD9 antibodies (mixed at an equal concentration of 30 μg / mL) were first captured via biotin-streptavidin interactions to all four FCs, and a capture level of approximately 2500 response units (RU) was obtained. Then, CD20 EVs were injected into FC2 or FC4 at a concentration of 0.25 μg / mL for 40 - 120 seconds (s). The resulting EV capture levels were in the range of 600 - 1800 RU. Anti-CD20 TDB at various concentrations was diluted in running buffer (0.01 M HEPES, 0.15 M NaCl, and 3 mM EDTA, pH 7.4) and then injected into the four FCs at a flow rate of 100 μL / min for 1 or 2 minutes (min); dissociation of anti-CD20 TDB from the antibody was allowed to proceed for 10 minutes for kinetic affinity measurements. The experiments were conducted at 37°C. The results are summarized in Figure 9B. Figure 9B also presents a representative Biacore Sensorgram of the binding of anti-CD20 TBD to CD20 EVs at 37°C.

[0209] In addition to the illustrated and claimed various embodiments, the subject matter of the present disclosure also encompasses other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein may be combined with each other in other ways within the scope of the subject matter of the present disclosure such that the subject matter of the present disclosure includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure has been presented for purposes of illustration and description. This description is not intended to be exhaustive or to limit the subject matter of the present disclosure to the disclosed embodiments.

[0210] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and methods of the subject matter of the present disclosure without departing from the spirit or scope of the subject matter of the present disclosure. Accordingly, it is intended that the subject matter of the present disclosure cover modifications and variations within the scope of the claims and their equivalents.

[0211] Various publications, patents, and patent applications are cited in this specification, and their contents are hereby incorporated by reference in their entirety.

Claims

1. 1. An assay for detecting a membrane associated protein in a sample, comprising: a) a capture antibody that binds to an extracellular vesicle containing a membrane-associated protein in a sample, thereby generating a capture antibody-extracellular vesicle complex; and b) a detection antibody that binds to the capture antibody-extracellular vesicle complex to form a detectable binding complex; wherein the signal from the detectable binding complex is calibrated to one or more known values ​​detected from extracellular vesicles containing the protein.

2. The assay of claim 1 , wherein the capture antibody does not compete with the detection antibody for binding.

3. 3. The assay of claim 1 or 2, wherein the capture antibody binds to a different epitope than the detection antibody.

4. 4. The assay of any one of claims 1 to 3, wherein the membrane bound protein is selected from human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof.

5. The assay of any one of claims 1 to 4, wherein the capture antibody is selected from the group consisting of rituximab, ocrelizumab, ofatumumab, obinutuzumab, and combinations thereof.

6. 6. The assay of any one of claims 1 to 5, wherein the detection antibody is selected from the group consisting of rituximab, ocrelizumab, ofatumumab, obinutuzumab, and combinations thereof.

7. The assay of any one of claims 1 to 6, further comprising an extracellular vesicle calibrator.

8. The assay of any one of claims 1 to 7, wherein the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof.

9. 1. A method for quantifying the concentration of a circulating protein in a sample, comprising: a) determining the level of a target protein in extracellular vesicles in a sample; and b) comparing the level of the target protein in the extracellular vesicles in the sample to a calibration curve generated using extracellular vesicles containing the target protein. The method includes:

10. 10. The method of claim 9, wherein the target protein is selected from the group consisting of human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof.

11. 11. The method of claim 9 or 10, wherein the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof.

12. The method according to any one of claims 9 to 11, wherein the concentration of the target protein and the calibration curve are determined using an immunoassay, ELISA and / or Western blot.

13. 13. The method of any one of claims 9 to 12, further comprising detecting the presence of an extracellular vesicle marker, wherein the extracellular marker is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

14. 1. A method for quantifying the concentration of a circulating protein in a sample, comprising: a) generating a calibration curve using extracellular vesicles containing the protein; and b) comparing the level of said protein in extracellular vesicles in the sample to a calibration curve to determine the amount of said protein in extracellular vesicles in the sample. The method includes:

15. 15. The method of claim 14, wherein the protein is selected from the group consisting of human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof.

16. 16. The method of claim 14 or 15, wherein the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof.

17. The method according to any one of claims 14 to 16, wherein the concentration and calibration curve of the circulating protein is determined using an immunoassay, ELISA and / or Western blot.

18. 18. The method of any one of claims 14 to 17, further comprising detecting the presence of an extracellular vesicle marker, wherein the extracellular marker is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

19. 1. A method for determining whether a patient having a B cell lymphoma is likely to respond to anti-CD20 therapy, comprising: a) obtaining a sample from a patient; b) determining the amount of circulating CD20 in extracellular vesicles in the sample; c) comparing the level of CD20 in the extracellular vesicles in the sample to a calibration curve generated using extracellular vesicles containing CD20; and d) determining whether the patient is likely to respond to CD20 therapy based on the amount of circulating CD20 in the extracellular vesicles determined in the sample; The method includes:

20. 20. The method of claim 19, wherein the anti-CD20 therapy comprises administration of an anti-CD20 antibody.

21. 21. The method of claim 20, wherein the anti-CD20 antibody is selected from the group consisting of rituximab, ocrelizumab, ofatumumab, obinutuzumab, CD20 T cell-dependent bispecific antibodies, and combinations thereof.

22. The method of any one of claims 19 to 21, wherein the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof.

23. The method according to any one of claims 19 to 22, wherein the concentration and calibration curve of the circulating protein is determined using an immunoassay, ELISA and / or Western blot.

24. 24. The method of any one of claims 19 to 23, further comprising detecting the presence of an extracellular marker, wherein the extracellular marker is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

25. A method for determining the affinity of an anti-CD20 antibody, comprising subjecting the anti-CD20 antibody to surface plasmon resonance (SPR) analysis, wherein the SPR analysis comprises the use of extracellular vesicles expressing CD20 as a ligand and the anti-CD20 antibody as an analyte.

26. 27. The method of claim 26, wherein the anti-CD20 antibody is selected from the group consisting of rituximab, ocrelizumab, ofatumumab, obinutuzumab, CD20 T cell-dependent bispecific antibodies, and combinations thereof.

27. 27. The method of claim 25 or 26, further comprising detecting the presence of an extracellular marker, wherein the extracellular marker is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

28. 1. A method for determining activation of T cells obtained from a patient, comprising: a) incubating extracellular vesicles expressing CD20 with T cells and a CD20 T cell-dependent bispecific antibody; and b) Determining T cell activation The method includes:

29. 30. The method of claim 28, further comprising detecting the presence of an extracellular marker, wherein the extracellular marker is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

30. 1. A method of treating a tumor in a subject in need thereof, comprising: a) obtaining a sample from a subject; b) generating a calibration curve using extracellular vesicles containing tumor antigens; c) comparing the level of the tumor antigen in the extracellular vesicles in the sample to a calibration curve to determine the amount of the target tumor antigen in the extracellular vesicles in the sample; d) determining whether the subject is likely to respond to antibody therapy based on the level of tumor antigen in the extracellular vesicles in the sample; and e) administering a treatment in response to the determination in d). The method includes:

31. 31. The method of claim 30, further comprising detecting the presence of an extracellular marker, wherein the extracellular marker is selected from the group consisting of CD81, CD63, CD9, and combinations thereof.

32. 32. The method of claim 30 or 31, wherein the antibody is selected from the group consisting of rituximab, ocrelizumab, ofatumumab, obinutuzumab, and combinations thereof.

33. 33. The method of any one of claims 30 to 32, wherein the target tumor antigen is selected from the group consisting of human CD20 antigen, mouse CD20 antigen, rat CD20 antigen, rabbit CD20 antigen, cynomolgus monkey CD20 antigen, human CD3 antigen, mouse CD3, rat CD3 antigen, rabbit CD3 antigen, cynomolgus monkey CD3 antigen, human FcRH5 antigen, human Ly6G6 antigen, human HER2 antigen, human EGFR antigen, human HER3 antigen, human HER4 antigen, human PSMA antigen, and combinations thereof.

34. The method of any one of claims 30 to 33, wherein the sample is selected from the group consisting of a plasma sample, a serum sample, a tissue culture supernatant sample, and combinations thereof.

35. The method according to any one of claims 30 to 34, wherein the concentration and calibration curve of the circulating tumor antigen is determined using an immunoassay, ELISA and / or Western blot.

36. The method of any one of claims 30 to 35, further comprising detecting the presence of extracellular vesicle markers, wherein the extracellular markers comprise CD81, CD63, and / or CD9.

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