Antibody titer test

Abstract

The present invention provides a cell-based test for measuring antibody titer. A surface-bound antigen is contacted with an antibody, which is then contacted with a reporter cell. Compositions and kits are also contemplated.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 835,960, filed April 18, 2019, the entire contents of which are incorporated herein by reference.

[0002] Submitting a sequence listing as an ASCII text file

[0002] The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (Filename: 146392046740SeqList.txt, Recording Date: April 17, 2020, Size: 1,112 bytes).

[0003]

[0003] The present invention provides methods for analyzing the titer of a polypeptide (eg, an antibody or immunoadhesin). Compositions and kits are also contemplated. [Background technology]

[0004]

[0004] An optimal antibody titer test should be accurate, precise, easy to use, have a fast turnaround time, and be amenable to automation and high-throughput scaling. Several conventional bioassays are available that reflect ADCPs and related mechanisms of action, such as ELISAs for secreted cytokines, PBMC-based methods, and FACS-based methods. Unfortunately, many of these tests have highly variable results and / or are time-consuming. The novel titer test described herein uses a cell-based approach with reporter cells that reflects ADCP activity and can be used to detect antibody-antigen binding interactions.

[0005]

[0005] All references cited herein, including patent applications and patent publications, are incorporated by reference in their entirety. Summary of the Invention

[0006]

[0006] In some aspects, the present invention provides a method for determining the activity of a polypeptide that binds to a target antigen and comprises an Fc receptor binding domain, comprising: a) contacting an immobilized target antigen with a polypeptide preparation to form an antigen-polypeptide complex; and b) contacting the antigen-polypeptide complex with a phagocyte, the phagocyte comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor; wherein expression of the reporter indicates activity of the polypeptide.

[0007] In some aspects, the present invention provides a method for quantifying the titer of a polypeptide preparation in which the polypeptide binds to a target antigen, the method comprising: a) contacting multiple populations of immobilized target antigen with different concentrations of the polypeptide preparation to form antigen-polypeptide complexes; b) contacting these antigen-polypeptide complexes with phagocytes, the phagocytes comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; c) measuring expression of the reporter; and d) measuring the EC of the polypeptide preparation. 50 Determine the EC of the polypeptide preparation 50 and the EC of a standard sample of a polypeptide with known titer. 50 In some embodiments, the method further comprises calculating an EC50-based potency of the polypeptide preparation using a multiparameter logistic fit to the standard. In some embodiments, the multiparameter logistic fit is a three-parameter, four-parameter, or five-parameter logistic fit. In some embodiments, the EC50 of the standard is calculated using a multiparameter logistic fit. 50 is the EC of the polypeptide preparation 50 is determined at the same time.

[0008] In some embodiments of the above aspects, the reporter is luciferase or a fluorescent protein. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the response element responsive to activation by an Fcγ receptor is an NFκB response element, an NFAT response element, an AP-1 response element, or an ERK-responsive transcription factor (e.g., Elk1).

[0009] In some embodiments of the above aspects, the phagocytes are monocytes. In some embodiments, the phagocytes are from a cell line. In some embodiments, the cell line is a THP-1 cell line or a U-937 cell line. In some embodiments, the Fcγ receptor is FcγRI (CD64), FcγRIIa (CD32a), or FcγRIII (CD16). In some embodiments, the phagocytes are engineered to overexpress the Fcγ receptor. In some embodiments, the phagocytes are engineered to overexpress FcγRIIa. In some embodiments, the phagocytes do not express FcγRIII.

[0010] In some embodiments of the above aspects, the target antigen is beta amyloid (Aβ) or CD20. In some embodiments, the target antigen is beta amyloid (Aβ). In some embodiments, the Aβ is human Aβ. In some embodiments, the Aβ comprises monomeric and / or oligomeric Aβ. In some embodiments, the human Aβ is Aβ1-40 or Aβ1-42. In some embodiments, the polypeptide comprises a full-length Fc domain, or an FcR-binding fragment of an Fc domain. In some embodiments, the polypeptide specifically binds to Aβ. In some embodiments, the polypeptide is an antibody or immunoadhesin. In some embodiments, the polypeptide is crenezumab.

[0011] In some embodiments of the above aspects, the target antigen is immobilized on a surface. In some embodiments, the surface is a plate. In some embodiments, the plate is a multiwell plate. In some embodiments, the antigen is immobilized on the surface at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus. In some embodiments, the target antigen is immobilized on the surface using a biotin-streptavidin system. In some embodiments, the target antigen is bound to biotin, and the surface comprises bound streptavidin. In some embodiments, the target antigen is bound to biotin at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus.

[0012]

[0012] In some embodiments of the above aspect, the reporter is detected after any one or more of about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, 24 hours or more than 24 hours have elapsed since the antigen-polypeptide complex was contacted with the phagocyte.

[0013]

[0013] In some aspects, the present invention provides a kit for determining the titer of a polypeptide preparation, wherein the polypeptide binds to a target antigen and comprises an Fc receptor binding domain, the kit comprising an immobilized target antigen and a phagocyte, wherein the phagocyte comprises an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor, and expression of the reporter indicates the titer of the polypeptide.

[0014]

[0014] In some aspects, the present invention provides a kit for quantifying the titer of a polypeptide preparation in which the polypeptide binds to a target antigen and comprises an Fc receptor binding domain, the kit comprising an immobilized target antigen, a phagocyte, and a standard sample, wherein the phagocyte comprises an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor, expression of the reporter indicating the titer of the polypeptide, and the standard sample comprises a preparation of the polypeptide of known titer.

[0015] In some embodiments of the kit, the reporter is luciferase or a fluorescent protein. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the response element responsive to activation by an Fcγ receptor is an NFκB response element, an NFAT response element, an AP-1 response element, or an ERK-responsive transcription factor (e.g., Elk1).

[0016] In some embodiments of the kit, the phagocytes are monocytes. In some embodiments, the phagocytes are from a cell line. In some embodiments, the cell line is a THP-1 cell line or a U-937 cell line. In some embodiments, the Fcγ receptor is FcγRI (CD64), FcγRIIa (CD32a), or FcγRIII (CD16). In some embodiments, the phagocytes are engineered to overexpress the Fcγ receptor. In some embodiments, the phagocytes are engineered to overexpress FcγRIIa. In some embodiments, the phagocytes do not express FcγRIII.

[0017] In some embodiments of the kit, the target antigen is beta amyloid (Aβ) or CD20. In some embodiments, the target antigen is beta amyloid (Aβ). In some embodiments, the Aβ is human Aβ. In some embodiments, the Aβ comprises monomeric and / or oligomeric Aβ. In some embodiments, the human Aβ is Aβ1-40 or Aβ1-42. In some embodiments, the polypeptide comprises a full-length Fc domain or an FcR-binding fragment of an Fc domain. In some embodiments, the polypeptide specifically binds to Aβ. In some embodiments, the polypeptide is an antibody or immunoadhesin. In some embodiments, the polypeptide is crenezumab.

[0018] In some embodiments of the kit, the target antigen is immobilized on a surface. In some embodiments, the surface is a plate. In some embodiments, the plate is a multiwell plate. In some embodiments, the antigen is immobilized on the surface at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus. In some embodiments, the target antigen is immobilized on the surface using a biotin-streptavidin system. In some embodiments, the target antigen is bound to biotin and the surface comprises bound streptavidin. In some embodiments, the target antigen is bound to biotin at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus. In some embodiments, the target antigen is immobilized on the surface using a biotin-streptavidin system. In some embodiments, the target antigen is bound to biotin and the surface comprises bound streptavidin. [Brief explanation of the drawings]

[0019] [Figure 1]

[0018] A map showing the construction of a CD32A expression vector. [Figure 2]

[0019] A map showing the construction of an NF-κB-luciferase expression vector. [Figure 3]

[0020] Figure 1 shows the expression of FcγR in phagocytosis reporter cells. Figure 2 shows the expression of CD16, CD32, and CD64 in parental U-937 cells, U-937 phagocytosis reporter cells, and THP-1 phagocytosis reporter cells. The shaded histograms represent unstained cells (contained only in U-937 cells), the solid line represents CD16 / CD32 / CD64, and the dashed line represents the isotype control. U937 and THP-1 cells were examined on different days using different instruments. [Figure 4A-C]

[0021] Figures 4A-C show the evaluation of different formats for incorporating Aβ peptides. THP-1 phagocytosis reporter cells (THP-1) were screened for activity using crenezumab and different forms of Aβ and test plates. Figure 4A shows soluble, non-biotinylated Aβ incubated with crenezumab and THP-1 cells. Figure 4B shows non-biotinylated Aβ adsorbed to a high-binding plate, then incubated with a dilution series of crenezumab and subsequently incubated with cells. Figure 4C compares the high-binding plate adsorbed with Aβ peptides with a streptavidin (SA) high-binding plate loaded with biotin-Aβ. An SA high-binding plate without Aβ was used as a negative control. In Figures 4A and 4B, different clones ("XXX strains") were evaluated. In Figure 4C, the THP-1 416 strain was utilized. [Figure 5]

[0022] FIG. 1 is a schematic diagram of the potency test. [Figure 6]

[0023] A representative calibration curve for crenezumab is shown. [Figure 7]

[0024] Figure 1 shows ocrelizumab activity in a phagocytosis reporter cell assay. A representative standard curve is presented showing the ability of ocrelizumab to activate U-937 phagocytosis reporter cells upon binding to CD20 peptide, as measured by luciferase reporter gene expression. [Figure 8]

[0025] Growth of THP-1 cells at different seeding densities is shown. Cells were seeded for a 3-day culture period and monitored using an Incucyte Zoom. Numbers represent seeding density x 10 cells / ml. [Figure 9]

[0026] Dose response of THP-1 clones to recombinant and synthetic Aβ is shown. Endotoxin testing of recombinant Aβ revealed bacterial lipopolysaccharide (LPS) levels of 912 EU / mg, while the synthetic peptide was below the detection limit. [Figure 10]

[0027] Factors that influence the EC50 are shown. [Figure 11]

[0028] The factors that affect the slope are shown. [Figure 12]

[0029] Factors that influence the response fold are shown. [Figure 13]

[0030] Factors affecting titers (mean and standard deviation) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0020]

[0031] In some embodiments, the invention provides a method for determining the activity of a polypeptide that binds to a target antigen and comprises an Fc receptor binding domain, comprising: a) contacting an immobilized target antigen with a polypeptide preparation to form an antigen-polypeptide complex; b) contacting the antigen-polypeptide complex with a phagocyte, the phagocyte comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; expression of the reporter indicates activity of the polypeptide. In some embodiments, the invention provides a method for quantifying the titer of a polypeptide preparation in which a polypeptide binds to a target antigen, comprising: a) contacting multiple populations of immobilized target antigen with different concentrations of the polypeptide preparation to form antigen-polypeptide complexes; b) contacting these antigen-polypeptide complexes with phagocytes, the phagocyte comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; c) measuring expression of the reporter; and d) measuring the EC of the polypeptide preparation. 50 Determine the EC of the polypeptide preparation 50 and the EC of a standard sample of a polypeptide with known titer. 50 In some embodiments, the polypeptide is an antibody or immunoadhesin. Compositions and kits are also provided.

[0021] definition

[0032] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, either naturally or by intervention; examples include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety or a toxin. Included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. The terms "polypeptide" and "protein," as used herein, specifically encompass antibodies.

[0022]

[0033] A "purified" polypeptide (e.g., an antibody or immunoadhesin) refers to an increase in the purity of the polypeptide, and is present in a more pure form than when originally synthesized and / or amplified in its natural environment or under laboratory conditions. Purity is a relative term and does not necessarily imply absolute purity.

[0023]

[0034] The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a native polypeptide. Similarly, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a native polypeptide. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of the native polypeptide, and the like. Methods for identifying agonists or antagonists of a polypeptide can include contacting the polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.

[0024]

[0035] A polypeptide that "binds" to an antigen of interest is one that binds to the antigen with sufficient affinity so that it is useful as a diagnostic and / or therapeutic agent in targeting cells or tissues that express the antigen, and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to "non-target" polypeptides will be less than about 10% of the binding of the polypeptide to its particular target polypeptide, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0025]

[0036] With respect to the binding of a polypeptide to a target molecule, the terms "specific binding" to or "specifically binding to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule in comparison with the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. Specific binding can be determined, for example, by competition between the target and a similar control molecule, such as excess unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

[0026]

[0037] The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies containing TDB), and antibody fragments so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0027]

[0038] Antibodies are naturally occurring immunoglobulin molecules with a variety of structures, all based on the immunoglobulin fold. For example, IgG antibodies have two "heavy" chains and two "light" chains that are disulfide-bonded to form a functional antibody. Each heavy and light chain itself contains a "constant" (C) and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and functions in non-antigen-specific interactions with immune effectors. The antigen-binding specificity of an antibody or antigen-binding fragment of an antibody refers to the ability of the antibody to specifically bind to a particular antigen.

[0028]

[0039] The antigen-binding specificity of an antibody is determined by structural features of the V regions. The variability is not uniformly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches of 15–30 amino acids called framework regions (FRs), separated by shorter, highly variable regions called "hypervariable regions" (HVRs), each 9–12 amino acids long. Naturally occurring heavy and light chain variable domains each contain four FRs that largely adopt a β-sheet structure, connected by three hypervariable regions that form loops that connect (and in some cases form part of) the β-sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., (1991)). The constant domains are not directly involved in binding the antibody to an antigen but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0029]

[0040] Each V region typically contains three HVRs, e.g., complementarity determining regions ("CDRs," each containing a "hypervariable loop"), and four framework regions. Thus, an antigen-binding site is the minimum structural unit required to bind to a specific desired antigen with sufficient affinity, and usually contains three CDRs and at least three, preferably four, framework regions interposed therebetween that hold and present the CDRs in a proper conformation. Classical four-chain antibodies are composed of V H Domains and V L The domains cooperate to define the antigen-binding site. Certain antibodies, such as camel and shark antibodies, lack light chains and rely on binding sites formed exclusively by heavy chains. H and V L It is possible to prepare single domain engineered immunoglobulins in which the binding site is formed by only the heavy or light chain, without cooperation between the heavy and light chains.

[0030]

[0041] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain four FRs that largely adopt a β-sheet structure, connected by three hypervariable regions that form loops that connect (and in some cases form part of) the β-sheet structure. The hypervariable regions in each chain are held in close proximity to each other by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0031]

[0042] The term "hypervariable region" (HVR), when used herein, refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region comprises amino acid residues of the "complementarity determining regions" or "CDRs" (e.g., V L In the case of V, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) are located around V. HIn the case of H1, the amino acid residues around 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)) and / or those in the "hypervariable loops" (e.g., V L In , residues 26–32 (L1), 50–52 (L2), and 91–96 (L3), and V H Examples of such sequences include 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0032]

[0043] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0033]

[0044] "Antibody fragments" include portions of intact antibodies, preferably including the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDbs); linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); one-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., but not limited to, Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv); and bispecific T-cell engagers (BiTEs).

[0034]

[0045] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0035]

[0046] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to form the V H -V L The antigen-binding site is formed on the surface of the dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0036]

[0047] Fab fragments also contain the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues in the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0037]

[0048] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0038]

[0049] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to these different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0039]

[0050] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V of an antibody. H Domain and V L In some embodiments, the scFv polypeptide comprises a V domain, and these domains are present in a single polypeptide chain. H Domains and V L The scFv further comprises a polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plueckthun, "The Pharmacology of Monoclonal Antibodies," vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0040]

[0051] The term "diabody" refers to a group of identical polypeptide chains (V H -V L ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V HDiabodies refer to small antibody fragments with two antigen-binding sites, each containing a 2-nucleotide polynucleotide (nucleotide) linked to a nucleotide sequence. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in further detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0041]

[0052] The term "multispecific antibody" is used in the broadest sense and specifically includes antibodies with polyepitopic specificity. Such multispecific antibodies include, but are not limited to, V H V L The heavy chain variable domain (V H ) and the light chain variable domain (V L ) antibodies containing each V H V L Two or more V units that bind to different epitopes L and V H These include antibodies with domains, antibodies with two or more single variable domains where each single variable domain binds a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies, and covalently linked or covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Monospecificity" refers to the ability to bind to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

[0042]

[0053] The phrase "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) is capable of conferring antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as recombinantly derived from human and mouse antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trends Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett (1994) 339:285-290; WO 00 / 29004; WO 02 / 051870).

[0043]

[0054] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may arise during monoclonal antibody production (typically, such variants are present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the methods provided herein may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0044]

[0055] As used herein, monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies containing variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey, such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780).

[0045]

[0056] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies have hypervariable region residues of a human immunoglobulin (recipient antibody) replaced by hypervariable region residues of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence, except for the FR substitution(s) noted above. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0046]

[0057] As used herein, an "intact antibody" comprises heavy and light variable domains, as well as an Fc region. The constant domains are native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0047]

[0058] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V) at one end. H ) followed by multiple constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at the other end. The light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0048]

[0059] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0049]

[0060] As used herein, the term "effector function" or "Fc-mediated effector function" refers to a biological activity attributable to the Fc region of an antibody (a native-sequence Fc region or an amino acid sequence variant Fc region) and varies depending on the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion.

[0050]

[0061] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells, subsequently causing lysis of the target cell. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such studies include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0051]

[0062] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. In some embodiments, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred.

[0052]

[0063] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., a polypeptide (e.g., an antibody)) complexed with a cognate antigen. To assess complement activation, a CDC test, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0053]

[0064] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to the process by which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, or dendritic cells) that bind to the Fc region of immunoglobulin.

[0054]

[0065] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native-sequence human FcR. Furthermore, preferred FcRs are those that bind IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. This term also includes the neonatal receptor, FcRn, 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)).

[0055]

[0066] The term "Aβ(XY)" as used herein refers to the amino acid sequence from amino acid position X to amino acid position Y of human amyloid β protein, comprising: X and Y both refer to the amino acid sequence from amino acid position X to amino acid position Y of the amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 1), or any naturally occurring variant thereof, particularly one having at least one mutation selected from the group consisting of A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T, and A42V, where the number refers to the start of the Aβ peptide, including both positions X and Y, or a sequence with up to three additional amino acid substitutions, none of which prevent globulomer formation. "Additional" amino acid substitutions are defined herein as deviations from the canonical sequence that do not occur in nature.

[0056]

[0067] More specifically, the term "Aβ(1-42)" as used herein refers to the amino acid sequence from amino acid position 1 to amino acid position 42 of human amyloid β protein, inclusive of both 1 and 42, and in particular refers to the amino acid sequence from amino acid position 1 to amino acid position 42 of the amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 1) (corresponding to amino acid positions 1 to 42) or a naturally occurring variant thereof. Such variants refer to those having at least one mutation selected from the group consisting of, for example, A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T, and A42V, where the numbers refer to the start of the Aβ peptide, including both amino acid positions 1 and 42, or a sequence with up to three additional amino acid substitutions, none of which prevent globulomer formation. Similarly, the term "Aβ(1-40)" herein refers to the amino acid sequence from amino acid position 1 to amino acid position 40 of human amyloid β protein, including both amino acid positions 1 and 40, and particularly refers to the amino acid sequence from amino acid position 1 to amino acid position 40 of the amino acid sequence DAEFRHDSGYEVHHQKLVFF AEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 2) or any naturally occurring variant thereof. Such variants refer to those having at least one mutation selected from the group consisting of, for example, A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), and D23N ("Iowa"), where the numbers refer to the start of the Aβ peptide including both amino acid positions 1 and 40, or a sequence with up to three additional amino acid substitutions, none of which prevent globulomer formation.

[0057]

[0068] "Contaminants" refer to substances that differ from the desired polypeptide product. In some embodiments of the invention, contaminants include charge variants of polypeptides. In some embodiments of the invention, contaminants include charge variants of antibodies or antibody fragments. In other embodiments of the invention, contaminants include, but are not limited to: host cell material, such as CHOP; leached Protein A; nucleic acids; variants, fragments, aggregates, or derivatives of the desired polypeptide; other polypeptides; endotoxins; viral contaminants; cell culture media components, etc. In some examples, contaminants may be host cell proteins (HCPs) derived from, for example, but not limited to, bacterial cells (such as E. coli cells), insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, or fungal cells.

[0058]

[0069] As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous polypeptide with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity (i.e., "heterologous") other than the antigen recognition and binding site of an antibody, with an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be derived from any immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

[0059]

[0070] As used herein, "reporter molecule" refers to a molecule that, by its chemical nature, provides an analytically distinguishable signal that allows for the detection of antibody activity. The most commonly used reporter molecules in this type of assay are enzymes, fluorophores, or radionuclide-containing molecules (i.e., radioisotopes), and chemiluminescent molecules.

[0060]

[0071] As used herein, "essentially the same" refers to a value or parameter that has not changed significantly. For example, the chromatographic mobile phase at the column outlet is essentially the same as the initial ionic strength of the mobile phase if the ionic strength has not changed significantly. For example, the ionic strength at the column outlet within 10%, 5%, or 1% of the initial ionic strength is essentially the same as the initial ionic strength.

[0061]

[0072] Reference herein to a value or parameter as "about" includes (and describes) variations on that value or parameter per se. For example, a reference to "about X" includes a description of "X."

[0062]

[0073] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Aspects and variations of the invention described herein include those "consisting of" and / or "consisting essentially of."

[0063] Cell-based potency assay

[0074] The present invention provides a cell-based assay for determining the activity or potency of a polypeptide preparation, wherein the polypeptide comprises an antigen-binding domain and an Fc receptor-binding domain. The antigen-binding domain of the polypeptide binds to an immobilized antigen and is then contacted with a phagocyte containing an Fc receptor. Upon binding of the Fc receptor to the Fc domain of the polypeptide, a reporter is activated. The reporter activity (correlated with reporter expression) is compared to the reporter activity activated by a polypeptide of known activity or potency. In some embodiments, the polypeptide is an antibody or immunoadhesin. Cell-based assays are useful, among other things, for detecting a polypeptide in a composition, quantifying the amount of a polypeptide in a composition, determining the specificity of a polypeptide in a composition, and / or determining the potency of a polypeptide composition.

[0064] Reporter

[0075] Reporter assays allow for the biological characterization of a given stimulus by monitoring the induction of reporter expression within cells. The stimulus induces an intracellular signaling pathway, resulting in a cellular response that typically involves the regulation of gene transcription. In some instances, stimulation of an intracellular signaling pathway regulates gene expression through the regulation and recruitment of transcription factors to upstream non-coding regions of DNA required for the initiation of RNA transcription leading to protein production. Regulation of gene transcription and translation in response to a stimulus is necessary to elicit most biological responses, such as cell proliferation, differentiation, survival, and immune response. These non-coding regions of DNA, also known as response elements, contain specific sequences that are recognition sequences for transcription factors that regulate the efficiency of gene transcription and, therefore, the amount and type of protein produced by the cell in response to the stimulus. In reporter assays, standard molecular biology techniques are used to engineer response elements and minimal promoters that respond to a given stimulus to drive reporter gene expression. This DNA is then transfected or transduced into cells that contain all the machinery required to specifically respond to the stimulus, and the level of reporter gene transcription, translation, or activity is measured as a surrogate measure of the biological response.

[0065]

[0076] In some embodiments, the invention provides methods for determining the activity of a polypeptide preparation, wherein the polypeptide binds to a target antigen and comprises an Fc receptor binding domain (e.g., an Fcγ receptor binding domain), comprising: a) contacting an immobilized target antigen with the polypeptide preparation to form an antigen-polypeptide complex; and b) contacting the antigen-polypeptide complex with a phagocyte, wherein the phagocyte comprises an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; wherein expression of the reporter indicates activity of the polypeptide. In some embodiments, the invention provides a method for quantifying the titer of a polypeptide preparation, wherein the polypeptide binds to a target antigen and comprises an Fc receptor binding domain (e.g., an Fcγ receptor binding domain), comprising: a) contacting multiple populations of immobilized target antigen with different concentrations of the polypeptide preparation to form antigen-polypeptide complexes; b) contacting these antigen-polypeptide complexes with phagocytes, wherein the phagocytes comprise an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; c) measuring expression of the reporter; and d) measuring the EC of the polypeptide preparation. 50 Determine the EC of the polypeptide preparation 50 and the EC of a standard sample of a polypeptide with known titer. 50In some embodiments, the polypeptide is an antibody or immunoadhesin. The reporter can be any molecule for which a test can be developed to measure the amount of the molecule produced by a cell in response to a stimulus. For example, the reporter can be a reporter protein encoded by a reporter gene that responds to a stimulus (e.g., binding of the polypeptide to an Fc receptor). Common examples of reporter molecules include, but are not limited to, photoproteins such as luciferase, which emit light that can be measured experimentally as a by-product of catalytic action of a substrate. Luciferase is a type of photoprotein available from many sources, including firefly luciferase (from the species Photinus pyralis), Renilla luciferase (from the sea pansy Renilla reniformis), click beetle luciferase (from Pyrearinus termitilluminans), Gaussia luciferase (from the marine copepod Gaussia princeps), and nanoluciferase (from the deep-sea shrimp Oplophorus gracilirostris). Firefly luciferase catalyzes the oxygenation of luciferin to oxyluciferin, while other luciferases, such as those from Renilla luciferase, catalyze the oxygenation of coelenterazine. The wavelengths of light emitted by different luciferase forms and variants can be read using different filter systems, facilitating multiplexing. The amount of luminescence is proportional to the amount of luciferase expressed in the cells, and the luciferase gene has been used as a highly sensitive reporter to quantitatively evaluate the effect of stimuli that induce biological responses.Reporter gene assays have been used for many years for a wide range of purposes, including basic research, HTS screening, and titer determinations (Brogan J, et al., 2012, Radiat Res. 177(4):508-513; Miraglia LJ, et al., 2011, Comb Chem High Throughput Screen. 14(8):648-657; Nakajima Y, and Ohmiya Y. 2010, Expert Opin Drug Discovery, 5(9):835-849; Parekh BS, et al., 2012, Mabs, 4(3):310-318; Svobodova K, and Cajtham L T., 2010, Appl Microbiol Biotechnol., 88(4): 839-847).

[0066]

[0077] In some embodiments, the present invention provides a cell-based assay for determining the activity and / or potency of a polypeptide, in which a polypeptide-antigen complex is contacted with an engineered phagocyte containing a reporter complex. In some embodiments, the reporter construct comprises a luciferase. In some embodiments, the luciferase is firefly luciferase (e.g., from Photinus pyralis), Renilla luciferase from sea pansy (e.g., from Renilla reniformis), click beetle luciferase (e.g., from Pyrearinus termitilluminans), marine copepod Gaussia luciferase (e.g., from Gaussia princeps), or deep-sea shrimp nanoluciferase (e.g., from Oplophorus gracilirostris). In some embodiments, expression of luciferase in the engineered phagocyte indicates the binding activity of the polypeptide or immunoadhesin to the phagocyte. In other aspects, the reporter construct encodes β-glucuronidase (GUS); a fluorescent protein or variant thereof, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP); chloramphenicol acetyltransferase (CAT); β-galactosidase; β-lactamase; or secreted alkaline phosphatase (SEAP).

[0067]

[0078] In some embodiments, engineered cells are provided that contain a nucleic acid encoding a reporter molecule (e.g., a reporter protein such as luciferase), operably linked to a regulatory sequence comprising a promoter and / or sequence that responds to Fc binding of an Fc domain to Fc on the surface of the cell. The promoter and / or sequence can be selected from those known in the art to respond to FcR activation. In some embodiments, the nucleic acid is stably integrated into the cell genome.

[0068]

[0079] In some embodiments, engineered cells (e.g., phagocytes) are provided that contain a nucleic acid encoding a reporter molecule under the control of a minimal promoter operably linked to one or more FcR activation response elements. In some embodiments, the minimal promoter is a thymidine kinase (TK) minimal promoter, a cytomegalovirus (CMV)-derived minimal promoter, an SV40-derived promoter, or an elongation factor 1 alpha (EF1α) minimal promoter. In some embodiments, the minimal promoter is a minimal TK promoter. In some embodiments, the minimal promoter is a minimal CMV promoter. In some embodiments, the activation response element comprises an NFAT (nuclear factor of activated T cells) response element, an AP-1 (Fos / Jun) response element, an NFAT / AP1 response element, an NFκB response element, a FOXO response element, a STAT3 response element, a STAT5 response element, or an IRF response element. In some embodiments, the FcR activation response element is arranged as tandem repeats (e.g., about 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). The FcR activation response element may be located 5' or 3' relative to the reporter coding sequence. In some embodiments, the FcR activation response element is located 5' from the minimal promoter. In some embodiments, the FcR activation response element is an NFκB response element. In some embodiments, the reporter molecule is a luciferase, such as firefly or Renilla luciferase. In some embodiments, the nucleic acid is stably integrated into the macrophage genome.

[0069] cell

[0080] In some embodiments, methods are provided for determining the activity and / or titer of a polypeptide preparation, wherein the polypeptide comprises an antigen-binding domain and an Fc receptor-binding domain (e.g., an FcγR-binding domain), by contacting the polypeptide-antigen complex with a population of cells comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor. In some embodiments, the cells are phagocytes. In some embodiments, the phagocytes are monocytes. In some embodiments, the phagocytes are from a cell line. In some embodiments, the phagocyte line is a THP-1 cell line or a U-937 cell line.

[0070]

[0081] In some embodiments, the reporter cells comprise an Fc receptor. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fcγ receptor is FcγRI (CD64), FcγRIIa (CD32a), and / or FcγRIII (CD16). In some embodiments, the reporter cells are engineered to express one or more of FcγRI (CD64), FcγRIIa (CD32a), or FcγRIII (CD16). In some embodiments, the reporter cells are engineered to overexpress one or more of FcγRI (CD64), FcγRIIa (CD32a), or FcγRIII (CD16). In some embodiments, the reporter cells are engineered to overexpress FcγRIIa. In some embodiments, the reporter cells do not express FcγRIII.

[0071]

[0082] In some embodiments, the reporter cell comprises a nucleic acid encoding a reporter operably linked to a response element responsive to activation by an Fcγ receptor. In some embodiments, the reporter comprises a polynucleotide encoding a luciferase. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the polynucleotide encoding the reporter (e.g., luciferase) is operably linked to an FcR activation-responsive regulatory sequence (e.g., an FcR activation-responsive promoter and / or sequence). In some embodiments, the promoter and / or sequence responsive to FcR activation is an NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter, or IRF promoter. In some embodiments, the reporter cell comprises a nucleic acid encoding a reporter operably linked to a response element responsive to activation by an Fcγ receptor, and comprises one or more of FcγRI, FcγRIIa, or FcγRIII.

[0072]

[0083] In some embodiments, the present invention provides compositions of cells engineered with an FcR activation reporter construct encoding a reporter molecule operably linked to a regulatory sequence comprising a promoter and / or sequence responsive to FcR activation. In some embodiments, the present invention provides compositions of cells engineered with an FcγR activation reporter construct encoding a reporter molecule operably linked to a regulatory sequence comprising a promoter and / or sequence responsive to FcγR activation. In some embodiments, the reporter molecule is luciferase, a fluorescent protein (e.g., GFP, YFP, etc.), alkaline phosphatase, or beta-galactosidase. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the promoter and / or sequence responsive to FcR (e.g., FcγR) activation is an NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter, or IRF promoter. In some embodiments, the sequence responsive to FcR signaling comprises an NFκB sequence.

[0073]

[0084] In some embodiments, the reporter cell is a phagocyte comprising one or more Fc receptors and further comprising a nucleic acid encoding a reporter under the control of a promoter and / or sequence activated by FcR signaling. In some embodiments, the reporter cell is a monocyte comprising one or more Fc receptors and further comprising a nucleic acid encoding a reporter under the control of a promoter and / or sequence activated by FcR signaling. In some embodiments, the reporter cell is a monocyte comprising one or more Fc receptors and further comprising a nucleic acid encoding a reporter under the control of a promoter and / or sequence activated by FcR signaling. In some embodiments, the reporter cell is a monocyte comprising one or more Fc receptors and further comprising a nucleic acid encoding a luciferase reporter under the control of an NF-κB promoter. In some embodiments, the reporter cell is a monocyte comprising one or more Fc receptors and further comprising a nucleic acid encoding a luciferase reporter under the control of an NF-κB promoter. In some embodiments, the reporter cells are THP-1 cells comprising FcγRI, FcγRIIa and / or FcγRIII and further comprising a nucleic acid encoding a luciferase reporter under the control of an NF-κB promoter. In some embodiments, the reporter cells are U-937 cells comprising FcγRI, FcγRIIa and / or FcγRIII and further comprising a nucleic acid encoding a luciferase reporter under the control of an NF-κB promoter.

[0074] Antibody activity or titer testing

[0085] In some embodiments, the present invention provides methods for determining the activity or titer of a polypeptide preparation, wherein the polypeptide comprises an antigen-binding domain and an Fc receptor-binding domain. In some embodiments, the method comprises contacting the polypeptide preparation with an immobilized antigen and then contacting the immobilized antigen-polypeptide complex with a population of cells comprising an Fc receptor and a nucleic acid encoding a reporter operably linked to a promoter and / or sequence responsive to Fc receptor activation. Expression of the reporter indicates the activity or titer of the polypeptide preparation. In some embodiments, the polypeptide is an antibody or immunoadhesin. In some embodiments, the reporter is luciferase, a fluorescent protein, alkaline phosphatase, beta-lactamase, or beta-galactosidase. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the promoter and / or sequence responsive to monocyte activation is an NFAT promoter, an AP-1 promoter, or an NFκB promoter. In some embodiments, the promoter and / or sequence responsive to Fc receptor activation comprises an Fc receptor activation-responsive sequence from any one or more of NFAT, AP-1, and NFκB. In some embodiments, the reporter cell is a phagocyte. In some embodiments, the reporter cell is a monocyte. In some embodiments, the reporter cell is from a cell line. In some embodiments, the cell line is a THP-1 cell line or a U-937 cell line. In some embodiments, the target antigen is beta amyloid (Aβ) or CD-20. In some embodiments, the Aβ is human Aβ. In some embodiments, the Aβ comprises monomeric and / or oligomeric Aβ. In some embodiments of the invention, the ratio of monomeric to oligomeric Aβ is about any of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9; or 1:10.In some embodiments, the human Aβ is Aβ1-40 or Aβ1-42. In some embodiments, the polypeptide is crenezumab.

[0075]

[0086] In some embodiments of the present invention, the antigen is immobilized on a surface. In some embodiments, the surface is a plate. In some embodiments, the surface is a plate with wells. In some embodiments, the surface is a plate with approximately 96, 182, 288, 384, 480, 576, or 672 wells. In some embodiments, the antigen is immobilized on the surface by adhesion. In some embodiments, the antigen is immobilized on the surface using the streptavidin-biotin system. In some embodiments, streptavidin is linked to the surface, biotin is linked to the antigen, and the antigen is then immobilized due to the high affinity of biotin for streptavidin. In some embodiments, the surface is a streptavidin-coated plate (e.g., a commercially available streptavidin-coated plate). In some embodiments, the surface is a streptavidin-coated 96-well plate.

[0076]

[0087] In some embodiments, the antigen is immobilized on the surface at or near its N-terminus. In some embodiments, the antigen is immobilized on the surface at or near its C-terminus. In some embodiments, the antigen is immobilized on the surface at or near its N-terminus and at or near its C-terminus, so that the antigen is oriented in opposite directions on the surface. In some embodiments, the antigen is immobilized on the surface at or near its N-terminus and at or near its C-terminus, so that the antigen forms a loop on the surface. In some embodiments, streptavidin is linked to the surface, the antigen contains biotin at its N-terminus, and the biotin binds to the streptavidin to immobilize the antigen at its N-terminus. In some embodiments, streptavidin is linked to the surface, the antigen contains biotin at its C-terminus, and the biotin binds to the streptavidin to immobilize the antigen at its C-terminus. In some embodiments, streptavidin is linked to the surface, and the antigen contains biotin at its N-terminus and C-terminus, so that the antigen is oriented in opposite directions on the surface. In some embodiments, streptavidin is linked to the surface and the antigen contains biotin at its N-terminus and its C-terminus, and both biotin moieties bind to the streptavidin, immobilizing the antigen by its N-terminus and C-terminus such that the antigen forms a loop on the surface.

[0077]

[0088] In some embodiments, the antigen is conjugated with biotin to form a biotinylated antigen. ... Biotinylated antigen at a concentration less than any of 5.0 μg / mL, 5.5 μg / mL, 6.0 μg / mL, 6.5 μg / mL, 7.0 μg / mL, 7.5 μg / mL, 8.0 μg / mL, 8.5 μg / mL, 9.0 μg / mL, 9.5 μg / mL, 10 μg / mL, 25 μg / mL or 50 μg / mL is contacted with a streptavidin-coated surface. In some embodiments, the biotinylated antigen is contacted with a streptavidin-coated multiwell plate to which each well has been added about 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, or more than 1.0 μg, or any value therebetween, of biotinylated antigen.

[0078]

[0089] In some embodiments, the immobilized antigen is administered at a concentration of about 0.01 ng / mL to about 30,000 ng / mL, about 0.01 ng / mL to about 20,000 ng / mL, about 0.01 ng / mL to about 10,000 ng / mL, about 0.05 ng / mL to about 10,000 ng / mL, about 0.1 ng / mL to about 10,000 ng / mL, about 0.5 ng / mL to about 10,000 ng / mL, about 1 ng / mL to about 10,000 ng / mL, about 5 ng / mL to about 10,000 ng / mL, about 10 ng / mL to about 10,000 ng / mL, about 0.01 ng / mL to about 5000 ng / mL, about 0.01 ng / mL to about 4000 ng / mL, about 0.01 ng / mL to about The subject is contacted with a composition comprising a polypeptide at a concentration within any of the following ranges: 3000 ng / mL, about 0.01 ng / mL to about 2000 ng / mL, about 0.01 ng / mL to about 1000 ng / mL, about 0.01 ng / mL to about 500 ng / mL, about 0.01 ng / mL to about 100 ng / mL, about 0.01 ng / mL to about 50 ng / mL, about 0.01 ng / mL to about 10 ng / mL, about 0.01 ng / mL to about 5 ng / mL, about 0.1 ng / mL to about 1000 ng / mL, about 0.5 ng / mL to about 1000 ng / mL, about 1 ng / mL to about 100 ng / mL, about 1 ng / mL to about 1000 ng / mL, or about 5 ng / mL to about 5000 ng / mL.

[0079]

[0090] In some embodiments, the immobilized antigen-polypeptide complex is contacted with reporter cells. In some embodiments, the immobilized antigen-polypeptide complex is contacted with about 1 x 10 4 , 5×10 4 , 7.5×10 4 , 1×10 5 , 1.25×10 5 , 1.5×10 5 , 1.75×10 5 , 2 × 10 5 , 2.25×10 5 , 2.5×10 5 , 2.75×10 5 , 3×10 5 , 3.25×10 5 , 3.5×10 5 , 3.75×10 5 , 4×105 , 4.25×10 5 , 4.5×10 5 , 4.75×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6,5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 , 9.5×10 5 , 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , or 5 x 10 6 In some embodiments, the immobilized antigen-polypeptide complex is contacted with about 1 x 10 reporter cells. 4 From 5 x 10 6 Up to 5 x 10 4 From 1×10 6 Up to 1 x 10 5 From 1×10 6 Up to 1 x 10 5 From 2 x 10 5 Up to 2 x 10 5 From 3 x 10 5 Up to 3 x 10 5 From 4×10 5 Up to 4 x 10 5 From 5 x 10 5 Up to 5 x 10 5 From 6 x 10 5 Up to 6 x 10 5 From 7 x 10 5 Up to 7 x 10 5 From 8 x 10 5 Up to 8 x 10 5 From 9 x 10 5 up to, or 9 x 10 5 From 1×10 6 In some embodiments, the immobilized antigen-polypeptide complex is contacted with up to about 1 x 10 reporter cells. 5 cells / ml, 2×10 5 cells / ml, 3×10 5cells / ml, 4×10 5 cells / ml, 5×10 5 cells / ml, 6×10 5 cells / ml, 7×10 5 cells / ml, 8×10 5 cells / ml, 9×10 5 cells / ml, 1×10 6 cells / ml, 2×10 6 cells / ml, 2.5×10 6 cells / ml, 3×10 6 cells / ml, 4×10 6 cells / ml, 5×10 6 cells / ml, 6×10 6 cells / ml, 7×10 6 cells / ml, 7.5×10 6 cells / ml, 8×10 6 cells / ml, 9×10 6 cells / ml, or 1 x 10 7 In some embodiments, the immobilized antigen-polypeptide complex is contacted with reporter cells at a concentration of less than about 1 x 10 cells / ml. 5 cells / ml to 1 x 10 7 cells / ml up to 1 x 10 5 cells / ml to 1 x 10 6 cells / ml up to 5 x 10 5 cells / ml to 5 x 10 6 cells / ml or up to 1 x 10 6 cells / ml to 1 x 10 7 The cells are contacted with reporter cells at a concentration anywhere from 1000 to 1000 cells / ml.

[0080]

[0091] In some embodiments, the reporter is detected after any of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 24 hours, 28 hours, 30 hours, or 36 hours or more after contacting the immobilized antigen-polypeptide complex with the reporter cell. In some embodiments, the reporter is detected between about 1 hour and about 36 hours, about 1 hour and about 24 hours, about 1 hour and about 12 hours, about 1 hour and about 8 hours, about 1 hour and about 6 hours, about 1 hour and about 4 hours, about 1 hour and about 2 hours, about 4 hours and about 24 hours, about 4 hours and about 12 hours, about 4 hours and about 8 hours, about 8 hours and about 24 hours, about 8 hours and about 12 hours, about 16 hours and about 24 hours, about 16 hours and about 20 hours, or about 20 hours and about 24 hours after contacting the immobilized antigen-polypeptide complex with the reporter cell.

[0081]

[0092] In some embodiments, the invention provides a method for quantifying the titer of a polypeptide preparation in which the polypeptide binds to a target antigen, the method comprising: a) contacting multiple populations of immobilized target antigen with different concentrations of the polypeptide preparation to form antigen-polypeptide complexes; b) contacting these antigen-polypeptide complexes with phagocytes, the phagocytes comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; c) measuring expression of the reporter; and d) measuring the EC of the polypeptide preparation. 50 Determine the EC of the polypeptide preparation 50 and the EC of a standard sample of a polypeptide with known titer. 50and comparing the reporter activity of a polypeptide preparation with the activity of a reporter gene. In some embodiments, the polypeptide is an antibody or immunoadhesin. In some embodiments, the reporter is luciferase, a fluorescent protein, alkaline phosphatase, beta-lactamase, or beta-galactosidase. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the promoter and / or sequence responsive to Fc receptor activation (e.g., Fcγ receptor activation) comprises an Fc receptor activation-responsive sequence for any one or more of NFAT, AP-1, or NFκB. In some embodiments, the reporter cell is a phagocyte. In some embodiments, the reporter cell is a phagocyte. In some embodiments, the reporter cell is a monocyte. In some embodiments, the reporter cell is from a cell line. In some embodiments, the cell line is a THP-1 cell line or a U-937 cell line. In some embodiments, the target antigen is beta amyloid (Aβ) or CD-20. In some embodiments, the Aβ is human Aβ. In some embodiments, the Aβ comprises monomeric and / or oligomeric Aβ. In some embodiments, the human Aβ is Aβ1-40 or Aβ1-42. In some embodiments, the polypeptide is crenezumab.

[0082]

[0093] In some embodiments, the EC of the polypeptide preparation 50 is compared with the EC of a polypeptide preparation of known activity or potency (e.g., a standard or standard preparation). 50 As used herein, EC 50 refers to the concentration of a polypeptide that elicits a response halfway between the baseline and maximum after a specified exposure time. In some embodiments, the EC50 of a polypeptide preparation of known activity or potency is determined by generating a standard curve of reporter activity after contacting immobilized antigen-standard polypeptide complexes with reporter cells. 50is determined. In some embodiments, the calibration curve is generated by contacting a population of cells with a standard polypeptide preparation at multiple concentrations ranging from about 0.01 ng / mL to about 30,000 ng / mL. In some embodiments, the calibration curve is generated by contacting a population of cells with a standard polypeptide preparation at multiple concentrations ranging from about 0.01 ng / mL to about 10,000 ng / mL. In some embodiments, the calibration curve is generated by contacting a population of cells with a standard polypeptide preparation at multiple concentrations ranging from about 0.01 ng / mL to about 15,000 ng / mL. In some embodiments, the calibration curve is generated by contacting a population of cells with a standard polypeptide preparation at multiple concentrations ranging from about 0.01 ng / mL to about 5,000 ng / mL. In some embodiments, the multiple concentrations of the standard polypeptide preparation include any one of about 0.01 ng / ml, 0.1 ng / ml, 1 ng / ml, 10 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 500 ng / ml, 750 ng / ml, 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 250 μg / ml, or 500 μg / ml. In some embodiments, the multiple concentrations of the standard polypeptide preparation include any one of about 10 μg / ml, 40 μg / ml, 100 μg / ml, 250 μg / ml, 750 μg / ml, 1000 μg / ml, 1600 μg / ml, 4000 μg / ml, or 10000 μg / ml. In some embodiments, the plurality of concentrations of the standard polypeptide preparation is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 concentrations.

[0083]

[0094] In some embodiments, the reporter is detected after any of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 26 hours, 28 hours, 30 hours, or 36 hours or more after contacting the cells with the composition. In some embodiments, the reporter is detected between about 1 hour and about 24 hours, between about 1 hour and about 12 hours, between about 1 hour and about 8 hours, between about 1 hour and about 6 hours, between about 1 hour and about 4 hours, between about 1 hour and about 2 hours, between about 4 hours and about 24 hours, between about 4 hours and about 12 hours, between about 4 hours and about 8 hours, between about 8 hours and about 24 hours, between about 8 hours and about 12 hours, between about 16 hours and about 24 hours, between about 16 hours and about 20 hours, or between about 20 hours and about 24 hours.

[0084]

[0095] In some embodiments of the present invention, the method comprises determining the EC of a polypeptide preparation using a multiparameter logistic fit to a standard sample. 50 In some embodiments, the multiparameter logistic fit is a three-parameter, four-parameter, or five-parameter logistic fit. Methods for such multiparameter fits are known in the art.

[0085]

[0096] In some embodiments, the potency of a polypeptide preparation is determined by the EC of the polypeptide preparation using a four-parameter logistic fit as follows: 50 Based on.

[0097] Luminescence values ​​measured in relative light units (RLU) for individual wells are used to calculate the mean well value for each standard (ST) and test article (control and sample(s); TA) concentration tested in replicate wells.

[0086]

[0098] Dose-response curves for standards, controls, and samples are generated by plotting the mean well values ​​for each concentration on the y-axis (linear scale) versus concentration on the x-axis (logarithmic scale).

[0087]

[0099] Separate curves for the ST and each TA are generated using a four-parameter logistic curve-fitting program. The four-parameter logistic curve-fitting equation is as follows: JPEG0007813582000001.jpg21170In the above formula, x = concentration of ST or TA y = average well value response (RLU) A = No dose response (Lower asymptote = LA): B=gradient C=EC 50 (Half the maximum effective concentration) D = maximum dose response (upper asymptote = UA)

[0088]

[0100] The coefficient of determination (R 2 ) is calculated.

[0089]

[0101] The fold response of the standard, control, and sample curves is calculated. Response ratio = UA ÷ LA

[0090]

[0102] The slope ratio is calculated as follows: JPEG0007813582000002.jpg16170

[0091]

[0103] The percentage difference in the upper asymptote is calculated as follows: JPEG0007813582000003.jpg17170

[0092]

[0104] The percentage difference of the lower asymptote is calculated as follows: JPEG0007813582000004.jpg17170

[0093]

[0105] The relative potency of the test article is calculated using a four-parameter parallel curve analysis. Constrained 4-P parallel curves for the ST and each TA are generated using a common set of parameters: slope (parameter B), upper asymptote (parameter D), and lower asymptote (parameter A). The resulting curve equations for the standard (ST) and test article (TA) are as follows: JPEG0007813582000005.jpg21170In the above formula, x = antibody concentration yST=Standard product RLU yTA = Test item RLU A = common lower asymptote B = common slope CST=Standard product EC 50 D = common upper asymptote ρ = relative titer of the sample (relative titer is the EC 50 ST's EC against 50 (ratio of

[0094]

[0106] Calculate the potency of the test article according to the following formula: Titer = ρ × activity of standard sample

[0095] kit

[0107] In some embodiments of the present invention, a kit or article of manufacture is provided for use in a test to determine the activity or potency of a polypeptide preparation, comprising a container containing a composition comprising engineered cells comprising a nucleic acid encoding a reporter operably linked to a promoter and / or sequence responsive to Fc receptor activation as described herein. In some embodiments, the kit further comprises a container containing a reference polypeptide preparation assay standard (a polypeptide preparation of known activity or potency) and / or a container containing a polypeptide preparation standard. In some embodiments, the kit further comprises a container or surface containing an immobilized antigen. In some embodiments, the reporter is luciferase, a fluorescent protein, alkaline phosphatase, beta-lactamase, or beta-galactosidase. In some embodiments, the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase. In some embodiments, the promoter and / or sequence responsive to Fc receptor activation comprises an Fc receptor activation-responsive sequence from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5, and IRF. In some embodiments, the reporter cell is a phagocyte. In some embodiments, the phagocyte is a monocyte. In some embodiments, the phagocyte is from a cell line. In some embodiments, the phagocyte line is a THP-1 cell line or a U-937 cell line.

[0096]

[0108] The container holds the formulation, and a label on or associated with the container may indicate instructions for use. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, culture supplies, reagents for detecting the reporter molecule, and package inserts with instructions for use.

[0097]

[0109] In some aspects of the present invention, a kit or article of manufacture is provided that includes a container containing a composition comprising an antigen conjugated with biotin and, optionally, provides instructions for use. In some embodiments, the kit further provides a standard polypeptide test sample (a polypeptide preparation of known activity or titer) and / or an antigen-binding control. The container contains the formulation, and a label on or associated with the container may indicate instructions for use. The article of manufacture may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, culture supplies, reagents for detecting reporter molecules, and a package insert with instructions for use.

[0098] Polypeptides

[0110] Polypeptides analyzed using the methods described herein are generally produced using recombinant techniques. Methods for producing recombinant proteins are described, for example, in U.S. Patent Nos. 5,534,615 and 4,816,567, which are specifically incorporated herein by reference. In some embodiments, the protein of interest is produced in CHO cells (see, e.g., WO 94 / 11026). In some embodiments, the polypeptide of interest is produced in E. coli cells. See, for example, U.S. Patent Nos. 5,840,523, 5,648,237, and 5,789,199, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of polypeptide fragments in E. coli. When using recombinant techniques, the polypeptide can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.

[0099]

[0111] Polypeptides may be recovered from the culture medium or host cell lysate. Cells used to express a polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. If the polypeptide is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating polypeptides secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the polypeptide is secreted into the culture medium, supernatants from such expression systems are generally first concentrated using commercially available polypeptide concentration filters, such as Amicon® or Millipore Pellicon® ultrafiltration units. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may also be included to prevent the growth of adventitious contaminants.

[0100]

[0112] In some embodiments, the polypeptide in the composition comprising the polypeptide and one or more contaminants is purified or partially purified by the methods of the present invention prior to analysis. For example, the polypeptide of the methods of the present invention is in the eluate from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed-mode chromatography, and hydrophobic interaction chromatography. In some embodiments, the polypeptide is in the eluate from Protein A chromatography.

[0101]

[0113] Examples of polypeptides that can be analyzed by the methods of the present invention include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, and interleukins.

[0102] (A) Antibody

[0114] In some embodiments of any of the methods described herein, the polypeptide for use in any of the methods of analyzing polypeptides and formulations comprising the polypeptides by the methods described herein is an antibody or immunoadhesin, hi some embodiments, the antigen target of the polypeptide of the invention is Abeta or CD20.

[0103]

[0115] Other exemplary antibodies include, but are not limited to, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 / neu antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis antibodies, X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD10 antibody, anti-CD11a antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-C D15 antibody, anti-CD19 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD4 Examples of the antibody include those selected from 5RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-S100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, anti-TebB2 antibody, anti-STEAP antibody, and anti-Tn antigen antibody.

[0104] (i) Monoclonal antibody

[0116] In some embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody (typically, such variants are present in minor amounts). Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete or polyclonal antibodies.

[0105]

[0117] For example, monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0106]

[0118] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0107]

[0119] Thus, the hybridoma cells are seeded and cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0108]

[0120] In some embodiments, the myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments, the myeloma cell line is a murine myeloma line, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0109]

[0121] The culture medium in which the hybridoma cells are growing is tested for production of monoclonal antibodies against the antigen. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0110]

[0122] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

[0111]

[0123] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, hybridoma cells may be grown in vivo as ascites tumors in an animal.

[0112]

[0124] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, polypeptide A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0113]

[0125] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In some embodiments, hybridoma cells serve as the source of such DNA. The isolated DNA can be placed into an expression vector and then transfected into host cells that do not otherwise produce immunoglobulin polypeptides, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Reviews on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Plueckthun, Immunol. Revs., 130:151-188 (1992).

[0114]

[0126] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0115]

[0127] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984)), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0116]

[0128] Typically, such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody, or for the variable domains of one antigen-binding site of an antibody, to create a chimeric bivalent antibody comprising one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0117]

[0129] In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

[0118] (ii) Humanized antibody

[0130] In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are commonly referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and colleagues (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting hypervariable region sequences with the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than intact human variable domains have been substituted by the corresponding sequences of a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0119]

[0131] To reduce antigenicity, the selection of human variable domains, both light and heavy, used in creating humanized antibodies is crucial. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies for a particular subgroup of light or heavy chain variable regions. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0120]

[0132] It is further important that antibodies be humanized with retention of high affinity for the antigen and other desirable biological properties. To achieve this goal, in some embodiments of the method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display predicted three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this manner, FR residues can be selected and combined from the recipient and import sequences to result in desired antibody characteristics, such as improved affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0121] (iii) human antibody

[0133] In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, in chimeric and germ-line mutant mice, the heavy chain joining region (J) of an antibody is expressed in a nucleotide sequence similar to that of a human antibody. HIt has been described that homozygous deletion of the .gt; (IgM) gene completely inhibits endogenous antibody production. Introduction of the human germ-line immunoglobulin gene array in these germ-line mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); and U.S. Patent Nos. 5,591,669; 5,589,369; and 5,545,807.

[0122]

[0134] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technology, antibody V domain genes are cloned in-frame into either a major or minor coat polypeptide gene of a filamentous bacteriophage (such as M13 or fd), and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, for reviews of which see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Multiple sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Repertoires of V genes from unimmunized human donors can be constructed, and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0123]

[0135] Human antibodies may also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0124] (iv) Antibody fragment

[0136] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is an antibody fragment comprising an Fc receptor binding domain. Various techniques have been developed to produce antibody fragments. Traditionally, such fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science 229:81 (1985)). However, such fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above.

[0125]

[0137] In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antibody fragment is an antigen-binding fragment comprising an Fc receptor binding domain. In some embodiments, the antibody fragment is an antigen-binding fragment comprising an Fcγ receptor binding domain.

[0126] (v) Bispecific antibodies

[0138] In some embodiments, the antibody is a bispecific antibody. A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes. Alternatively, the binding arm of the bispecific antibody can be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3) or an IgG Fc receptor (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus cellular defense mechanisms on the cell. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0127]

[0139] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, with the two chains having different specificities (Millstein et al., Nature 305:537-539 (1983)). Because the immunoglobulin heavy and light chains are randomly assorted, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography steps, which are quite tedious and result in low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0128]

[0140] In a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy chain constant region (CH1), containing the site necessary for light chain binding, is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and cotransfected into a suitable host organism. This allows for great flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, if expression of at least two polypeptide chains in equal ratios results in high yields, or if the ratios are not particularly critical, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector.

[0129]

[0141] In some embodiments of this approach, the bispecific antibody is composed of a composite immunoglobulin heavy chain with a first binding specificity in one arm and a composite immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It has been found that this asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule facilitates separation. This approach is disclosed in WO 94 / 04690. For further details on generating bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986).

[0130]

[0142] According to another approach described in U.S. Patent No. 5,731,168, the intermolecular interface of a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. In some embodiments, the interface is located between the C and C regions of the antibody constant domains. H The antibody comprises at least a portion of domain 3. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), compensatory "cavities" of identical or similar size to the large side chain(s) are created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products such as homodimers.

[0131]

[0143] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be conjugated to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and as a treatment for HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 0308936). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0132]

[0144] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0133]

[0145] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provided an alternative mechanism for making bispecific antibody fragments. These fragments consist of light-chain variable domains (V) connected by a linker that is too short to allow pairing between the two domains on the same chain. L ) connected to the heavy chain variable domain (V H ) is included. Therefore, the V H and V L The domain is a complementary V L and V H The Fv domains are forced to pair to form two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0134]

[0146] Trivalent or higher antibodies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0135] (v) multivalent antibody

[0147] In some embodiments, the antibody is a multivalent antibody. Multivalent antibodies may be internalized (and / or catabolized) more quickly by cells expressing the antigen to which the antibody binds than bivalent antibodies. The antibodies provided herein may be multivalent antibodies (other than IgM class antibodies) having three or more antigen-binding sites (e.g., tetravalent antibodies), and such antibodies can be easily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. A multivalent antibody may comprise a dimerization domain and three or more antigen-binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or hinge region. In this case, the antibody comprises an Fc region and three or more antigen-binding sites at the amino terminus of the Fc region. A preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen-binding sites. A multivalent antibody comprises at least one polypeptide chain (preferably two polypeptide chains), and the polypeptide chain(s) comprise two or more variable domains. For example, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc (wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1). For example, the polypeptide chain(s) may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain; or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (preferably four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptide contemplated herein comprises a light chain variable domain and optionally further comprises a CL domain.

[0136]

[0148] In some embodiments, the antibody is a multispecific antibody. Examples of multispecific antibodies include, but are not limited to, V H V L The heavy chain variable domain (V H) and the light chain variable domain (V L ) antibodies containing each V H V L Two or more V units that bind to different epitopes L and V H Examples of multispecific antibodies include antibodies having domains, antibodies having two or more single variable domains where each single variable domain binds to a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, and scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and covalently or covalently linked antibody fragments. In some embodiments, the antibody is polyepitopic; e.g., capable of specifically binding to two or more different epitopes on the same or different target(s). In some embodiments, the antibody is monospecific, e.g., an antibody that binds to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

[0137] (vi) Other antibody modifications

[0149] Modifications of the antibodies provided herein may be desirable with respect to effector function, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or additionally, cysteine ​​residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ, Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has two Fc regions and may thereby have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0138]

[0150] To increase the serum half-life of the antibody, amino acid modifications can be made to the antibody as described in U.S. Patent Application Publication No. 2006 / 0067930, which is incorporated herein by reference in its entirety.

[0139] (B) Polypeptide variants and modifications

[0151] The amino acid sequence modification(s) of the polypeptides (including antibodies) described herein may be used in methods for purifying the polypeptides (e.g., antibodies) described herein.

[0140] (i) variant polypeptide

[0152] "Polypeptide variant" refers to a polypeptide, preferably an active polypeptide, as defined herein, having at least about 80% amino acid sequence identity with the full-length native sequence of the polypeptide, the polypeptide sequence lacking the signal peptide, or the extracellular domain of the polypeptide (with or without the signal peptide). Such polypeptide variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence. Typically, a TAT polypeptide variant has at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to the full-length native sequence polypeptide sequence, the polypeptide sequence lacking the signal peptide, or the extracellular domain of the polypeptide (with or without the signal peptide). Optionally, the variant polypeptide has only one conservative amino acid substitution compared to the native polypeptide sequence, or has no more than about 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to the native polypeptide sequence.

[0141]

[0153] Variant polypeptides may be truncated, for example, at the N- or C-terminus, or may lack internal residues compared to the full-length native polypeptide. Certain variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides with truncations, deletions, and insertions can be prepared by any of several conventional techniques. The desired variant polypeptide may also be chemically synthesized. Another suitable technique involves isolating and amplifying a nucleic acid fragment encoding the desired variant polypeptide by polymerase chain reaction (PCR). The 5' and 3' primers of the PCR use oligonucleotides that determine the desired termini of the nucleic acid fragment. Preferably, the variant polypeptide shares at least one biological and / or immunological activity with the native polypeptide described herein.

[0142]

[0154] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionine residue or an antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0143]

[0155] For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide. Amino acid sequence variants of the polypeptide are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the polypeptide. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes may also alter post-translational processing of the polypeptide (e.g., antibody), such as changing the number or position of glycosylation sites.

[0144]

[0156] Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting a desired activity can be found by comparing the sequence of a polypeptide with the sequences of known homologous polypeptide molecules and minimizing the number of amino acid sequence changes made in areas of high homology.

[0145]

[0157] A useful method for identifying specific residues or regions of a polypeptide (e.g., an antibody) that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science 244:1081-1085 (1989). In this method, a target residue or residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, Glu, etc.) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect antigen-amino acid interactions. Those amino acid locations demonstrating functional sensitivity to the substitution are then refined by introducing further or other variants at or for the substitution sites. In this way, while the site for introducing an amino acid sequence variant is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0146]

[0158] Another type of variant is an amino acid substitution variant. Such substitution variants have at least one amino acid residue in the antibody molecule replaced with a different residue. The most interesting sites for substitutional mutagenesis include hypervariable regions, although FR alterations are also contemplated. Conservative substitutions are shown under the heading "exemplary substitutions" in Table 1. More drastic changes, referred to as "substitutions" in Table 1 or further described below in relation to amino acid classes, can then be introduced and the products screened if such substitutions result in changes in biological activity. JPEG0007813582000006.jpg170170

[0147]

[0159] Substantial alterations in the biological properties of a polypeptide can be achieved by selecting substitutions that significantly differ in (a) the structure of the polypeptide backbone in the region of the substitution, e.g., sheet or helix structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the effectiveness of maintaining the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties as follows (AL Lehninger, Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0148]

[0160] Alternatively, naturally occurring residues can be divided into the following groups based on 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 that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0149]

[0161] Non-conservative substitutions entail exchanging a member of one of these classes for a member of another class.

[0150]

[0162] Cysteine ​​residues not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to a polypeptide to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0151]

[0163] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they were generated. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues significantly contributing to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and target. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants can be screened as described herein and antibodies with superior properties in one or more relevant tests selected for further development.

[0152]

[0164] Another type of amino acid variant of a polypeptide alters the original glycosylation pattern of the antibody. The polypeptide may contain non-amino acid moieties. For example, the polypeptide may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in a host cell or host organism, or may be a deliberate modification resulting from human intervention. By altering is meant deleting one or more carbohydrate moieties found in the polypeptide and / or adding one or more glycosylation sites that are not present in the polypeptide.

[0153]

[0165] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0154]

[0166] Addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0155]

[0167] Removal of carbohydrate moieties present on a polypeptide can be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved through the use of a variety of endo- and exoglycosidases.

[0156]

[0168] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0157] (ii) chimeric polypeptide

[0169] The polypeptides described herein may be modified to form chimeric molecules comprising the polypeptide fused to another, heterologous polypeptide or amino acid sequence. In some embodiments, a chimeric molecule comprises a fusion of the polypeptide with a tag polypeptide providing an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the polypeptide. The presence of such epitope-tagged forms of the polypeptide can be detected using an antibody against the tag polypeptide. Provision of the epitope tag also allows the polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

[0158]

[0170] In an alternative embodiment, the chimeric molecule may comprise a fusion of the polypeptide with an immunoglobulin or a particular region of an immunoglobulin. The bivalent form of the chimeric molecule is referred to as an "immunoadhesin."

[0159]

[0171] As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous polypeptide with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity (i.e., "heterologous") other than the antigen recognition and binding site of an antibody, with an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, e.g., IgG1, IgG2, IgG3, or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

[0160]

[0172] The Ig fusions preferably involve the substitution of a soluble (transmembrane domain deleted or inactivated) form of a polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or hinge, CH2 and CH3 regions of an IgG1 molecule.

[0161] (iii) Polypeptide conjugates

[0173] Polypeptides for use in polypeptide formulations may be conjugated to a cytotoxic agent, such as a chemotherapeutic agent; a growth inhibitory agent; a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof); or a radioactive isotope (i.e., a radioconjugate).

[0162]

[0174] Chemotherapeutic agents useful for generating such conjugates may be used. In addition, enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Jatropha forsi protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides may be utilized for the production of radioconjugated polypeptides. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Conjugates of polypeptides and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., 2,6-triylene diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating radionucleotides to polypeptides.

[0163]

[0175] Conjugates of a polypeptide and one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a trichothecene, and CC1065, and derivatives of these toxins that have toxin activity, are also contemplated herein.

[0164]

[0176] Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata. It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and its derivatives and analogs are also contemplated. Numerous linking groups are known in the art for preparing polypeptide-maytansinoid conjugates, including those disclosed in U.S. Pat. No. 5,208,020. Linking groups include disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, or esterase-labile groups, as disclosed in the above-identified patents; disulfide groups and thioether groups are preferred.

[0165]

[0177] The linker can be attached to the maytansinoid molecule at various positions, depending on the type of linkage. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. This reaction can occur at the C-3 position, which bears a hydroxyl group, the C-14 position, which is modified with hydroxymethyl, the C-15 position, which is modified with a hydroxyl group, and the C-20 position, which bears a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

[0166]

[0178] Another conjugate of interest comprises a polypeptide conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see, e.g., U.S. Patent No. 5,712,374. Structural analogs of calicheamicin that can be used include, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG, and θ1 I Another antitumor drug that can be conjugated to an antibody is the antifolate drug QFA. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, when cells take up these agents through polypeptide (e.g., antibody)-mediated internalization, their cytotoxic effects are greatly enhanced.

[0167]

[0179] Other anti-tumor agents that can be conjugated to the polypeptides described herein include BCNU, streptozoicin, vincristine, and 5-fluorouracil, a family of drugs known collectively as LL-E33288 complexes, and esperamicin.

[0168]

[0180] In some embodiments, the polypeptide may be a conjugate between a polypeptide and a compound with nucleolytic activity (eg, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

[0169]

[0181] In yet another embodiment, a polypeptide (e.g., an antibody) may be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pre-targeting, and the polypeptide-receptor conjugate is administered to a patient, followed by the use of a clearing agent to remove unbound conjugate from the circulation, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0170]

[0182] In some embodiments, the polypeptide may be conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent) into an active anti-cancer drug. The enzyme component of the immunoconjugate includes any enzyme capable of acting on a prodrug in such a way as to convert it into its more active, cytotoxic form.

[0171]

[0183] Useful enzymes include, but are not limited to, alkaline phosphatases, which are useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, which are useful for converting the non-toxic 5-fluorocytosine to the anti-cancer drug 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs to free drugs; D-amino Examples include D-alanylcarboxypeptidases, which are useful for converting prodrugs containing acid substituents; carbohydrate-cleaving enzymes (such as β-galactosidase and neuraminidase), which are useful for converting glycosylated prodrugs to free drugs; β-lactamases, which are useful for converting β-lactam-derivatized drugs to free drugs; and penicillin amidases (such as penicillin V amidase or penicillin G amidase), which are useful for converting drug substances derivatized at the amine nitrogen with a phenoxyacetyl or phenylacetyl group, respectively. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert prodrugs to free active drugs.

[0172] (iv) Other

[0184] Another type of covalent modification of polypeptides involves linking the polypeptide to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Polypeptides may also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization methods, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro, AR, Ed., (1990).

[0173] Obtaining Polypeptides for Use in Formulations and Methods

[0185] Polypeptides used in the analytical methods described herein can be obtained using methods well known in the art, including recombinant methods. The following section provides guidance regarding these methods.

[0174]

[0186] (A) Polynucleotide

[0187] "Polynucleotide" or "nucleic acid," used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.

[0175]

[0188] Polynucleotides encoding polypeptides can be obtained from any source, including, but not limited to, cDNA libraries prepared from tissues believed to have polypeptide mRNA and express it at detectable levels. Thus, polynucleotides encoding polypeptides can be conveniently obtained from cDNA libraries prepared from human tissue. Genes encoding polypeptides may also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0176]

[0189] For example, a polynucleotide can encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes a heavy chain variable region (V H ), as well as a heavy chain constant region (C), which typically consists of three constant domains: CH1, CH2, and CH3, and a "hinge" region. H In some situations, the presence of a constant region is desirable. In some embodiments, the polynucleotide encodes one or more immunoglobulin molecule chains of TDB.

[0177]

[0190] Other polypeptides that can be encoded by the polynucleotides include antigen-binding antibody fragments, such as single domain antibodies ("dAbs"), Fvs, scFvs, Fab' and F(ab')2, and "minibodies." Minibodies are fragments of C H 1 and C K or C LMinibodies are domain-excised (typically) bivalent antibody fragments. Because minibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical / diagnostic applications, but because they are bivalent, they should retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context indicates otherwise, the term "antibody" as used herein encompasses not only whole antibody molecules but also the above-mentioned types of antigen-binding antibody fragments. Preferably, each framework region present in the encoded polypeptide contains at least one amino acid substitution relative to the corresponding human acceptor framework. Thus, for example, the framework region may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the acceptor framework region.

[0178] Exemplary Implementations

[0191] 1. A method for determining the activity of a polypeptide that binds to a target antigen and comprises an Fc receptor binding domain, comprising: a) contacting an immobilized target antigen with a polypeptide preparation to form an antigen-polypeptide complex; b) contacting the antigen-polypeptide complex with a phagocyte, the phagocyte comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; Includes; A method for determining the activity of a polypeptide, wherein expression of a reporter is indicative of activity of the polypeptide.

[0179]

[0192] 2. A method for quantifying the titer of a polypeptide preparation in which the polypeptide binds to a target antigen, comprising: a) contacting multiple populations of immobilized target antigens with different concentrations of a polypeptide preparation to form antigen-polypeptide complexes; b) contacting these antigen-polypeptide complexes with phagocytes, the phagocytes comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; c) measuring the expression of the reporter; and d) EC of the polypeptide preparation 50 Determine the EC of the polypeptide preparation 50 and the EC of a standard sample of a polypeptide with known titer. 50 and A method for quantifying the titer of a polypeptide preparation.

[0180]

[0193] 3. EC of Polypeptide Preparations Using Multiparameter Logistic Fit to Standard Samples 50 3. The method of embodiment 2, further comprising calculating the titer based on:

[0181]

[0194] 4. The method of embodiment 3, wherein the multiparameter logistic fit is a three-parameter, four-parameter, or five-parameter logistic fit.

[0182]

[0195] 5. The EC50 of the standard is the EC50 of the polypeptide preparation. 50 The method of any one of embodiments 2 to 4, wherein the aforesaid conditions are determined simultaneously.

[0183]

[0196] 6. The method of any one of embodiments 1 to 5, wherein the reporter is luciferase or a fluorescent protein.

[0184]

[0197] 7. The method of embodiment 6, wherein the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase.

[0185]

[0198] 8. A method according to any one of embodiments 1 to 7, wherein the response element that responds to activation by an Fcγ receptor is an NFκB response element, an NFAT response element, an AP-1 response element or an ERK-responsive transcription factor (e.g., Elk1).

[0186]

[0199] 9. The method of any one of embodiments 1 to 8, wherein the phagocyte is a monocyte.

[0187]

[0200] 10. The method of any one of embodiments 1 to 9, wherein the phagocytes are from a cell line.

[0188]

[0201] 11. The method of embodiment 10, wherein the cell line is a THP-1 cell line or a U-937 cell line.

[0189]

[0202] 12. The method of any one of embodiments 1 to 11, wherein the Fcγ receptor is FcγRI (CD64) or FcγRIIa (CD32a) or FcγRIII (CD16).

[0190]

[0203] 13. The method of any one of embodiments 1 to 12, wherein the phagocytes are engineered to overexpress Fcγ receptors.

[0191]

[0204] 14. The method of embodiment 13, wherein the phagocytes are engineered to overexpress FcγRIIa.

[0192]

[0205] 15. A method according to any one of embodiments 1 to 14, wherein the phagocyte does not express FcγRIII.

[0193]

[0206] 16. The method of any one of embodiments 1 to 15, wherein the target antigen is beta amyloid (Aβ) or CD20.

[0194]

[0207] 17. The method of embodiment 16, wherein the target antigen is beta amyloid (Aβ).

[0195]

[0208] 18. The method of embodiment 17, wherein Aβ is human Aβ.

[0196]

[0209] 19. The method of embodiment 17 or 18, wherein the Aβ comprises monomeric and / or oligomeric Aβ.

[0197]

[0210] 20. The method of embodiment 17, wherein the human Aβ is Aβ1-40 or Aβ1-42.

[0198]

[0211] 21. The method of any one of embodiments 1 to 20, wherein the polypeptide comprises a full-length Fc domain or an FcR-binding fragment of an Fc domain.

[0199]

[0212] 22. The method of any one of embodiments 1 to 21, wherein the polypeptide specifically binds to Aβ.

[0200]

[0213] 23. The method of any one of embodiments 1 to 22, wherein the polypeptide is an antibody or immunoadhesin.

[0201]

[0214] 24. The method of embodiment 22 or 23, wherein the polypeptide is crenezumab.

[0202]

[0215] 25. A method according to any one of embodiments 1 to 24, wherein the target antigen is immobilized on a surface.

[0203]

[0216] 26. The method of embodiment 25, wherein the surface is a plate.

[0204]

[0217] 27. The method of embodiment 26, wherein the plate is a multiwell plate.

[0205]

[0218] 28. The method of any one of embodiments 25 to 27, wherein the antigen is immobilized to the surface at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus.

[0206]

[0219] 29. A method according to any one of embodiments 25 to 28, wherein the target antigen is immobilized on the surface using a biotin-streptavidin system.

[0207]

[0220] 30. The method of embodiment 29, wherein the target antigen is bound to biotin and the surface comprises bound streptavidin.

[0208]

[0221] 31. The method of embodiment 29 or 30, wherein the target antigen is conjugated to biotin at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus.

[0209]

[0222] 32. The method of any one of embodiments 1 to 31, wherein the reporter is detected after any one or more of about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, 24 hours or more than 24 hours from contacting the antigen-polypeptide complex with the phagocyte.

[0210]

[0223] 33. A kit for determining the titer of a polypeptide preparation, wherein the polypeptide binds to a target antigen and comprises an Fc receptor binding domain, comprising an immobilized target antigen and a phagocyte, wherein the phagocyte comprises an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor; Expression of the reporter indicates the titer of the polypeptide; kit.

[0211]

[0224] 34. A kit for quantifying the titer of a polypeptide preparation, wherein the polypeptide binds to a target antigen and comprises an Fc receptor binding domain, the kit comprising an immobilized target antigen, phagocytes, and a standard sample; the phagocyte comprises an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor, wherein expression of the reporter indicates titer of the polypeptide; and The standard sample comprises a preparation of a polypeptide of known titer; kit.

[0212]

[0225] 35. The kit of embodiment 33 or 34, wherein the reporter is luciferase or a fluorescent protein.

[0213]

[0226] 36. The kit of embodiment 35, wherein the luciferase is firefly luciferase, Renilla luciferase, or nanoluciferase.

[0214]

[0227] 37. The kit of any one of embodiments 33 to 36, further comprising a reagent for detecting expression of the reporter.

[0215]

[0228] 38. A kit described in any one of embodiments 33 to 37, wherein the response element that responds to activation by an Fcγ receptor is an NFκB response element, an NFAT response element, an AP-1 response element, or an ERK-responsive transcription factor (e.g., Elk1).

[0216]

[0229] 39. A kit described in any one of embodiments 33 to 38, wherein the phagocytes are from a cell line.

[0217]

[0230] 41. The kit of embodiment 39, wherein the cell line is a THP-1 cell line or a U-937 cell line.

[0218]

[0231] 41. The kit of any one of embodiments 33 to 40, wherein the Fcγ receptor is FcγRI (CD64), FcγRIIa (CD32a), or FcγRIII (CD16).

[0219]

[0232] 42. A kit described in any one of embodiments 33 to 41, in which the phagocytes are engineered to overexpress Fcγ receptors.

[0220]

[0233] 43. The kit of embodiment 42, wherein the phagocytes are engineered to overexpress FcγRIIa.

[0221]

[0234] 44. A kit described in any one of embodiments 33 to 43, in which the phagocytes do not express FcγRIII.

[0222]

[0235] 45. A kit described in any one of embodiments 33 to 44, wherein the target antigen is beta amyloid (Aβ) or CD20.

[0223]

[0236] 46. ​​A kit described in any one of embodiments 33 to 45, wherein the target antigen is beta amyloid (Aβ).

[0224]

[0237] 47. The kit of embodiment 46, wherein the Aβ is human Aβ.

[0225]

[0238] 48. The kit of embodiment 46 or 47, wherein the Aβ comprises monomeric and / or oligomeric Aβ.

[0226]

[0239] 49. The kit of embodiment 48, wherein the human Aβ is Aβ1-40 or Aβ1-42.

[0227]

[0240] 50. A kit described in any one of embodiments 33 to 49, wherein the polypeptide comprises a full-length Fc domain or an FcR-binding fragment of an Fc domain.

[0228]

[0241] 51. A kit described in any one of embodiments 33 to 50, wherein the polypeptide specifically binds to Aβ.

[0229]

[0242] 52. The kit of any one of embodiments 33 to 51, wherein the polypeptide is an antibody or immunoadhesin.

[0230]

[0243] 53. The kit of embodiment 52, wherein the polypeptide is crenezumab.

[0231]

[0244] 54. A kit described in any one of embodiments 33 to 53, wherein the target antigen is immobilized on a surface.

[0232]

[0245] 55. The kit of embodiment 54, wherein the surface is a plate.

[0233]

[0246] 56. The kit of embodiment 55, wherein the plate is a multiwell plate.

[0234]

[0247] 57. The kit of embodiment 55 or 56, wherein the target antigen is conjugated to biotin at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus.

[0235]

[0248] 58. A kit described in any one of embodiments 54 to 57, wherein the target antigen is immobilized on the surface using a biotin-streptavidin system.

[0236]

[0249] 59. The kit of embodiment 58, wherein the target antigen is bound to biotin and the surface comprises bound streptavidin.

[0237]

[0250] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0238]

[0251] Further details of the present invention are illustrated by the following non-limiting examples, the disclosures of all references cited herein are expressly incorporated herein by reference. [Example]

[0239]

[0252] The following examples are intended to be merely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0240] Example 1 Materials and Methods Phagocytosis reporter cell generation

[0253] Human FCGR2A (CD32A) cDNA (protein_id=NP_067674.2; coded by=NM_021642.3; HIS variant) was first chemically synthesized (GeneArt TM Gene Synthesis). Restriction sites (EcoRI, 5' end; NotI, 3' end) were added to the cDNA template, and a Kozak sequence (GCCACC) was added immediately 5' of the ATG start codon. Using EcoRI and NotI, the cDNA was subcloned into the lentiviral vector pCDH-CMV-MCS-IRES-Puro (Figure 1). The resulting construct, pCDH-CMV-CD32A-IRES-Puro, was sequenced to confirm the entire cDNA insert. Reporter constructs were generated by cloning a nuclear factor-κB (NF-κB) response element (RE) upstream of the firefly luciferase (Luc) gene into the lentiviral vector (Figure 2). These constructs were used to generate lentiviral particles, which were then used to transduce U-937 and THP-1 monocytic cell lines. A parental pool was generated by selection with 1 μg / mL puromycin (Clontech). Luminescence activity was confirmed using TNFα, which also activates NF-κB, as a positive control. Clones were isolated by limiting dilution and screened for activity using crenezumab and amyloid-β (Aβ). Cells were cultured in RPMI (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) (Gibco), 1x Glutamax (Gibco), and 1x penicillin-streptomycin (Gibco), and then frozen in 90% HI FBS, 10% dimethyl sulfoxide (DMSO) (ATCC).

[0241] Reagents and buffers

[0254] Non-biotinylated Aβ peptide (rPeptide or Anaspec) and biotin beta amyloid 1-42 peptide (biotin-Aβ) (Anaspec) were reconstituted by first adding 40 μL of room-temperature DMS to a vial of peptide (0.5 mg each). After washing the walls of the vial 2-3 times, 960 μL of phosphate-buffered saline (PBS) adjusted to pH 8.0 was added. The vial was vortexed for approximately 1 minute until the reagents were dissolved, after which the reagents were pooled, aliquoted, and stored at -60°C or below until use. Additional peptides included a 51-amino acid peptide of CD20 with biotin at each end (CD20-biotin) (CPC Scientific). This peptide was similarly reconstituted in DMSO and brought to a stock concentration of 1 mg / mL in PBS.

[0242]

[0255] The TBS binding buffer consisted of Tris-buffered saline (10 mM Tris pH 8.0, 150 mM NaCl). The wash buffer consisted of PBS containing 1 mM CaCl2 and 1 mM MgCl2. The test medium was RPMI (Gibco) containing 10% HI-FBS (Gibco), 1x Glutamax (Gibco), and 1x penicillin-streptomycin (Gibco). For early development and testing of the ocrelizumab version, low-IgG HI FBS (Hyclone Ultra-Low IgG or Gibco) was used instead of HI-FBS. Luciferase expression was quantified using a luminescence reagent (Promega, Steady-Glo® Luciferase Assay System). The ELISA blocking buffer was Dulbecco's phosphate-buffered saline (DPBS) containing 1 mM CaCl2 and 1 mM MgCl2, plus 0.5% bovine serum albumin (BSA). The ELISA test diluent was PBS, 0.5% BSA, 0.05% polysorbate 20.

[0243]

[0256] The crenezumab standard and samples were manufactured by Genentech. The formulation buffer was 200 mM arginine succinate (0.05% (w / v)), polysorbate 20 (pH 5.5 ± 0.3). To prepare light-stressed samples, 25 mL of crenezumab was placed in a glass vial and placed in a calibrated light box, where it was exposed to 2.4 million lux hours of cumulative light over a 16-hour period. A light control was provided by wrapping the sample in aluminum foil.

[0244] Flow cytometry

[0257] Cells were washed with PBS or FACS Wash (PBS containing 0.5% bovine serum albumin and 0.1% sodium azide) and resuspended in FACS Wash. For U-937 experiments, cells were first stained with vital dye (Invitrogen) and preincubated with Fc-blocking antibodies (eBioscience, anti-CD16: 16-0166-85, anti-CD32: 16-0329-85, anti-CD64: 14-0649-82) for 10–15 min. Cells were then stained for 30–60 min with the following anti-FcγR antibodies or isotype controls: CD16-phycoerythrin (PE) (eBio, 12-0167-42), CD32-PE (BD Pharmingen, 550586), CD64-PE (eBio, 12-0649), CD64-FITC (eBio, 11-0649-42), FITC-mouse IgG1κ (eBio, 11-4714-42), PE-mouse IgG1κ (eBio, 12-4714-42), and PE-mouse IgG2κ (BD Pharmingen, 555743). Cells were washed, resuspended in FACS Wash, and fluorescence was detected using a flow cytometer (BD LSR II or FACSCaliber).

[0245] Evaluation of Aβ peptides and plate formats

[0258] Soluble non-biotinylated Aβ (25 μL) at 5 μg / mL was incubated with a 1:3 dilution series of crenezumab (25 μL, starting concentration 600,000 ng / mL) and THP-1 phagocytosis reporter cells (50 μL, 500,000 cells / mL) in test medium in tissue-culture-treated white test plates (Costar) for 5 hours at 37°C. After adding the luminescence reagent Steady-Glo® (100 μL) (Promega) and shaking for 20 minutes, luminescence was detected using a luminescence plate reader (Perkin-Elmer, EnVision). Alternatively, 100 μL of non-biotinylated Aβ at 1 μg / mL in PBS was adsorbed overnight at 4°C to a high-binding white plate (Thermo, Maxisorp). The plate was washed with PBS, blocked with 200 μL of test medium for 30 minutes, and washed again. The plates were then incubated with 100 μL of crenezumab (starting concentration 50,000 ng / mL) in a 1:3 dilution series in test medium for 30 minutes at 37°C. The plates were washed again, and 100 μL of THP-1 phagocytosis reporter cells at 200,000 cells / mL were added and incubated for 5 hours at 37°C. The luminescent reagent Steady-Glo® (Promega) (100 μL) was added, and after 20 minutes of shaking, luminescence was detected using a luminescence plate reader (Perkin-Elmer, EnVision). This variation of the procedure was also used to initially evaluate biotin-Aβ and streptavidin in a high-binding capacity 96-well white plate (Figure 4).

[0246] Crenezumab Aβ binding ELISA

[0259] Recombinant human amyloid β1-42 peptide (rPeptide) was reconstituted in DMSO and frozen in single-use aliquots. For testing, the peptide was diluted to 1 μg / mL in DPBS, and 100 μL was added to a high-binding polystyrene plate (Nunc). The plate was incubated at 2-8°C for 16-72 hours, then dumped and blocked with 200 μL of ELISA block buffer at 25°C for 1-2 hours. The plate was washed with PBS + 0.05% polysorbate 20, and 100 μL of crenezumab standard and sample diluents diluted in ELISA test diluent were added. The plate was incubated at 25°C for 1 hour and then washed again. 2 ng / mL goat anti-human IgG-horseradish peroxidase (HRP) solution (Jackson Immunoresearch) was added to the plate, incubated at 25°C for 40 minutes, and then washed. Colorimetric TMB detection reagent (SureBlue Reserve, KPL) was added, and the plate was developed for 10–30 min with shaking, followed by the addition of 0.6 N sulfuric acid. Absorbance was measured at 450 nm using a plate reader (Molecular Devices), and absorbance at 650 nm was used as the standard absorbance. Potency compared to crenezumab standards was calculated using a parallel line analysis curve-fitting program.

[0247] Crenezumab phagocytosis reporter assay

[0260] Biotin-Aβ was diluted to a concentration of 1.5 μg / mL in TBS binding buffer and bound to a streptavidin-coated, high-binding capacity 96-well white plate (Pierce, Thermo Scientific) at 25°C for 16–72 hours. The plate was washed three times with wash buffer using a plate washer (Biotek) and equilibrated in warm test medium at 37°C for 1–2.5 hours in a humidified incubator with 5% CO2, covered with a breathable plate sealer (Aeraseal, Sigma) or lid. For protein quantification by UV SpecScan, standards and samples were diluted in formulation buffer. Eight-point dilution curves of standards, test controls (independently diluted standards), and samples in warm test medium were prepared, targeting concentrations of 10,000, 4000, 1600, 750, 250, 100, 40, and 10 ng / mL. Phagocytic reporter cells were harvested from the flasks by centrifugation, resuspended in warm test medium, and counted to yield 2.5 x 10 6 The plates were then diluted to 100 cells / mL. The plates were washed again, and 50 μL each of sample dilutions and cell preparations were added. The plates were then incubated at 37°C in a humidified incubator with 5% CO2, covered with a breathable plate sealer or lid, for 3–5 hours. The test plates were then cooled in a 25°C incubator for 15–20 minutes, followed by the addition of 100 μL of luminescence reagent. The plates were then shaken at room temperature on a benchtop shaker, and the luminescence signal was detected using a luminescence plate reader (Molecular Devices, Paradigm or i3x with a LUM96 cartridge). Titers were calculated based on the EC50 ratio using a 4P constrained fit to the crenezumab standard. Plate reading and titer calculations were performed using software (Molecular Devices, SoftMax® Pro v6.5).

[0248] Ocrelizumab phagocytosis reporter assay

[0261] The ocrelizumab test method was similar to the crenezumab test method with the following modifications. CD20-biotin peptide was diluted to 8 μg / mL in PBS (pH 6.5) and allowed to bind to the plate for 16 to 72 hours at 2 to 8°C. The wash buffer was PBS + 0.05% polysorbate 20. After peptide binding, the plate was washed six times, equilibrated with test medium, washed, and incubated with 100 μL of ocrelizumab dilutions at 37°C for 1.5 hours. The ocrelizumab concentrations were 100,000, 30,000, 15,000, 8000, 4000, 2000, 1000, and 100 ng / mL. The plate was then washed and 1.5 × 10 6 100 μL of U-937 phagocytosis reporter cells were added at a concentration of 100 cells / mL. The plates were incubated at 37°C for 2 hours and 40 minutes. Low IgG HI FBS was used as the test medium.

[0249] result Phagocytosis reporter cells

[0262] The phagocytosis reporter assay was first developed for crenezumab, which binds to soluble Aβ oligomers and promotes the uptake of immune complexes by microglia (Adolfsson et al.). This mechanism is similar to antibody-dependent cellular phagocytosis (ADCP) in that it involves phagocytes and is mediated by Fcγ receptors (FcγRs). To best reflect the biological properties of ADCP, the phagocytic human monocyte cell lines THP-1 and U-937 were selected as parent cell lines to generate phagocytosis reporter cell lines. The THP-1 and U-937 cell lines were engineered to express the firefly luciferase gene under the control of the NF-κB response element (NF-κB) as described in Materials and Methods. NF-κB is a transcription factor induced by signaling through FcγRs, among other immune receptors. While the specific Fcγ receptors involved in crenezumab-mediated microglial clearance of Aβ are unknown, crenezumab, an IgG4, binds with highest affinity to CD64 (FcγRI). CD32A (also known as FcγRIIa) is thought to be related to ADCP due to its preference for immune complexes over monomeric IgG. This receptor is also sensitive to Fc galactosylation, making it a potential antibody therapeutic. Therefore, cells were engineered with additional CD32A constructs to maximize sensitivity to potential product variants. U-937 and THP-1 cells also express CD64 but little to no CD16 (FcγRIIIa) (Figure 3). Both U-937 and THP-1 reporter cells represent the phagocytic mechanism of action, and titer testing for each specific antibody and target, including cell line selection, was optimized. THP-1 cells were ultimately chosen for crenezumab potency testing due to their superior testing accuracy and consistency for this antibody / target.

[0250] Evaluation of Aβ peptides and plate formats

[0263] Three approaches were evaluated to introduce Aβ peptide oligomers into the assay (Figure 4). The first utilized a soluble Aβ peptide preparation, which oligomerizes in aqueous solution, mixed with crenezumab and reporter cells. This approach did not generate a luminescent signal, likely due to incomplete or inefficient formation of Aβ oligomer complexes. To mimic and / or seed Aβ complexes, a plate-binding format was explored in which crenezumab and reporter cells were layered onto plates coated with Aβ peptides. Aβ peptides adsorbed to high-binding plates showed a positive but inconsistent signal within reporter cells. To improve the signal and consistency of Aβ binding to the plate surface, a streptavidin (SA)-biotin system was utilized, in which biotinylated Aβ was bound to streptavidin-coated plates.

[0251] Test format and crenezumab calibration curve

[0264] The format of the phagocytosis reporter cell assay involves binding of a biotinylated peptide to a streptavidin-coated plate (Figure 5). Peptide-specific antibodies bind to the peptide target, triggering FcγR clustering and activation. This activates NF-κB, leading to expression of the reporter gene luciferase, which can be quantified upon addition of substrate. A representative dose-response curve for crenezumab standard is shown in Figure 6.

[0252] Example of crenezumab titer determination: Degraded sample

[0265] This assay was used to determine the potency of crenezumab samples. To demonstrate that the phagocytosis reporter cell assay can detect changes in potency due to product degradation, stressed samples of crenezumab from light stress studies were tested for activity. These samples showed a loss of Aβ-binding activity as measured by ELISA, and a similar loss was observed using the phagocytosis reporter cell assay (Table 2), demonstrating that the reporter cell assay can detect loss of potency due to loss of Aβ-binding activity. JPEG0007813582000007.jpg39170The results are the relative potency % assigned to the crenezumab reference sample as 100% and are the average of three independent tests.

[0253] Adapting the test format to other products / targets

[0266] To determine whether the phagocytosis reporter assay could be applied to other antibody products, we adapted the format to other peptide target / antibody combinations. Ocrelizumab is a CD20-binding antibody with ADCP as its proposed mechanism of action. Therefore, we bound a biotinylated CD20 peptide to a streptavidin plate and then conjugated ocrelizumab to this peptide to mimic the binding of ocrelizumab to the surface of CD20-expressing cells. Using U-937 phagocytosis reporter cells, we observed the luminescence signal and generated a dose-response curve. This allowed us to assess the potency of ADCP relative to ocrelizumab (Figure 7).

[0254] overview

[0267] We developed an assay to measure crenezumab potency using a reporter cell line and plate-bound peptide (Figure 5). This assay serves as a surrogate for FcγR-mediated immune complex uptake / ADCP. It mirrors the mechanism of action in that it utilizes a phagocytic monocyte cell line to measure FcγR engagement and activation by immune complexes of crenezumab and Aβ peptide. This assay was demonstrated to be sensitive to loss of potency using stress samples of crenezumab. Furthermore, this assay format can be applied to other targets and products, as demonstrated with ocrelizumab (CD20 binding).

[0255] Example 2

[0268] The development of the phagocytosis reporter cells and the assay format have been described previously. Here, we provide additional data regarding the optimization of assay conditions for crenezumab testing.

[0256] Cell line optimization

[0269] THP-1 and U-937 cell lines were engineered to overexpress CD32 and express the firefly luciferase gene under the control of a nuclear factor-κB (NF-κB) response element to maximize sensitivity to potential product variants.

[0257]

[0270] The engineered U-937 cell line was initially selected for crenezumab testing based on its higher fold response and faster proliferation. However, after further comparison of crenezumab testing performance, THP-1 phagocytosis reporter cells were selected. Experiments were conducted to evaluate the effect of cell seeding density on the proliferation of THP-1 phagocytosis reporter cells, improving cell proliferation and yield in the test. Because THP-1 cells grew slower at lower cell seeding densities (Figure 8), a relatively high seeding density was incorporated into the cell culture procedure.

[0258] Reagent selection and optimization

[0271] Because NF-κB is activated downstream of several immune receptors, off-target activation of reporter cells by contaminants such as bacterial lipopolysaccharide (LPS) in recombinant Aβ peptide preparations was a potential concern. Therefore, recombinant and synthetic sources of Aβ peptide were compared for their ability to activate reporter cells in the absence of crenezumab (Figure 9). Synthetic Aβ peptide was chosen to minimize the possibility of endotoxin-mediated activation of reporter cells.

[0259] Experimental Design Optimization of Test Parameters

[0272] A Plackett-Burman design was used to further optimize the assay and evaluate the impact of test factors on assay readout. Test factors evaluated included test cell concentration, Aβ peptide concentration, incubation time, cell growth concentration (seeding density in flasks), SteadyGlo® incubation time, and FBS type (HI or low IgG FBS) (Table 3). Additionally, two batches of Aβ peptide preparation and multiple analysts were incorporated into the design. Test factors were evaluated in main-effects analyses for their impact on EC50, slope, fold response, mean titer, and titer standard deviation (SD) (Figures 10-13). JPEG0007813582000008.jpg133170

Claims

1. 1. A method for determining the activity of a polypeptide that binds to a target antigen and comprises an Fc receptor binding domain, comprising: a) contacting an immobilized target antigen with a polypeptide to form an antigen-polypeptide complex; b) contacting the antigen-polypeptide complex with a phagocyte, the phagocyte comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element responsive to activation by the Fcγ receptor, the reporter being a luciferase or a fluorescent protein, the luciferase being firefly luciferase, Renilla luciferase, or nanoluciferase, and the response element being an NFκB response element, an AP-1 response element, or an ERK-responsive transcription factor; Including; the target antigen is beta amyloid (Aβ), and the Aβ is Aβ1-40 or Aβ1-42; Expression of the reporter indicates activity of the polypeptide; Methods for determining the activity of a polypeptide.

2. The method of claim 1 , wherein the reporter is luciferase.

3. The method of claim 1 or 2, wherein the phagocyte is a monocyte.

4. 4. The method of any one of claims 1 to 3, wherein the phagocytes are from a cell line.

5. The method according to claim 4, wherein the cell line is a THP-1 cell line or a U-937 cell line.

6. 6. The method of any one of claims 1 to 5, wherein the Fcγ receptor is FcγRI (CD64), FcγRIIa (CD32a), or FcγRIII (CD16).

7. 7. The method of any one of claims 1 to 6, wherein the phagocytes are engineered to overexpress Fcγ receptors.

8. The method of claim 7, wherein the phagocyte is engineered to overexpress FcγRIIa.

9. The method of any one of claims 1 to 8, wherein the phagocyte does not express FcγRIII.

10. 10. The method of any one of claims 1 to 9, wherein Aβ is human Aβ.

11. The method of claim 10, wherein the Aβ comprises monomeric and / or oligomeric Aβ.

12. 12. The method of any one of claims 1 to 11, wherein the polypeptide comprises a full-length Fc domain or an FcR-binding fragment of an Fc domain.

13. 13. The method of any one of claims 1 to 12, wherein the polypeptide specifically binds to Aβ.

14. 14. The method of any one of claims 1 to 13, wherein the polypeptide is an antibody or an immunoadhesin.

15. 15. The method of claim 13 or 14, wherein the polypeptide is crenezumab.

16. 16. The method of any one of claims 1 to 15, wherein the target antigen is immobilized on a surface.

17. 17. The method of claim 16, wherein the surface is a plate.

18. 18. The method of claim 17, wherein the plate is a multi-well plate.

19. 19. The method of any one of claims 16 to 18, wherein the target antigen is immobilized to the surface at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus.

20. 20. The method of any one of claims 16 to 19, wherein the target antigen is immobilized on the surface using a biotin-streptavidin system.

21. 21. The method of claim 20, wherein the target antigen is bound to biotin and the surface comprises bound streptavidin.

22. 22. The method of claim 20 or 21, wherein the target antigen is conjugated to biotin at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and at or near the C-terminus.

23. 23. The method of any one of claims 1 to 22, wherein the reporter is detected after any one or more of the following: 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, 24 or more hours after contacting the antigen-polypeptide complex with the phagocyte.

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