Neutralizing antibody assays for therapeutic proteins

Abstract

The present invention relates generally to methods of testing for the presence of neutralizing antibodies (NAbs) against therapeutic proteins. Specifically, the present invention relates to the use of mitigants for interfering competitive drugs in ligand binding assays or cell-based assays for the detection of neutralizing antibodies to therapeutic proteins.SOLUTION: A method for detecting a neutralizing agent to a therapeutic protein in a sample, comprising: (a) contacting the sample having the neutralizing agent and a competing drug with the therapeutic protein, a target of the therapeutic protein, and a mitigant; (b) measuring binding of the therapeutic protein to the target; and (c) comparing the result of (b) to a control measurement to detect the neutralizing agent.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 018,821, filed May 1, 2020, U.S. Provisional Patent Application No. 63 / 041,768, filed June 19, 2020, and U.S. Provisional Patent Application No. 63 / 172,488, filed April 8, 2021, each of which is incorporated herein by reference.

[0002] Field The present application relates to assay methods, modules, and kits for performing diagnostic assays for the detection of neutralizing antibodies against therapeutic proteins. [Background technology]

[0003] background Administration of biological therapeutic agents to patients can induce unwanted immunogenic responses in patients, which can lead to the development of anti-drug antibodies (ADAs) (Mire-Sluis, AR, et al., J Immunol Methods, 289(1):1-16 (2004)). Neutralizing antibodies (NAbs) are a subset of ADAs that inhibit the binding of drugs to their targets, rendering them biologically inactive. By definition, NAbs neutralize the effects of drugs and potentially reduce their clinical activity. In addition, if the drug is a biological mimic of an endogenous protein, NAbs may cross-react with the endogenous analogue of the drug, which may have important consequences for drug safety (Finco, D., et al., J Pharm Biomed Anal, 54(2):351-358(2011) (Non-Patent Document 2); Hu, J., et al., J Immunol Methods, 419:1-8(2015) (Non-Patent Document 3)).

[0004] Detection of immunogenic responses involves a stepwise approach, in which samples are first tested for the presence of ADA, typically using a bridging immunoassay (Mire-Sluis, AR, et al., J Immunol Methods, 289(1):1-16(2004)). Further characterization of the ADA response can include titer assays to determine the relative amount of ADA and assays to determine whether the antibody response is neutralizing (Wu, B., et al., AAPS Journal, 18(6):1335-1350(2016) (Non-Patent Document 5); Shankar, G, et al., J Pharm Biomed Anal 48(5):1267-1281(2008) (Non-Patent Document 6); Gupta, S., et al., J Pharm Biomed Anal 55(5):878-888(2011) (Non-Patent Document 7)).

[0005] NAb assays can be subject to interferences that prevent accurate quantification of neutralization against therapeutic proteins. For example, if the endogenous drug target is soluble, it may be present in the subject sample and competitively bind with the therapeutic agent, resulting in a false-positive NAb signal. Also, residual drug from previous administration of the therapeutic agent may be present in the subject sample, which may competitively bind with the NAb and result in a false-negative NAb signal. To address these sources of interference and obtain accurate quantification of NAbs, various techniques have been developed (Xu, W., et al., J Immunol Methods, 462:34-41(2018) (Non-Patent Document 8); Xu, W., et al., J Immunol Methods, 416:94-104(2015) (Non-Patent Document 9); Xiang, Y., et al., AAPS Journal, 21(1):4(2019) (Non-Patent Document 10); Sloan, JH, et al., Bioanalysis, 8(20):2157-2168(2016) (Non-Patent Document 11)).

[0006] An additional source of potential interference that has not yet been characterized is interference from residual drugs that are different from the therapeutic protein being tested and competitively bind to the same drug target as the therapeutic, resulting in false-positive NAb signals. Therefore, no strategies have been developed to date to mitigate this type of interference.

[0007] It will therefore be appreciated that a need exists for methods to identify and mitigate interference from competing drugs in ligand binding assays or cell-based assays for the detection of neutralizing antibodies to therapeutic proteins. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Mire-Sluis, AR, et al., J Immunol Methods, 289(1):1-16(2004) [Non-patent document 2] Finco, D., et al., J Pharm Biomed Anal, 54(2):351-358(2011) [Non-patent document 3] Hu, J., et al., J Immunol Methods, 419:1-8(2015) [Non-patent document 4] Mire-Sluis, AR, et al., J Immunol Methods, 289(1):1-16(2004) [Non-Patent Document 5] Wu, B., et al., AAPS Journal, 18(6):1335-1350(2016) [Non-patent document 6] Shankar, G, et al., J Pharm Biomed Anal48(5):1267-1281(2008) [Non-Patent Document 7] Gupta, S., et al., J Pharm Biomed Anal, 55(5):878-888(2011) [Non-patent document 8] Xu,W.,et al.,J Immunol Methods,462:34-41(2018) [Non-Patent Document 9] Xu, W., et al., J Immunol Methods, 416:94-104(2015) [Non-Patent Document 10] Xiang, Y., et al., AAPS Journal, 21(1):4(2019 [Non-Patent Document 11] Sloan, JH, et al.,Bioanalysis,8(20):2157-2168(2016) Summary of the Invention

[0009] overview The present disclosure provides methods for detecting a neutralizing agent of a therapeutic protein in a sample. In some exemplary embodiments, the methods include (a) contacting the sample having the neutralizing agent and a competing drug with (i) the therapeutic protein, (ii) a target of the therapeutic protein, and (iii) a mitigating agent, (b) measuring binding of the therapeutic protein to the target, and (c) comparing the results of (b) with a control measurement to detect the neutralizing agent.

[0010] In one embodiment, the control measurement is obtained by measuring binding of the therapeutic protein to the target in the absence of a neutralizing agent, hi another embodiment, the neutralizing agent is a neutralizing antibody.

[0011] In one embodiment, the therapeutic protein is an antibody, a soluble receptor, an antibody-drug conjugate, or an enzyme. In a specific embodiment, the therapeutic protein is a monoclonal antibody. In yet another specific embodiment, the monoclonal antibody is an anti-PD-1 antibody, an anti-TNF antibody, an anti-PD-L1 antibody, an anti-EGFR antibody, an anti-CD20 antibody, an anti-CD38 antibody, or an anti-LAG3 antibody.

[0012] In one embodiment, the therapeutic protein is a bispecific antibody. In a specific embodiment, the bispecific antibody is a CD20xCD3 antibody, a BCMAxCD3 antibody, an EGFRxCD28 antibody, or a CD38xCD28 antibody.

[0013] In one aspect, the therapeutic protein is immobilized on a solid support. In another aspect, the therapeutic protein is labeled for detection. In a particular aspect, the label is detectable by fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, or affinity purification. In yet another particular aspect, the label comprises ruthenium.

[0014] In one embodiment, the target is an antigen, receptor, ligand, or enzyme substrate. In another embodiment, the target is a cell surface protein. In yet another embodiment, the target is a recombinant protein. In yet another embodiment, the target is expressed by a cell. In particular embodiments, the cell is a HEK293 cell, a MOLP-8 cell, a Jurkat cell, or modified versions thereof.

[0015] In one embodiment, the target is immobilized on a solid support. In another embodiment, the target is labeled for detection. In certain embodiments, the label is detectable by fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, or affinity purification. In another embodiment, the target is an enzyme substrate. In yet another embodiment, the target is CD20, CD3, BCMA, PD-1, EGFR, CD28, CD38, TNF, PD-L1, or LAG3. In yet another embodiment, the method additionally includes a second target.

[0016] In one aspect, the competing drug is a monoclonal antibody. In a specific aspect, the competing drug is rituximab, pembrolizumab, nivolumab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ublituximab, cetuximab, daratumumab, or adalimumab. In another aspect, the competing drug is a bispecific antibody.

[0017] In one embodiment, the mitigating agent is a monoclonal antibody. In another embodiment, the method comprises using two, three, four, or more mitigating agents.

[0018] In one aspect, binding of the therapeutic protein to the target is measured by measuring receptor phosphorylation, phosphorylation of downstream proteins in a signaling pathway, cytokine release, cell proliferation, cell death, or production of secondary proteins. In another aspect, binding of the therapeutic protein to the target is measured by expression of a reporter gene. In a particular aspect, the reporter gene is luciferase.

[0019] In one aspect, the method further comprises a pretreatment step of contacting the sample with the mitigating agent prior to contacting the sample with the therapeutic protein or the target.

[0020] The present disclosure also provides kits for carrying out the methods of the invention. In some exemplary embodiments, the kits include a therapeutic protein, a target of the therapeutic protein, a neutralizer for the therapeutic protein, a competitor, and a mitigator.

[0021] In one aspect, the kit further comprises cells expressing the target. In a particular aspect, the kit further comprises cells that produce a measurable activity or signal in response to binding of the therapeutic protein to the target. In another particular aspect, the activity is expression of luciferase.

[0022] In one aspect, the target is immobilized on a solid support. In another aspect, the kit further comprises a label attached to the therapeutic protein. In a particular aspect, the label comprises ruthenium.

[0023] These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the present invention. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1A shows a diagram of a cell-based neutralizing antibody (NAb) assay according to an exemplary embodiment. Figure 1B shows an increase in luciferase activity with increasing concentrations of a bispecific CD20xCD3 drug antibody, while a negative control antibody does not induce a luciferase signal, according to an exemplary embodiment. Figure 1C shows an increase in luciferase activity with increasing concentrations of two bispecific BCMAxCD3 drug antibodies, according to an exemplary embodiment. [Figure 2A] FIG. 1 shows a diagram of a cell-based NAb assay with the addition of neutralizing antibodies against each arm of a therapeutic antibody, according to an exemplary embodiment. [Figure 2B] 1 shows the decrease in luciferase activity with increasing concentrations of a surrogate neutralizing antibody against either the CD20 arm or the CD3 arm of a bispecific CD20xCD3 drug antibody, according to an exemplary embodiment. [Figure 2C] 1 shows the decrease in luciferase activity with increasing concentrations of a surrogate neutralizing antibody against the BCMA arm of a bispecific BCMAxCD3 drug antibody, according to an exemplary embodiment. [Figure 2D] 1 shows the decrease in luciferase activity with increasing concentrations of a surrogate neutralizing antibody against the CD3 arm of a bispecific BCMAxCD3 drug antibody, according to an exemplary embodiment. [Figure 2E] 1 shows no change in luciferase activity with the addition of an isotype control antibody for an NAb assay for a bispecific BCMAxCD3 drug antibody according to an exemplary embodiment. [Figure 2F]1 shows the decrease in luciferase activity with increasing concentrations of a surrogate neutralizing antibody against the BCMA arm of a second bispecific BCMAxCD3 drug antibody according to an exemplary embodiment. [Figure 2G] 1 shows the decrease in luciferase activity with increasing concentrations of a surrogate neutralizing antibody against the CD3 arm of a second bispecific BCMAxCD3 drug antibody according to an exemplary embodiment. [Figure 2H] 10 shows no change in luciferase activity with the addition of an isotype control antibody for an NAb assay for a second bispecific BCMAxCD3 drug antibody according to an exemplary embodiment. [Figure 3A] 1 shows the reduction of luciferase activity in an NAb assay for a bispecific CD20xCD3 drug antibody by the addition of a competing antibody against the drug target CD20, according to an exemplary embodiment. [Figure 3B] 1 shows the reduction of luciferase activity in an NAb assay for a bispecific CD20xCD3 drug antibody by the addition of a competing antibody against the drug target CD3, according to an exemplary embodiment. [Figure 3C] 1 shows the reduction of luciferase activity in an NAb assay for a bispecific BCMAxCD3 drug antibody by the addition of a competing antibody against the drug targets BCMA or CD3, according to an exemplary embodiment. [Figure 3D] 1 shows the reduction of luciferase activity in an NAb assay for a bispecific BCMAxCD3 drug antibody by the addition of a competing antibody against the drug targets BCMA or CD3, according to an exemplary embodiment. [Figure 3E] FIG. 10 shows the reduction of luciferase activity in an NAb assay for a second bispecific BCMAxCD3 drug antibody upon addition of a competing antibody against the drug targets BCMA or CD3, according to exemplary embodiments. [Figure 3F] FIG. 10 shows the reduction of luciferase activity in an NAb assay for a second bispecific BCMAxCD3 drug antibody upon addition of a competing antibody against the drug targets BCMA or CD3, according to exemplary embodiments. [Figure 4] Figure 4A shows an increase in luciferase activity in an NAb assay with increasing concentrations of a therapeutic antibody, according to an exemplary embodiment. The addition of naive human serum did not affect the luciferase activity. Figure 4B shows quantification of the NAb assay signal by comparing luciferase activity in the presence of a drug control with luciferase activity in the presence of an experimental sample, according to an exemplary embodiment. [Figure 5] 1 shows cell-based NAb assay results from 60 drug-naive clinical samples according to an exemplary embodiment. [Figure 6] 1 shows the correlation between the concentration of rituximab in clinical samples and the NAb assay signal, according to an exemplary embodiment. [Figure 7] Figure 7A shows a diagram of a cell-based NAb assay with the addition of rituximab, Figure 7B shows a diagram of an NAb assay with the addition of rituximab and a milder antibody to rituximab, and Figure 7C shows the recovery of luciferase activity in an NAb assay with the addition of a milder antibody to rituximab, according to an exemplary embodiment. [Figure 8] 1 shows the reduction of false positive NAb assay signals in drug-naive clinical samples with the addition of a mitigating antibody to rituximab, according to an exemplary embodiment. [Figure 9] Figure 9A shows a diagram of a target capture ligand-binding NAb assay according to an exemplary embodiment, Figure 9B shows a diagram of a target capture ligand-binding NAb assay with the addition of an NAb against an arm of a therapeutic protein according to an exemplary embodiment, and Figure 9C shows a diagram of a drug capture ligand-binding NAb assay according to an exemplary embodiment. [Figure 10] Figure 10A shows a diagram of a ligand-binding NAb assay with the addition of a competing drug, according to an exemplary embodiment. Figure 10B shows the increase in false-positive signal inhibition in a ligand-binding NAb assay with increasing concentrations of a competing drug, according to an exemplary embodiment. [Figure 11]Figure 11A shows a diagram of a ligand-binding NAb assay with the addition of a competing drug and a mild antibody against the competing drug, according to an exemplary embodiment. Figure 11B shows the elimination of false-positive NAb assay signals with the addition of a mild antibody against the competing drug, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description Therapeutic proteins are an important class of drugs used to treat various human diseases. However, therapeutic proteins can induce immune responses in recipients, generating anti-drug antibodies (ADAs). Neutralizing antibodies (NAbs) are a subpopulation of ADAs that can potentially impact patient safety and mediate loss of efficacy by blocking the biological activity of therapeutic proteins. Therefore, characterization and monitoring of NAbs is an important aspect of immunogenicity assessment and requires sensitive and reliable methods that reflect the therapeutic mechanism of action (Wu, B., et al., AAPS Journal, 18(6):1335-1350 (2016)).

[0026] NAb assays are expected to reliably detect NAbs with adequate sensitivity, specificity, selectivity, and accuracy. Both cell-based and non-cell-based assays are options for NAb evaluation. Generally, NAb assays present the target of a therapeutic protein and the mechanism of signal output in response to the binding of the therapeutic protein to its target, allowing for quantification of binding. If NAbs are present in a co-incubated sample, they inhibit the binding of the therapeutic protein to its target and reduce the signal output, allowing for quantification of NAbs in the sample.

[0027] The sample matrix may contain interfering substances that prevent accurate quantification of NAbs, for example, by directly interacting with the NAb, the therapeutic protein, or the target. Matrix components that may interfere by interacting with and occupying the NAb include, for example, residual drugs from previous administration of a therapeutic protein. Another component that may interfere by interacting with and occupying the therapeutic protein includes, for example, a soluble drug target. These interfering substances have been characterized in the prior art, and techniques have been developed to address these sources of interference and obtain accurate quantification of NAbs (Xu, W., et al., J Immunol Methods, 462:34-41(2018); Xu, W., et al., J Immunol Methods, 416:94-104(2015); Xiang, Y., et al., AAPS Journal, 21(1):4(2019); Sloan, JH, et al., Bioanalysis, 8(20):2157-2168(2016)).

[0028] However, another possible interfering substance that has not yet been characterized or addressed is residual competing drugs in the subject sample that are different from the therapeutic protein being tested, which may interact with and occupy the therapeutic protein target, resulting in false-positive quantification of NAbs.

[0029] To address the challenge of accurately measuring neutralizing antibodies to a therapeutic protein, methods and kits are described herein for using mitigating agents against competing drugs to prevent interference in neutralizing antibody assays. Also disclosed herein is the detection of interference in NAb assays from drugs that competitively bind to the target of a therapeutic protein. This interference can result in a reduction in the therapeutic protein binding signal or activity in the NAb assay and a false-positive NAb assay signal. To overcome this interference, mitigating agents can be used that reduce binding of the competing drug to the target, allowing the therapeutic protein to bind to its target and restoring an accurate NAb assay signal.

[0030] Interference from residual competing drugs is a significant challenge in accurately assessing NAbs when testing therapeutic proteins for clinical use, as demonstrated, for example, in Examples 5 and 6. Novel therapeutics may be tested after patients have already received first-line therapy that may competitively interact with the same target. In these cases, interference from competing drugs must be identified and mitigated. For example, numerous drug candidates with shared targets of B-cell maturation antigen (BCMA) or CD3 are listed in Table 1. Other therapeutic targets for which many competing drugs may exist include, for example, epidermal growth factor receptor (EGFR), which can be targeted by drugs or drug candidates such as cetuximab; CD28; CD38, which can be targeted by drugs or drug candidates such as daratumumab; lymphocyte-activation gene 3 (LAG3); programmed cell death protein 1 (PD-1), which can be targeted by drugs or drug candidates such as cemiplimab, pembrolizumab, or nivolumab; programmed death-ligand 1 (PD-L1); tumor necrosis factor (TNF), which can be targeted by drugs or drug candidates such as adalimumab; or CD20, which can be targeted by drugs or drug candidates such as rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, or ublituximab. The present disclosure teaches methods suitable for mitigating NAb assay interference from these and other drugs and drug candidates.

[0031] Table 1. Examples of drug candidates with shared targets TIFF2026012788000002.tif170163

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, particular methods and materials are now described.

[0033] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variation as understood by one of ordinary skill in the art, and when ranges are provided, the endpoints are included. As used herein, the terms "include," "includes," and "including" are intended to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0034] As used herein, the terms "protein" or "protein of interest" can include any amino acid polymer having covalently linked amide bonds. Proteins comprise one or more amino acid polymer chains, generally known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof linked via peptide bonds, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein can comprise one or more polypeptides to form a single functional biomolecule. Proteins can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. The protein of interest may include any of the following: biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. Proteins may be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells), etc.For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation" (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings of which are incorporated herein). Proteins can be classified based on composition and solubility and thus can include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

[0035] In some exemplary embodiments, the protein of interest may be a recombinant protein, an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a fusion protein, and combinations thereof.

[0036] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., an IgG1 or IgG4 immunoglobulin), or alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0037] As used herein, the term "antibody" includes immunoglobulin molecules, including four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, namely, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present invention, the FRs of an anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs. The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. Antibody-binding fragments of antibodies can be obtained from complete antibody molecules using any suitable standard technique, such as proteolytic digestion involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains, or recombinant genetic engineering techniques. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized.The DNA can be sequenced and chemically manipulated by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0038] As used herein, "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of the antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are connected by a peptide linker. In some exemplary embodiments, an antibody fragment contains the full amino acid sequence of a parent antibody such that the fragment binds to the same antigen as the parent antibody, and in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and / or it may be recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single-chain antibody fragment. Alternatively or additionally, an antibody fragment may comprise multiple chains linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multimolecular complex. Functional antibody fragments typically comprise at least about 50 amino acids, more typically at least about 200 amino acids.

[0039] The term "bispecific antibody" includes antibodies capable of selectively binding to two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, each of which specifically binds to a different epitope, either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least one to two, or three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells that express immunoglobulin light chains.

[0040] A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, hinge, CH2 domain, and CH3 domain, and an immunoglobulin light chain, which does not confer antigen-binding specificity but can associate with each heavy chain, or with each heavy chain and associate with one or more of the epitopes bound by the heavy chain antigen-binding region, or with each heavy chain and enable one or both of the heavy chains to bind one or both epitopes. BsAbs can be divided into two major classes: those that retain the Fc region (IgG-like) and those that lack the Fc region, the latter usually being smaller than Fc-containing IgG and IgG-like bispecific molecules. IgG-like bsAbs can have different formats such as, but not limited to, triomab, knob-into-hole IgG (kih IgG), crossMab, ortho-Fab IgG, dual variable domain Ig (DVD-Ig), two-in-one or dual acting Fab (DAF), IgG-single chain Fv (IgG-scFv), or κλ-body. Different non-IgG-like formats include tandem scFvs, diabody formats, single-chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DARTs), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014) (the teachings of which are incorporated herein in their entirety)).

[0041] As used herein, a "multispecific antibody" refers to an antibody that has binding specificities for at least two different antigens. Such molecules typically bind only two antigens (i.e., bispecific antibodies, bsAbs), but antibodies with additional specificities, such as trispecific antibodies and KIH trispecifics, may be subject to the systems and methods disclosed herein.

[0042] The term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure may be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0043] In some exemplary embodiments, the protein of interest may be produced from mammalian cells. Mammalian cells may be of human or non-human origin, and may include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their derivatives (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells, and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 cells, and the like). VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PKi cells, PK(15) cells, GHi cells, G H3 cells, L2 cells, LLC-RC256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells, or derivatives thereof), any Fibroblasts derived from tissues or organs (including, but not limited to, heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphatic tissue (lymph glands, adenoids, tonsils, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, D BS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells The cells may include, for example, mouse L cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, 2071 strain (mouse L) cells, LM strain (mouse L) cells, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, and Jensen cells, Sp2 / 0, NS0, NS1 cells, or derivatives thereof.

[0044] As used herein, the term "therapeutic protein" refers to any protein that can be administered to a subject for the treatment of a disease or disorder. In some exemplary embodiments, the therapeutic protein is intended for the treatment of cancer. A therapeutic protein may be any protein with a pharmacological effect, such as an antibody, a soluble receptor, an antibody-drug conjugate, or an enzyme. In some exemplary embodiments, the therapeutic protein may be a bispecific CD20xCD3 antibody. In some exemplary embodiments, the therapeutic protein may be a bispecific BCMAxCD3 antibody. In some exemplary embodiments, the therapeutic protein may be a monoclonal antibody against programmed cell death protein 1 (PD-1), such as cemiplimab. In other embodiments, the therapeutic protein may be a bispecific EGFRxCD28 antibody, a bispecific CD38xCD28 antibody, a monoclonal anti-TNF antibody, a monoclonal anti-PD-L1 antibody, a monoclonal anti-EGFR antibody, a monoclonal anti-CD20 antibody, a monoclonal anti-CD38 antibody, or a monoclonal anti-LAG3 antibody.

[0045] As used herein, the term "target" refers to any molecule that can specifically interact with a therapeutic protein to achieve a pharmacological effect. For example, the target of an antibody may be the antigen to which the antibody is directed, the target of a ligand may be the receptor to which the ligand selectively binds, or vice versa, the target of an enzyme may be the substrate to which the enzyme selectively binds, etc. A single therapeutic protein may have two or more targets. Various targets are suitable for use in the methods of the present invention depending on the particular application. The target may be, for example, present on the cell surface, soluble, cytoplasmic, or immobilized on a solid surface. The target may be a recombinant protein. In some exemplary embodiments, the target may be CD20, CD3, BCMA, PD-1, EGFR, CD28, CD38, TNF, PD-L1, or LAG3.

[0046] As used herein, the term "anti-drug antibody" or "ADA" refers to an antibody produced by a subject's immune system that targets an epitope on a therapeutic protein. A subset of ADAs are "neutralizing antibodies" or "NAbs," which can bind to a therapeutic protein in a manner that inhibits or neutralizes its pharmacological activity. NAbs can affect the clinical efficacy of a therapeutic protein and therefore must be monitored when a therapeutic protein is administered to a subject.

[0047] As used herein, the term "neutralizer" refers to a molecule that can interact with a therapeutic protein in a manner that inhibits or neutralizes its pharmacological activity. A neutralizer can be, for example, an oligonucleotide such as an aptamer, or a protein such as an antibody. A neutralizer can arise from a variety of sources, for example, by chemical synthesis, by recombinant production, or from the subject's immune system. For simplicity, neutralizing antibodies (NAbs) produced by the subject's immune system are the primary neutralizers discussed herein, although it should be understood that the methods of the present invention can be applied to the detection of any neutralizer.

[0048] NAbs can be monitored using a variety of assays. NAb assays can be broadly classified as cell-based or non-cell-based. The choice of cell-based versus non-cell-based assay depends on the therapeutic protein, target, and application in question, and those skilled in the art will be able to select an assay according to their needs.

[0049] Cell-based assays involve at least one cell type. A therapeutic protein may bind to a target such that a cellular event is affected, which can then be measured as an output of therapeutic protein binding. Useful cellular events that result in a measurable signal or activity may include, for example, receptor phosphorylation, phosphorylation of a downstream protein in a signal transduction pathway, cytokine release, cell proliferation, cell death, production of a secondary protein, or any other cellular activity. Additionally or alternatively, a reporter gene may be used that is expressed in response to a cellular event caused by a therapeutic protein binding to a target, for example, a fluorescent protein such as luciferase, green fluorescent protein (GFP), or any variant thereof.

[0050] Measuring a signal generated by a therapeutic protein binding to a target and measuring inhibition of that signal by an NAb can be referred to as a "direct" cell-based assay. Conversely, in an "indirect" cell-based assay, binding of the therapeutic protein to a target inhibits a measurable signal, and restoration of that signal is used to detect an NAb. For simplicity, the discussion will be limited to direct cell-based assays, although the methods described herein can be applied to indirect cell-based assays as well.

[0051] Disclosed herein is a cell-based NAb assay comprising two cell types that produce measurable cellular events when crosslinked by a therapeutic bispecific antibody. Each cell type may present a target on its cell surface, the antigen recognized by one arm of the bispecific antibody. Simultaneous binding of both targets crosslinks the two cells, resulting in downstream cellular events that can be measured as an indicator of therapeutic protein binding. Examples of cells used in cell-based NAb assays include HEK293 / hCD20 cells, which express human CD20, MOLP-8 cells, which endogenously express BCMA, and Jurkat / NFAT-Luc cells. Jurkat / NFAT-Luc cells express CD3 and a T cell receptor (TCR) on their cell surface. When a bispecific antibody, such as a bispecific CD20xCD3 antibody or a bispecific BCMAxCD3 antibody, crosslinks this cell with a second cell, the TCR initiates a signaling pathway that leads to the expression of a luciferase reporter, generating a measurable signal. This signal can be reduced by the presence of NAbs in the assay or by competing drugs, as further described in the Examples.

[0052] It should be understood that many cell types can be used in the cell-based assays of the invention, depending on the therapeutic protein and target being tested, provided that the cells express or can be modified to express the target and / or can respond to binding of the therapeutic protein and target by producing a measurable signal or activity. Non-limiting examples of cells that can be used in the methods of the invention include HEK293 cells, HEK293 / hCD20 cells, HEK293 / MfBCMA cells, HEK293 / hBCMA cells, NCI-H929 cells, MOLP-8 cells, Jurkat cells, Jurkat / NFAT-Luc cells, Jurkat / NFAT-Luc / MfCD3 cells, and modified versions thereof.

[0053] Non-cell-based assays can detect the presence of NAbs in the absence of cells. One type of non-cell-based assay is called a competitive ligand binding (CLB) assay. A CLB assay, or as referred to herein, a ligand binding assay, measures the binding of a therapeutic protein to a target, which can be, for example, a purified recombinant protein or a natural target associated with a prepared cell membrane. The target can be immobilized on a solid support, such as a microplate or beads, to allow capture of a labeled therapeutic protein, and detection of the label can be used to measure binding. NAbs in the sample block the binding of the therapeutic protein to the target, reducing the signal. Alternatively, the therapeutic protein can be immobilized on a solid surface while a soluble target is labeled, and the same principle applies in another way. The label can be detectable, for example, by fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, or affinity purification, and / or can provide a signal or activity.

[0054] Measuring a signal generated by a therapeutic protein binding to a target and measuring the inhibition of that signal by an NAb can be referred to as a direct binding assay. Conversely, in an indirect binding assay, binding of a therapeutic protein to a target inhibits a measurable signal, and restoration of that signal is used to detect an NAb. For simplicity, the discussion will be limited to direct binding assays, but the methods described herein can be applied to indirect binding assays as well.

[0055] Disclosed herein is a ligand-binding NAb assay comprising a biotinylated target, e.g., PD-1, immobilized on an avidin-coated microplate and co-incubated with a ruthenylated therapeutic protein, e.g., cemiplimab. Binding of labeled cemiplimab to immobilized PD-1 allows for detection of a signal that can be used to measure this binding. As discussed further in the Examples, the presence of an NAb or a competing drug in the assay can reduce this signal.

[0056] The second type of non-cell-based assay is called an enzyme activity-based assay. Enzyme activity-based assays measure the ability of an enzyme preparation to catalyze a reaction biologically relevant to its mechanism of action by converting a suitable substrate into a product. Enzyme activity can be measured by directly measuring the binding of the enzyme to its substrate or by measuring the amount of product produced. The presence of an NAb or a competing drug in the assay can be indicated by reduced binding or reduced production of the product. Therefore, the methods disclosed herein are also applicable to the accurate quantification of NAbs in enzyme activity-based assays.

[0057] To detect the presence of NAb in a sample, an NAb assay should include an experimental condition and a control condition. The experimental condition includes the sample being tested for the presence of NAb. The control condition can be, for example, a negative control condition known to contain no NAb. A signal or activity is generated in the NAb assay as a measure of the therapeutic protein binding to the target, and a reduction in the signal in the experimental condition compared to the control condition is a measure of neutralization of the therapeutic protein and, therefore, the presence of NAb in the experimental condition, as shown, for example, in FIG. 4B.

[0058] Conversely, the positive control condition can be known to contain an NAb or another neutralizing agent and can be used, for example, to validate an NAb assay or to calibrate its signal.

[0059] The change in signal between experimental and control conditions can also be caused by interference from interfering substances. Disclosed herein are methods for reducing such interference so that the presence of NAbs in a sample can be accurately detected.

[0060] As used herein, the term "interfering substance" refers to any molecule present in an NAb assay or sample matrix that may interfere with the accurate measurement of an NAb. Interference may be caused by association with an NAb, a therapeutic protein, a therapeutic protein target, or any component of an NAb assay. Examples of interfering substances may include a soluble target of a therapeutic protein, a protein with a similar sequence to a therapeutic protein targeted by the same NAb, or residual drugs from a previous administration of a therapeutic protein.

[0061] A particular class of interfering substances may be "competing drugs" present in the sample matrix that are not therapeutic proteins but can competitively bind to a component of the NAb assay, such as a therapeutic protein target. Competing drugs may be residual drugs previously administered to a subject. In some exemplary embodiments, competing drugs may competitively bind to therapeutic targets, including, for example, CD20, CD3, BCMA, PD-1, EGFR, CD28, CD38, TNF, PD-L1, or LAG3. In some exemplary embodiments, competing drugs may be any of the drugs or drug candidates listed in Table 1. In some exemplary embodiments, competing drugs may be rituximab, pembrolizumab, nivolumab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ublituximab, cetuximab, daratumumab, or adalimumab.

[0062] As used herein, the term "mitigating agent" refers to any molecule capable of binding to an interfering substance to reduce or prevent interference in an NAb assay and enable accurate detection of NAb in a sample. Any molecule capable of specifically interacting with an interfering substance and preventing its interference with an NAb, therapeutic protein, target, or other components of an NAb assay may be a suitable mitigating agent. The mitigating agent may be, for example, an oligonucleotide such as an aptamer, or a protein such as an antibody. In some exemplary embodiments, the mitigating agent may be a blocking antibody against a competing drug, for example, an anti-rituximab blocking antibody, an anti-pembrolizumab blocking antibody, or an anti-nivolumab blocking antibody.

[0063] Kits for carrying out the methods of the present invention are also disclosed herein. The kits of the present invention allow users to accurately detect the presence of NAb in a sample by mitigating interference from competing drugs. The kits of the present invention may include, for example, a therapeutic protein, a target of the therapeutic protein, a mitigating agent, a means for producing a signal or activity as a measure of binding between the therapeutic protein and the target, and instructions for use of the kit. They may also include a neutralizing substance that can be used as a positive control. They may additionally include a competing drug that can be used as a positive control.

[0064] The kits may be directed to cell-based or non-cell-based NAb assays, or both. Kits directed to cell-based NAb assays may include cells suitable for target expression and for providing a signal or activity as a measure of therapeutic protein binding to the target, such as HEK293 / hCD20 cells, Jurkat / NFAT-Luc cells, MOLP-8 cells, or any other cells capable of expressing a target and / or responding to binding of a therapeutic protein to the target by providing a measurable signal or activity. Suitable targets in kits directed to cell-based NAb assays may be, for example, CD20, CD3, BCMA, EGFR, CD28, CD38, or combinations thereof. Suitable therapeutic proteins may be, for example, a bispecific CD20xCD3 antibody, a bispecific BCMAxCD3 antibody, a bispecific EGFRxCD28 antibody, or a bispecific CD38xCD28 antibody.

[0065] Kits for non-cell-based NAb assays may include a solid support, such as a microplate or beads, capable of binding a target and / or therapeutic protein, for example, by being coated with avidin. They may additionally include a target and / or therapeutic protein that can be bound to the solid support, for example, by being conjugated to biotin. They may further include a labeled target and / or therapeutic protein, for example, a ruthenium-labeled target and / or therapeutic protein. Suitable targets in kits for non-cell-based NAb assays may be, for example, PD-1, TNF, PD-L1, EGFR, CD20, CD38, or LAG3. Suitable therapeutic proteins may be, for example, cemiplimab or a monoclonal antibody against any of the aforementioned targets.

[0066] It is understood that the present invention is not limited to any of the aforementioned therapeutic proteins, targets, neutralizers, cell-based assays, cell types, non-cell-based assays, reporters, labels, interferents, competing drugs, or mitigators, and that any therapeutic protein, target, neutralizer, cell-based assay, cell type, non-cell-based assay, reporter, label, interferent, competing drug, or mitigator may be selected by any suitable means.

[0067] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention. [Example]

[0068] Materials and Methods. The present invention can be practiced by those skilled in the art using methods such as those described in, for example, Sambrook et al. "Molecular Cloning: A Laboratory Manual," 3 rd ed.2001, Ausubel et al. “Short Protocols in Molecular Biology”,5 th ed.1995, “Methods in Enzymology”, Academic Press, Inc., MacPherson, Hames and Taylor (eds.). th ed. 2000, "Methods in Molecular Biology" vol. 149 ("The ELISA Guidebook" by John Crowther) Humana Press 2001, and later editions of these articles (e.g., "Molecular Cloning" by Michael Green (4 thEd.2012) and “Culture of Animal Cells” by Freshney (7 th Ed., 2015), and the latest electronic edition.

[0069] Reagents for practicing embodiments of the methods and kits of the invention include biotinylated PD-1 (target); anti-rituximab antibodies α-Ritux Ab1, α-Ritux Ab2, and α-Ritux Ab3; anti-pembrolizumab and anti-nivolumab antibodies (palliatives); anti-CD3, anti-CD20, anti-BCMA, anti-PD-1 antibodies, rituximab, pembrolizumab, and nivolumab (competitive drugs); and bispecific antibodies CD20xCD3, BCMAxCD3, and cemiplimab (therapeutic proteins), see, e.g., U.S. Patent Nos. 9,657,102 and 10,550,193, the entire teachings of which are incorporated herein by reference. Negative control antibodies, e.g., hIgG1, hIgG4, are available from several commercial sources.

[0070] Cells suitable for practicing embodiments of the methods and kits of the invention include HEK293 / hCD20, MOLP-8, Jurkat / NFAT-Luc, and Jurkat / NFAT-Luc / MfCD3 cells, all of which are available from several commercial sources.

[0071] Luciferase assays are performed according to guidelines from the manufacturer, see for example Promega and ThermoFisher.

[0072] Example 1. Cell-based assay design for detecting neutralizing antibodies (NAb) against a therapeutic protein This example demonstrates the experimental design of a cell-based neutralizing antibody (NAb) assay of the present invention for evaluating therapeutic protein candidates. Briefly, human immortalized B cells engineered to express the cell surface human antigen CD20 were prepared (designated HEK293 / hCD20). These cells represent the "target cells" of the assay, mimicking human cancer cells expressing CD20. In addition, human immortalized T cells expressing the T cell receptor (TCR) and the cell surface antigen CD3 were prepared and engineered to express a reporter gene (luciferase) under the control of a TCR / CD3-inducible promoter (nuclear factor of activated T cells (NFAT)). As shown in Figure 1A, these Jurkat / NFAT-Luc cells represent the "reporter cells" of the assay, mimicking patient immune cells that, when crosslinked with a drug antibody such as a bispecific CD20xCD3 antibody, can bind to and potentially eliminate CD20-expressing cancer cells via a cell-mediated cytotoxic response.

[0073] As shown in Figure 1B, addition of an antibody that binds to CD20 and CD3 (bispecific CD20xCD3 drug antibody), which mediates clustering of T cell receptors (TCRs) on reporter cells, results in expression of the luciferase reporter gene and a robust dose-dependent luciferase signal. Addition of a hIgG4 isotype control antibody did not result in luciferase activity, as indicated by the open square in Figure 1B.

[0074] Another cell-based NAb assay was designed using Jurkat / NFAT-Luc cells as reporter cells, as described above, in combination with MOLP-8 cells as target cells. MOLP-8 is a multiple myeloma cell line that endogenously expresses the cell surface protein B-cell maturation antigen (BCMA). As shown in Figure 1C, the bispecific BCMAxCD3 antibody can crosslink reporter and target cells, mediating TCR clustering on the reporter cells and resulting in expression of a luciferase reporter gene and a dose-dependent luciferase signal. Two BCMAxCD3 antibodies were tested; the dotted lines indicate the concentrations used in subsequent assays.

[0075] These results demonstrate that the cell-based assay of the present invention provides a robust dose-response curve and responds predictably to positive and negative controls.

[0076] Example 2. Detection of NAbs to therapeutic proteins using a cell-based NAb assay This example demonstrates further proof-of-concept of the experimental design of the NAb assay of the present invention. In a cell-based NAb assay, an NAb against a therapeutic protein inhibits binding of the therapeutic protein to its target cells and / or reporter cells, thereby eliminating the reporter signal. A reduction in reporter signal or activity in the NAb assay is a measure of the presence of an NAb in the sample.

[0077] For example, Figure 2A shows the effect of NAbs on a bispecific CD20xCD3 drug antibody. Binding of NAbs to the anti-CD20 or anti-CD3 arms of the bispecific antibody prevents binding to CD20 or CD3, respectively, and abolishes luciferase activity. To further validate this cell-based NAb assay, surrogate NAbs were added to the NAb assay, targeting either the anti-CD20 or anti-CD3 arms of the bispecific CD20xCD3 drug antibody. As shown in Figure 2B, addition of NAbs caused a dose-dependent decrease in luciferase activity.

[0078] The validity of this cell-based NAb assay was further validated for use with two bispecific BCMA×CD3 drug antibodies. Surrogate NAbs were added to the NAb assay, targeting either the anti-BCMA or anti-CD3 arms of the two bispecific BCMA×CD3 drug antibodies. As shown in Figures 2C, 2D, 2F, and 2G, addition of the NAbs caused a dose-dependent decrease in luciferase activity. As shown in Figures 2E and 2H, addition of an isotype control had no effect on luciferase activity.

[0079] These results demonstrate that the assay of the present invention reliably measures the presence of neutralizing antibodies to a therapeutic protein in a dose-dependent manner.

[0080] Example 3. Interference of cell-based NAb assays by competing drugs NAb assays can be prone to false-positive or false-negative results due to interference from matrix components. One potential source of interference is a second drug that competitively binds to the target of the therapeutic protein being tested. As proof of concept for this type of interference, NAb assays of bispecific CD20 × CD3 drug antibodies were performed with the addition of a competing antibody against either CD20 or CD3, as shown in Figures 3A and 3B. Addition of the competing drug caused a dose-dependent reduction in luciferase activity, mimicking the reduction in luciferase activity caused by the surrogate NAb and thus generating a false-positive result.

[0081] Interference from competing drugs was also seen in the NAb assays for the two bispecific BCMA x CD3 drug antibodies. As shown in Figures 3C and 3E, addition of bivalent parent antibodies against BCMA or CD3 resulted in a decrease in luciferase signal in both assays. As shown in Figures 3D and 3F, addition of various clinical candidate antibodies against BCMA also caused a decrease in luciferase signal in both assays.

[0082] These results demonstrate proof-of-concept that the presence of a competing second drug can lead to false-positive results in cell-based NAb assays.

[0083] Example 4. Addition of human serum to a cell-based NAb assay As discussed above, NAb assays can be susceptible to interference from matrix components. To test the resilience of the NAb assay of the present invention against potential interference, we performed an NAb assay of the bispecific CD20 × CD3 drug antibody with the addition of drug-naive human serum, as shown in Figure 4A. Luciferase activity was not affected by the addition of human serum, demonstrating the resilience of the NAb assay of the present invention against interference from human serum components and therefore its suitability for clinical application.

[0084] Figure 4B shows a simple representation of the "NAb assay signal." The relative presence of NAb in a sample is quantified by dividing the luciferase activity induced in the drug control by the luciferase activity induced in the experimental sample. Luciferase activity is reduced in a dose-dependent manner in the presence of NAb, resulting in a higher NAb assay signal.

[0085] Example 5. Interference of cell-based NAb assays in clinical samples The NAb assay of the present invention was used to test 60 drug-naive human samples from a clinical trial for the presence of NAbs against the bispecific CD20xCD3 drug antibody, as shown in Figure 5. Although the patients tested had not been exposed to the drug antibody, many samples showed false-positive results for NAbs.

[0086] As discussed in Example 3, one possible cause of false-positive signals in NAb assays is a competing second drug. Many patients in this clinical trial had a history of anti-CD20 therapy. To evaluate whether competing anti-CD20 drugs could contribute to false-positive results in NAb assays, a subset of 17 human samples was tested for the presence of the anti-CD20 antibody, rituximab, using a commercially available ELISA. As shown in Figure 6, the presence of rituximab correlated with false-positive NAb assay signals.

[0087] These results demonstrate that interference from residual competing drugs can lead to false-positive results in NAb assays in clinical applications and must be addressed to accurately detect NAbs to therapeutic proteins.

[0088] Example 6. Mitigation of cell-based NAb assay interference by competitive drugs As mentioned above, the presence of a competing drug can interfere with the binding of a therapeutic protein to its target in an NAb assay, resulting in reduced reporter activity and a false-positive NAb assay signal. This is shown in Figure 7A using the example of a bispecific CD20xCD3 drug antibody as the therapeutic protein and the anti-CD20 antibody rituximab as the competing drug. To accurately detect NAbs to a therapeutic protein in the presence of a competing drug, binding of the competing drug to the common target must be mitigated. This is shown in Figure 7B using the example of an anti-rituximab antibody as the mitigating agent, preventing interference from the competing drug and enabling accurate detection of NAbs to a therapeutic protein.

[0089] Blocking antibodies against rituximab were tested for their ability to alleviate interference in the NAb assay of the present invention. As shown in Figure 7C, anti-rituximab antibodies were co-incubated in serum spiked with rituximab and added to the NAb assay. Addition of anti-rituximab antibodies restored luciferase activity and eliminated the false-positive NAb assay signal caused by rituximab.

[0090] These results demonstrate that the use of a mitigating agent against a competing drug can eliminate false-positive NAb assay signals and allow accurate detection of NAbs against therapeutic proteins.

[0091] Example 7. Mitigation of cell-based NAb assay interference by competing drugs in clinical samples As shown in Example 5, many drug-naive human samples from clinical trials, when tested for NAb against the bispecific CD20×CD3 drug antibody, resulted in false-positive NAb assay signals, potentially due to the presence of the competing drug, the anti-CD20 antibody rituximab. As shown in Figure 8, NAb assays were performed using clinical samples to which an anti-rituximab blocking antibody was added to mitigate interference from rituximab. Sample No. 1 is a control sample with a low NAb assay signal. Samples No. 2 and No. 3 showed high false NAb assay signals. The addition of the anti-rituximab antibody eliminated the false-positive NAb assay signals.

[0092] These results confirm that residual competing drugs, in this case rituximab, in clinical samples can interfere with NAb assays and lead to inaccurate results. They further demonstrate that mitigation agents for competing drugs can eliminate false-positive NAb assay signals in clinical applications. The use of mitigation agents for competing drugs allows for accurate detection of NAb against the therapeutic protein being tested.

[0093] Example 8. Ligand binding assay design for detecting NAbs against therapeutic proteins This example illustrates the experimental design of a ligand-binding NAb assay of the present invention for evaluating therapeutic protein candidates. An exemplary embodiment of the present invention involves a target capture ligand-binding NAb assay. Briefly, a sample is incubated with a biotinylated target and transferred to an avidin-coated microplate. A ruthenylated drug is added to the microplate in a subsequent step. As shown in Figure 9A, in the absence of an NAb, the ruthenium-labeled drug binds to the immobilized biotin target, generating a signal in the assay. As shown in Figure 9B, in the presence of an NAb, the ruthenium-labeled drug is unable to bind to the biotin target, resulting in inhibition of the assay signal.

[0094] Additional ligand-binding NAb assays may be suitable for evaluating NAbs against therapeutic proteins. For example, instead of target capture design, ligand-binding assays may be designed for drug capture, in which the therapeutic protein of interest is immobilized and the target is labeled to generate an assay signal. Figure 9C shows a ligand-binding assay design in which a biotinylated drug is immobilized on an avidin-coated microplate, a ruthenylated target generates an assay signal, and an NAb against the immobilized drug blocks binding to the target, thereby inhibiting the assay signal.

[0095] Example 9. Interference of Ligand Binding NAb Assays by Competitive Drugs Similar to the cell-based NAb assay described above, ligand-binding NAb assays can be prone to false-positive or false-negative results due to interference from matrix components. As shown in Figure 10A, one potential source of interference is a second drug that competitively binds to the target of the therapeutic protein being tested. For example, the drug antibodies cemiplimab, pembrolizumab, and nivolumab share the same drug target, PD-1. When testing clinical samples for NAbs against cemiplimab, if the binding of ruthenylated cemiplimab to biotinylated PD-1 is used to generate a signal, any residual pembrolizumab, nivolumab, or unlabeled cemiplimab in the clinical sample will competitively bind to the target, inhibiting the assay signal and causing a false-positive result for the presence of an NAb.

[0096] As proof of concept, increasing concentrations of cemiplimab, pembrolizumab, or nivolumab were added to a target capture NAb assay of NAbs to cemiplimab, as shown in Figure 10B. Concentrations of cemiplimab, pembrolizumab, or nivolumab greater than 125 ng / mL inhibited the signal from ruthenylated cemiplimab, resulting in a false-positive NAb assay signal.

[0097] These results demonstrate that the presence of competing drugs can result in false-positive ligand-binding NAb assay signals and must be addressed to accurately detect NAbs against therapeutic proteins.

[0098] Example 10. Mitigation of Ligand Binding NAb Assay Interference by Competitive Drugs As discussed above, the presence of a competing drug can interfere with the binding of a therapeutic protein to its target in a ligand-binding NAb assay, resulting in a reduced signal and a false-positive NAb assay signal. To accurately detect NAbs to a therapeutic protein in the presence of a competing drug, the binding of the competing drug to the common target must be mitigated. This is shown in Figure 11A, using the example of anti-pembrolizumab or anti-nivolumab antibodies as mitigating agents that prevent interference from the competing drug and allow accurate detection of NAbs to a therapeutic protein.

[0099] Blocking antibodies against pembrolizumab and nivolumab were tested for their ability to mitigate interference in the NAb assay of the present invention. As shown in Figure 11B, anti-pembrolizumab or anti-nivolumab antibodies were co-incubated in samples spiked with pembrolizumab or nivolumab, respectively, and added to the ligand-binding NAb assay. The addition of the mitigating agent to the competing drug eliminated false-positive NAb assay signals caused by competitive binding to the target.

[0100] These results demonstrate that the use of a mitigating agent against a competing drug can eliminate false-positive NAb assay signals in ligand binding assays, allowing accurate detection of NAbs against therapeutic proteins.

Claims

1. 1. A method for detecting a neutralizing substance to a therapeutic protein in a sample, comprising: (a) contacting the sample having the neutralizing agent and the competing drug with the therapeutic protein, a target of the therapeutic protein, and a mitigating agent; (b) measuring binding of the therapeutic protein to the target; (c) comparing the results of (b) with control measurements to detect said neutralizing substance; A method comprising:

2. The control measurement measuring binding of the therapeutic protein to the target in the absence of a neutralizing agent. The method of claim 1 , comprising:

3. The method of claim 1 , wherein the neutralizing substance is a neutralizing antibody.

4. 10. The method of claim 1, wherein the therapeutic protein is selected from the group consisting of an antibody, a soluble receptor, an antibody-drug conjugate, and an enzyme.

5. 10. The method of claim 1, wherein the therapeutic protein is a monoclonal antibody.

6. 6. The method of claim 5, wherein the monoclonal antibody is selected from the group consisting of an anti-PD-1 antibody, an anti-TNF antibody, an anti-PD-L1 antibody, an anti-EGFR antibody, an anti-CD20 antibody, an anti-CD38 antibody, and an anti-LAG3 antibody.

7. 10. The method of claim 1, wherein the therapeutic protein is a bispecific antibody.

8. 8. The method of claim 7, wherein the bispecific antibody is selected from the group consisting of a CD20xCD3 antibody, a BCMAxCD3 antibody, an EGFRxCD28 antibody, and a CD38xCD28 antibody.

9. 10. The method of claim 1, wherein the therapeutic protein is immobilized on a solid support.

10. The method of claim 1 , wherein the therapeutic protein is labeled for detection.

11. 11. The method of claim 10, wherein the label is detectable by fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, or affinity purification.

12. The method of claim 11 , wherein the label comprises ruthenium.

13. The method of claim 1 , wherein the target is an antigen, a receptor, a ligand, or an enzyme substrate.

14. The method of claim 1 , wherein the target is a cell surface protein.

15. The method of claim 1 , wherein the target is a recombinant protein.

16. The method of claim 1 , wherein the target is expressed by a cell.

17. 17. The method of claim 16, wherein the cells are HEK293 cells, MOLP-8 cells, Jurkat cells, or modified forms thereof.

18. The method of claim 1 , wherein the target is immobilized on a solid support.

19. The method of claim 1 , wherein the target is labeled for detection.

20. 20. The method of claim 19, wherein the label is detectable by fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, or affinity purification.

21. The method of claim 1 , wherein the target is an enzyme substrate.

22. 2. The method of claim 1, wherein the target is CD20, CD3, BCMA, PD-1, EGFR, CD28, CD38, TNF, PD-L1, or LAG3.

23. The method of claim 1, additionally comprising a second target.

24. The method of claim 1 , wherein the competing drug is a monoclonal antibody.

25. 25. The method of claim 24, wherein the competing drug is rituximab, pembrolizumab, nivolumab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ublituximab, cetuximab, daratumumab, or adalimumab.

26. The method of claim 1 , wherein the competing drug is a bispecific antibody.

27. 10. The method of claim 1, wherein the palliative is a monoclonal antibody.

28. 10. The method of claim 1, comprising using two, three, four, or more emollients.

29. 10. The method of claim 1, wherein binding of the therapeutic protein to the target is measured by measuring receptor phosphorylation, phosphorylation of a downstream protein in a signal transduction pathway, cytokine release, cell proliferation, cell death, or production of a secondary protein.

30. 10. The method of claim 1, wherein binding of the therapeutic protein to the target is measured by expression of a reporter gene.

31. 31. The method of claim 30, wherein the reporter gene is luciferase.

32. a pretreatment step of contacting the sample with the mitigating agent prior to contacting the sample with the therapeutic protein or the target. The method of claim 1 further comprising:

33. (a) a therapeutic protein; and (b) a target of said therapeutic protein; and (c) a neutralizing substance against the therapeutic protein; and (d) a competing drug; and (e) a mitigating agent; Includes a kit.

34. Cells expressing said target 34. The kit of claim 33, further comprising:

35. A cell that produces a measurable activity or signal in response to said binding of said therapeutic protein to said target.

35. The kit of claim 34, further comprising:

36. 36. The kit of claim 35, wherein the activity is expression of luciferase.

37. 34. The kit of claim 33, wherein the target is immobilized on a solid support.

38. 34. The kit of claim 33, further comprising a label attached to the therapeutic protein.

39. 39. The kit of claim 38, wherein the label comprises ruthenium.

Images