Pharmacokinetic immunoassays that suppress interference

Abstract

Herein is reported a method for determining a bispecific antibody in a serum-containing sample, the method comprising the steps of: first incubating a solid phase having immobilized a capture antibody that specifically binds to the bispecific antibody with the sample to form an immobilized capture antibody-bispecific antibody-complex, thereafter, the solid phase is incubated with a replacement antibody that competes with the capture antibody for binding to the bispecific antibody and thereby forms a solution comprising a replacement antibody-bispecific antibody-complex, and the replacement antibody-bispecific antibody-complex is determined in the solution.

Description

[0001] This application is a divisional application of PCT application PCT / EP2020 / 062965, filed on May 11, 2020, with the invention name “Pharmacokinetic Immunoassay with Suppressed Interference”. The date on which the PCT application entered the Chinese national phase is November 11, 2021, and the application number is 202080035191.9. Technical Field

[0002] The present invention belongs to the field of immunoassays, more specifically to the field of pharmacokinetic immunoassays. Reported herein are, among other things, interference suppression immunoassays for detecting bispecific antibodies in serum-containing samples in the presence of interfering monospecific antibodies. Background Art

[0003] In order to analyze therapeutic monoclonal antibodies (tmAbs) in samples derived in vitro or in vivo, corresponding assays are needed, especially in the early stages of tmAb development. This requires the provision of specific binding reagents, such as anti-idiotypic antibodies for determining tmAbs.

[0004] Especially for a complete pharmacokinetic evaluation of a tmAb, it is important to know the soluble tmAb concentration in plasma / serum samples after administration under treatment-like conditions.

[0005] Lee, JW, et al. (AAPS J. 13 (2011) 99-110) reported that the main driver for bioanalysis in support of drug development is the intended use of the data. Reliable methods for measuring circulating mAbs and their antigenic ligands (L) are essential for assessing exposure-response relationships to support efficacy and safety evaluations and dose selection.

[0006] De Zwart et al. (Bioanal. 8 (2016) 2065-2070) reported in a white paper on interference testing of co-medication in bioanalytical method validation. Based on survey data used from previous experience, de Zwart et al. concluded that for both planned and unplanned co-medication, there were no cases of co-medication interfering with the LC-MS / MS assay of the investigational drug (data from 389 observations), whereas for LBA-based assays, the survey data identified only one reported interference in 36 assays.

[0007] Ligand binding assays (LBAs) are widely used to analyze protein biotherapeutics and antigen-ligand (L) to support pharmacokinetic / pharmacodynamic (PK / PD) and safety assessments. Summary of the invention

[0008] An immunoassay for determining the presence or amount of at least a bispecific (therapeutic) antibody in a serum-containing sample is reported herein. In more detail, the assay is a two-step assay comprising a substitution step for reducing interference from a monospecific (therapeutic) antibody also present in the sample. Thus, interference caused by the presence of a second antibody comprising one binding site of the at least bispecific antibody in the immunoassay can be reduced. In an immunoassay according to the invention, this intermediate substitution step is employed for the first time to reduce immunoassay interference.

[0009] According to one aspect of the present invention is a method for determining a multispecific therapeutic antibody, preferably a bispecific antibody, in a serum-containing sample, the method comprising the following steps:

[0010] a) incubating a solid phase on which a capture antibody that specifically binds to the therapeutic antibody has been immobilized with the sample to form an immobilized capture antibody-therapeutic antibody-complex,

[0011] b) optionally washing the solid phase, wherein the capture antibody-therapeutic antibody-complex remains bound to the solid phase,

[0012] c) incubating the solid phase with a displacement antibody that sterically interferes with the binding of the capture antibody to the therapeutic antibody, or competes with the capture antibody for binding to the therapeutic antibody, or binds to the same epitope on the therapeutic antibody as the capture antibody, thereby separating the therapeutic antibody from the solid phase and forming a solution comprising the displacement antibody-therapeutic antibody-complex,

[0013] d) determining said replacement antibody-therapeutic antibody-complex in said solution obtained in step c), thereby determining said therapeutic antibody in the serum-containing sample.

[0014] According to a further aspect of the present invention is a method for enriching therapeutic antibodies, preferably multispecific, most preferably bispecific antibodies, from a serum-containing sample, the method comprising the following steps:

[0015] a) incubating a solid phase on which a capture antibody that specifically binds to the therapeutic antibody has been immobilized with the sample to form an immobilized capture antibody-therapeutic antibody-complex,

[0016] b) optionally washing the solid phase, wherein the capture antibody-therapeutic antibody-complex remains bound to the solid phase,

[0017] c) incubating the solid phase with a replacement antibody that competes with the capture antibody for binding to the therapeutic antibody and thereby separates the therapeutic antibody from the solid phase and forms a solution comprising the replacement antibody-therapeutic antibody-complex, thereby enriching the therapeutic antibody from the serum-containing sample.

[0018] In one embodiment of all aspects and embodiments, the replacement antibody is an antibody with the same specificity as the capture antibody and with (slightly) higher affinity. In one embodiment, the replacement antibody has a Kp1 binding affinity to the therapeutic antibody. D value (as a measure of binding affinity) that is greater than the K of the capture antibody. D An order of magnitude lower.

[0019] In one embodiment of all aspects and embodiments, the capture antibody is biotinylated, the solid phase is conjugated to streptavidin, and immobilization of the capture antibody is performed by interaction of the biotin of the antibody and the streptavidin of the solid phase.

[0020] In one embodiment of all aspects and embodiments, the detection of the replacement antibody-therapeutic antibody-complex is performed in an immunoassay. In one embodiment, the immunoassay is a bridging immunoassay, preferably a bridging ELISA.

[0021] In one embodiment of all aspects and embodiments, the replacement antibody is labeled and the determination of the replacement antibody-therapeutic antibody-complex is performed by detecting the label. In one embodiment, the replacement antibody is conjugated to digoxigenin and the detection is performed by an anti-digoxigenin antibody.

[0022] In one embodiment of all aspects and embodiments, the therapeutic antibody comprises a first binding site that specifically binds to a first antigen and a second binding site that specifically binds to a second antigen. In one embodiment, the therapeutic antibody is a bispecific trivalent antibody having one binding site that specifically binds to a first antigen and two binding sites that specifically bind to a second antigen.

[0023] In one embodiment of all aspects and embodiments, the capture antibody or the replacement antibody or both are anti-idiotypic antibodies. In one embodiment, the capture antibody or the replacement antibody or both are anti-idiotypic antibodies that specifically bind to the first binding site of the therapeutic antibody.

[0024] In a preferred embodiment of all aspects and embodiments, the capture antibody is an anti-idiotypic antibody directed against / specifically binding to the first binding site of the bispecific therapeutic antibody.

[0025] In a preferred embodiment of all aspects and embodiments, the replacement antibody is an anti-idiotypic antibody directed against / specifically binding to the first binding site of the bispecific therapeutic antibody.

[0026] In a preferred embodiment of all aspects and embodiments, the capture antibody and the replacement antibody are different anti-idiotypic antibodies directed against / specifically binding to the first binding site of the bispecific therapeutic antibody.

[0027] In one embodiment of all aspects and embodiments, capture of the replacement antibody-therapeutic antibody soluble complex is performed via an immobilized anti-idiotypic antibody that specifically binds to a second binding specificity of the therapeutic antibody and detection is performed via a detectable label conjugated to the replacement antibody.

[0028] In one embodiment of all aspects and embodiments, step d) is incubating the solution obtained in step c) with a second solid phase to form an immobilized second capture molecule-therapeutic antibody-replacement antibody complex, a second capture molecule that specifically binds to a therapeutic antibody (e.g., a second antigen or an anti-idiotypic antibody that specifically binds to a second binding site of a therapeutic antibody or an antibody that specifically binds to a constant region / constant domain of a human antibody), preferably a second capture antibody (which is different from the first capture antibody) has been immobilized to the second solid phase, wherein the solution contains a replacement antibody-therapeutic antibody-complex, and the determination is performed by determining the immobilized second capture molecule-therapeutic antibody-replacement antibody complex. In one embodiment, the second capture molecule competes with the different replacement antibodies for binding to the therapeutic antibody.

[0029] In one embodiment of all aspects and embodiments, the replacement antibody is an antibody with the same binding specificity (same epitope) as the capture antibody or the capture antibody itself.

[0030] In one embodiment of all aspects and embodiments, the solid phase in step c) is incubated with the replacement antibody at a concentration of about 6.67 nM or higher.

[0031] In one embodiment of all aspects and embodiments, the serum-containing sample comprises a therapeutic antibody (which is a multispecific antibody) and a monospecific antibody comprising one binding site of the therapeutic antibody, and the method is a method of reducing interference of the monospecific antibody when determining the presence or amount of the therapeutic antibody. In one embodiment, the sample is obtained from an individual who has been administered a monospecific antibody prior to administration of the therapeutic antibody.

[0032] In one embodiment of all aspects and embodiments, the therapeutic antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody. In a preferred embodiment, the therapeutic antibody is a bispecific (therapeutic) antibody. In one embodiment, the bispecific (therapeutic) antibody is a TCB.

[0033] In one embodiment of all aspects and embodiments, the bispecific (therapeutic) antibody comprises - a first Fab fragment and a second Fab fragment, wherein each binding site of the first Fab fragment and the second Fab fragment specifically binds to a second antigen,

[0034] - a third Fab fragment, wherein the binding site of the third Fab fragment specifically binds to the first antigen, and wherein the third Fab fragment comprises a domain crossover such that the variable light chain domain (VL) and the variable heavy chain domain (VH) are replaced with each other, and

[0035] - an Fc region comprising a first Fc region polypeptide and a second Fc region polypeptide,

[0036] wherein the first Fab fragment and the second Fab fragment each comprise a heavy chain fragment and a full-length light chain,

[0037] wherein the C-terminus of the heavy chain fragment of the first Fab fragment is fused to the N-terminus of the first Fc region polypeptide,

[0038] The C-terminus of the heavy chain fragment of the second Fab fragment is fused to the N-terminus of the variable light chain domain of the third Fab fragment, and the C-terminus of the heavy chain constant domain 1 of the third Fab fragment is fused to the N-terminus of the second Fc region polypeptide.

[0039] In one embodiment of all aspects and embodiments, the bispecific (therapeutic) antibody is an anti-CD3 / CD20 bispecific antibody. In one embodiment, the anti-CD3 / CD20 bispecific antibody is a TCB with CD20 as the second antigen. In one embodiment, the bispecific anti-CD3 / CD20 antibody is RG6026.

[0040] In one embodiment of all aspects and embodiments, the monospecific antibody is an anti-CD20 antibody. In one embodiment, the anti-CD20 antibody is obinutuzumab.

[0041] In one embodiment of all aspects and embodiments, the method is used to determine a multispecific (bispecific) antibody having target binding capability.

[0042] In one embodiment of all aspects and embodiments, the method is used to determine the target binding capabilities of two (two) binding sites of a multispecific (bispecific) antibody.

[0043] In one embodiment of all aspects and embodiments, the sample comprises at least 10% (v / v) serum. In one embodiment, the sample comprises about 50% (v / v) serum.

[0044] In one embodiment of all aspects and embodiments, the solid phase is a well of a multiwell plate (MTP). In one embodiment, the MTP is a 96-well multiwell plate. In one embodiment, the wells of the multiwell plate have a total volume of 320 μL-360 μL. In one embodiment, the wells of the multiwell plate have a working volume of 100 μL-200 μL.

[0045] In one embodiment of all aspects and embodiments, the replacement antibody has a concentration of 0.75 μg / mL or more. In a preferred embodiment, the replacement antibody has a concentration of about 1 μg / mL or more.

[0046] In one embodiment of all aspects and embodiments, more than 100 μL of the solution comprising the replacement antibody is added to the solid phase. In one embodiment, more than 115 μL of the solution comprising the replacement antibody is added to the solid phase. In a preferred embodiment, 135 μL or more of the solution comprising the replacement antibody is added.

[0047] In one embodiment of all aspects and embodiments, the incubation with the replacement antibody is 1 hour or longer. In one embodiment, the incubation with the replacement antibody is 1.5 hours or longer. In one embodiment, the incubation with the replacement antibody is 2 hours or longer. In a preferred embodiment, the incubation with the replacement antibody is about 2.5 hours or longer.

[0048] In one embodiment of all aspects and embodiments, more than 100 μL of a solution comprising a replacement antibody at a concentration of about 0.75 μg / mL or more is added to the solid phase and incubated for about 1.5 hours or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Immunoassay protocol for determination of bispecific antibodies: immobilization of the bispecific antibody to a solid phase (SA-MTP; streptavidin-coated microtiter plate) via a capture reagent and determination of bound bispecific antibody via a detection reagent.

[0050] Figure 2 Biased immunoassay protocol: The monospecific antibodies interact directly with the solid phase and thereby bias the specific capture step.

[0051] Figure 3Concentration-response curves of a bridging immunoassay using an anti-idiotypic anti-CD3 binding site antibody as capture antibody and an anti-idiotypic anti-CD20 binding site antibody as detection antibody for determination of bispecific CD20-TCBs in the presence of the corresponding monospecific anti-CD20 antibodies.

[0052] Figure 4 Concentration-response curves of a bridging immunoassay using an anti-idiotypic anti-CD3 binding site antibody as capture antibody and an anti-idiotypic anti-CD20 binding site antibody as detection antibody for determination of bispecific CD20-TCBs in the absence of the corresponding monospecific anti-CD20 antibody.

[0053] Figure 5 Concentration-response curves of a bridging immunoassay using anti-idiotypic anti-CD3 binding site antibody as capture antibody and anti-P329G antibody as detection antibody were used to determine bispecific CD20-TCBs in the presence and absence of the corresponding monospecific anti-CD20 antibodies.

[0054] Figure 6 The concentration-response curves of the bridging immunoassay according to the invention comprise an additional displacement step in the presence and absence of the respective monospecific anti-CD20 antibody. DETAILED DESCRIPTION

[0055] Herein is reported an immunoassay for determining at least a bispecific antibody in a serum-containing sample using a displacement step. Thus, interferences in the immunoassay caused by the presence of a second antibody comprising one binding site of the at least bispecific antibody can be reduced. In an immunoassay according to the invention, for the first time such an intermediate displacement step is employed to reduce immunoassay interferences.

[0056] The present invention is based at least in part on the discovery that the detection of multispecific therapeutic antibodies in serum-containing samples in immunoassays can be improved (nonspecific interference caused by the presence of a second antibody comprising one binding site of the multispecific antibody is reduced or even eliminated) by inserting an additional displacement step after the capture of the multispecific therapeutic antibody on a solid phase. In the displacement step, the capture antibody-therapeutic antibody-complex is incubated with a replacement antibody that competes (sterically) with the capture antibody for binding to the therapeutic antibody, for example by binding to the same epitope or a closely related epitope on the therapeutic antibody. The displacement of the capture antibody by the replacement antibody affects the desorption of the captured therapeutic antibody from the solid phase to form an intermediate soluble complex. This intermediate complex can then be detected in a subsequent step with reduced or even eliminated interference caused by the antibody comprising one binding site of the therapeutic antibody.

[0057] definition

[0058] The term "about" means a range of + / -20% of the value that follows. In one embodiment, the term "about" means a range of + / -10% of the value that follows. In one embodiment, the term "about" means a range of + / -5% of the value that follows.

[0059] The terms "therapeutic (monoclonal) antibody" and "drug" are used interchangeably herein. These terms are used in the broadest sense and encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0060] Antibodies generally comprise two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each of the heavy chain polypeptides and the light chain polypeptides contains a variable domain (variable region) (usually the amino terminal portion of the polypeptide chain), which contains a binding region that can interact with an antigen. Each of the heavy chain polypeptides and the light chain polypeptides comprises a constant region (usually the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of antibodies i) to cells (such as phagocytes) carrying Fcγ receptors (Fc R) or ii) to cells carrying neonatal Fc receptors (FcRn) (also referred to as Brambell receptors). It also mediates the binding to some factors, including factors of the classical complement system, such as components (C1q). The constant domain of the heavy chain of an antibody comprises a CH1 domain, a CH2 domain, and a CH3 domain, while the light chain comprises only one constant domain CL, which can be a κ isotype or a λ isotype.

[0061] The variable domain of an immunoglobulin light or heavy chain comprises different segments, namely four framework regions (FR) and three hypervariable regions (HVR).

[0062] As used herein, the term "hypervariable region" or "HVR" refers to the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0063] (a) present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32

[0064] (H1), 53-55 (H2), and 96-101 (H3) hypervariable loops (Chothia and Lesk, J.

[0065] Mol.Biol.196:901-917(1987));

[0066] (b) present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b

[0067] (H1), 50-65 (H2), and 95-102 (H3) CDRs (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0069] (c) present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b

[0070] Antigenic contact points at (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0071] (d) a combination of (a), (b) and / or (c) comprising HVR amino acid residues 46-56 (L2), 47-56

[0072] (L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65

[0073] (H2), 93-102(H3) and 94-102(H3).

[0074] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al.

[0075] The term "valency" as used in this application indicates the presence of a specified number of binding sites in a (antibody) molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" indicate the presence of two binding sites, four binding sites and six binding sites in a (antibody) molecule, respectively. A bispecific antibody as reported herein is a preferred embodiment of "bivalent".

[0076] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0077] The term "binding (to an antigen)" means that an antibody binds to its antigen in an in vitro assay (in one embodiment, in a binding assay), wherein the antibody is bound to a surface and the binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). Binding means measurement of, for example, the binding ability of an antibody to target A or target B or the binding ability of an antibody to a capture molecule (e.g., anti-human Fab capture against the antibody).

[0078] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production process of monoclonal antibody preparations, such variants are usually presented in small amounts), each antibody comprising the population is identical and / or binds to the same epitope. Contrary to the polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0079] The term "sample" includes, but is not limited to, any amount of material from an organism or formerly an organism. Such organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. In one embodiment, the sample is obtained from a monkey, particularly a cynomolgus monkey, or a rabbit, or a mouse, or a rat, or a human. In a preferred embodiment, the sample is a human sample. In one embodiment, such material includes, but is not limited to, whole blood, plasma, or serum from an individual, which are the most widely used sample sources in clinical routine.

[0080] The term "solid phase" refers to non-fluid substances and includes: particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic particles, ferromagnetic particles), glass and ceramics; gel substances such as silica gel, alumina gel and polymer gel; capillaries, which can be made of polymers, metals, glass and / or ceramics; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, tubes or other spectrometer sample containers. The solid phase component is distinguished from an inert solid surface in that the "solid phase" contains at least one portion on its surface that is intended to interact with substances in the sample. The solid phase can be a fixed component such as a tube, strip, cuvette or microtiter plate, or it can be a non-fixed component such as beads and microparticles. A variety of microparticles that allow non-covalent or covalent attachment of proteins and other substances can be used. Such particles include polymer particles such as polystyrene and poly (methyl methacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica particles, glass particles and metal oxide particles. See, for example, Martin, CR, et al., Analytical Chemistry—News & Features, 70 (1998) 322A-327A or Butler, JE, Methods 22 (2000) 4-23.

[0081] The term "immunoassay" refers to any technique that utilizes specific binding molecules (e.g., antibodies) to capture and / or detect a specific target for qualitative or quantitative analysis. Typically, an immunoassay is characterized by the following steps: 1) immobilizing or capturing the analyte and 2) detecting and measuring the analyte. The analyte can be captured, i.e., bound to any solid surface, such as a membrane, a plastic plate, or some other solid surface.

[0082] Multispecific Antibodies

[0083] In certain embodiments, the drug is a bispecific antibody. In one embodiment, the drug is a bispecific trivalent antibody. In a preferred embodiment, the drug is a monoclonal, bispecific, trivalent antibody.

[0084] In certain embodiments, the drug is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different antigens. In certain embodiments, one of the binding specificities is for a first antigen, and the other is for a different second antigen. Bispecific antibodies can be made into full-length antibodies or antibody fragments. In one embodiment, the antibody is a bispecific antibody that specifically binds to a first antigen and a second antigen. In one embodiment, the bispecific antibody has i) a first binding specificity that specifically binds to a first antigen, and ii) a second binding specificity that specifically binds to a second antigen. In one embodiment, the antibody is a bispecific trivalent antibody. In a preferred embodiment, the antibody is a monoclonal, bispecific, trivalent antibody.

[0085] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" engineering (see, e.g., US 5,731,168). Multispecific antibodies can also be prepared by the following methods: engineering electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553); using the "diabody" technology to prepare bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); using single-chain Fv (scFv) dimers (Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

[0086] Multispecific antibodies are described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 or WO 2010 / 145793.

[0087] Different bispecific antibody formats are known.

[0088] An exemplary bispecific antibody format in which the methods as reported herein can be used is

[0089] -Domain exchange format: a multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment

[0090] a) Only the CH1 and CL domains are replaced with each other (i.e. the light chain of the first Fab fragment contains

[0091] VL and CH1 domains, and the heavy chain of the first Fab fragment comprises VH and

[0092] CL domain);

[0093] b) Only the VH and VL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises

[0094] VH and CL domains, and the heavy chain of the first Fab fragment comprises VL and

[0095] CH1 domain); or

[0096] c) the CH1 and CL domains are replaced with each other and the VH and VL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises the VH and CH1 domains, and the heavy chain of the first Fab fragment comprises the VL and CL domains); and

[0097] The second Fab fragment comprises: a light chain comprising VL and CL domains and a heavy chain comprising VH and CH1 domains;

[0098] The domain exchange antibody may comprise a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, wherein the two CH3 domains are engineered in a complementary manner by respective amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain, for example, as described in WO

[0099] 96 / 27011、WO 98 / 050431、EP 1870459、WO 2007 / 110205、WO 2007 / 147901、WO2009 / 089004、WO 2010 / 129304、WO

[0100] 2011 / 90754、WO 2011 / 143545、WO 2012 / 058768、WO

[0101] 2013 / 157954, or WO 2013 / 096291 (incorporated herein by reference);

[0102] - One-armed single-chain form (= one-armed single-chain antibody): contains a first binding site and a second binding site

[0103] The first binding site specifically binds to a first antigen and the second binding site specifically binds to a second antigen, wherein the individual chains are as follows:

[0104] -Light chain (variable light chain domain + light chain kappa constant domain)

[0105] - Combined light chain / heavy chain (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation)

[0106] - Heavy chain (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation);

[0107] - Two-armed single-chain form (= two-armed single-chain antibody): contains a first binding site and a second binding site

[0108] The first binding site specifically binds to a first antigen and the second binding site specifically binds to a second antigen, wherein the individual chains are as follows:

[0109] - Combined light chain / heavy chain 1 (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation)

[0110] - Combined light chain / heavy chain 2 (variable light chain domain+light chain constant domain+peptide linker+variable heavy chain domain+CH1+hinge+CH2+CH3 with knob mutation);

[0111] - Common light chain bispecific format (= common light chain bispecific antibody): comprising a first

[0112] An antibody having a binding site and a second binding site, wherein the first binding site specifically binds to a first antigen and the second binding site specifically binds to a second antigen, wherein the single

[0113] The unique link is as follows:

[0114] -Light chain (variable light chain domain + light chain constant domain)

[0115] -Heavy chain 1 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation)

[0116] -Heavy chain 2 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation)

[0117] - Dual-targeting Fab: Fab contains two (non-recombinant) complementary pairs of VH and VL domains.

[0118] The first paratope comprises (or consists of) amino acid residues from CDR1 and CDR3 of the VL domain and CDR2 of the VH domain, and

[0119] The second paratope comprises (consists of) residues from CDR1 and CDR3 of the VH domain and CDR2 of the VL domain; the term "non-overlapping" in this context means that the amino acid residues comprised in the first paratope of DutaFab are not comprised in the second paratope, and the amino acids comprised in the second paratope of DutaFab are not comprised in the second paratope.

[0120] One complementary position

[0121] -T cell bispecific format (TCB): a bispecific antibody comprising

[0122] - a first Fab fragment and a second Fab fragment, wherein each binding site of the first Fab fragment and the second Fab fragment specifically binds to a second antigen,

[0123] - a third Fab fragment, wherein the binding site of the third Fab fragment specifically binds to the first antigen, and wherein the third Fab fragment comprises a domain crossover such that the variable light chain domain (VL) and the variable heavy chain domain (VH) are replaced with each other, and

[0124] - an Fc region comprising a first Fc region polypeptide and a second Fc region polypeptide,

[0125] wherein the first Fab fragment and the second Fab fragment each comprise a heavy chain fragment and a full-length light chain,

[0126] wherein the C-terminus of the heavy chain fragment of the first Fab fragment is fused to the N-terminus of the first Fc region polypeptide,

[0127] The C-terminus of the heavy chain fragment of the second Fab fragment is fused to the N-terminus of the variable light chain domain of the third Fab fragment, and the C-terminus of the heavy chain constant domain 1 of the third Fab fragment is fused to the N-terminus of the second Fc region polypeptide.

[0128] In one embodiment, the bispecific antibody is a domain swapped antibody.

[0129] In one embodiment, the bispecific antibody is a one-armed single chain antibody.

[0130] In one embodiment, the bispecific antibody is a two-armed single chain antibody.

[0131] In one embodiment, the bispecific antibody is a common light chain bispecific antibody.

[0132] In one embodiment, the bispecific antibody is a dual targeting Fab.

[0133] In one embodiment, the bispecific antibody is a consensus T cell bispecific antibody.

[0134] Multivalent, multispecific antibodies specifically bind to different targets, most likely with different affinities and complex stabilities for each target. Only fully active multivalent, multispecific antibodies can bind to all targets and show full biological activity in the corresponding assays.

[0135] The term "CD20-TCB" as used herein refers to a TCB targeting CD20 (CD20-TCB; RG6026; anti-CD3 / CD20 antibody in TCB format) that has a long half-life and high potency, which is achieved by high-affinity bivalent binding to CD20 and the head-to-tail orientation of the B and T cell binding domains in a previously characterized 2:1 TCB molecule format (see, e.g., Bacac, M. et al. Clin. Cancer Res. 22 (2016)) 3286-3297; Bacac, M. et al. Oncoimmunology 5 (2016) e1203498).

[0136] Immunoassay

[0137] The principles of different immunoassays are described in the art. For example, Hage, DS (Anal. Chem. 71 (1999) 294R-304R). Lu, B. et al. (Analyst 121 (1996) 29R-32R) reported on the orientation of antibodies for immunoassays. For example, Wilchek, M. and Bayer, EA, reported on avidin-biotin mediated immunoassays in Methods Enzymol. 184 (1990) 467-469.

[0138] Monoclonal antibodies and their constant domains contain many reactive amino acid side chains for conjugation with binding pairs, such as members of polypeptides / proteins, polymers (such as PEG, cellulose or polystyrene) or enzymes. The chemically reactive groups of amino acids are, for example, amino groups (lysine, α-amino), thiol groups (cystine, cysteine ​​and methionine), carboxylic acid groups (aspartic acid, glutamic acid) and sugar alcohol groups. This method is described, for example, by Aslam M. and Dent, A. in "Bioconjugation", MacMillan Ref. Ltd. 1999, pages 50-100.

[0139] One of the most common reactive groups of antibodies is the aliphatic ε-amine of the amino acid lysine. In general, almost all antibodies are rich in lysine. Lysine amine is a fairly good nucleophile above pH 8.0 (pKa = 9.18), so it reacts easily and cleanly with various reagents to form stable bonds. Amine-reactive reagents react mainly with α-amino groups of lysine and proteins. Reactive esters, especially N-hydroxy-succinimide (NHS) esters, are the most commonly used amine-modifying reagents. The optimal pH for reaction in an aqueous environment is pH 8.0 to 9.0. Isothiocyanates are amine-modifying reagents and form thiourea bonds with proteins. They react with protein amines in aqueous solution (optimal pH is 9.0 to 9.5). Aldehydes react with aliphatic and aromatic amines, hydrazines and hydrazides under mild aqueous conditions to form imine intermediates (Schiff bases). Schiff bases can be selectively reduced with mild or strong reducing agents (such as sodium borohydride or sodium cyanoborohydride) to derive stable alkylamine bonds. Other reagents that have been used to modify amines are anhydrides. For example, diethylenetriamine pentaacetic anhydride (DTPA) is a bifunctional chelating agent containing two amine-reactive anhydride groups. It can react with the N-terminus and the ε-amine group of amino acids to form an amide bond. The anhydride ring opens to form a multivalent metal chelating arm that is able to bind tightly to the metal in the coordination complex.

[0140] Another common reactive group in antibodies is the thiol residue from the sulfur-containing amino acid cystine and its reduction product cysteine ​​(or half cystine). Cysteine ​​contains a free thiol group, which is more nucleophilic than amines and is generally the most reactive functional group in proteins. Thiols are generally reactive at neutral pH and can therefore selectively couple to other molecules in the presence of amines. Since free sulfhydryl groups are relatively reactive, proteins with these groups are generally present with them in their oxidized form as disulfide groups or disulfide bonds. In such proteins, reduction of disulfide bonds with reagents such as dithiothreitol (DTT) is required to generate reactive free thiols. Thiol-reactive reagents are those that will couple to thiol groups on polypeptides to form thioether coupling products. These reagents react rapidly at slightly acidic to neutral pH and can therefore selectively react in the presence of amine groups. The literature reports that the use of several thiolation cross-linking reagents such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio) acetate (SATA) and sulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamide] hexanoate (Sulfo-LC-SPDP) provides an efficient way to introduce multiple sulfhydryl groups via reactive amino groups. Haloacetyl derivatives, such as iodoacetamide, form thioether bonds and are also reagents for thiol modification. Another useful reagent is maleimide. The reaction of maleimide with thiol-reactive reagents is essentially the same as that of iodoacetamide. Maleimide reacts rapidly at weakly acidic to neutral pH.

[0141] Another common reactive group in antibodies is the carboxylic acid. Antibodies contain carboxylic acid groups at the C-terminal position and within the side chains of aspartic acid and glutamic acid. The relatively low reactivity of carboxylic acids in water generally makes it difficult to use these groups to selectively modify peptides and antibodies. Once this is accomplished, the carboxylic acid group is typically converted to a reactive ester using a water-soluble carbodiimide and reacted with a nucleophilic reagent such as an amine, hydrazide, or hydrazine. Amine-containing reagents should be weakly basic in order to react selectively with the activated carboxylic acid in the presence of the more basic ε-amine of lysine to form a stable amide bond. When the pH rises above 8.0, protein cross-linking may occur.

[0142] Sodium periodate can be used to oxidize the alcohol portion of the sugar in the carbohydrate portion of the antibody attached to the aldehyde. Each aldehyde group can react with amines, hydrazides or hydrazines as described for carboxylic acids. Since the carbohydrate portion is mainly present in the crystallizable fragment region (Fc-region) of the antibody, conjugation can be achieved by site-specific modification of carbohydrates away from the antigen binding site. A Schiff base intermediate is formed, which can be reduced to an alkylamine by reducing the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents.

[0143] The conjugation of the tracing and / or capturing and / or detecting antibodies to their conjugation partners can be performed by different methods, such as chemical binding, or via binding of a binding pair. As used herein, the term "conjugation partner" means, for example, a solid support, a polypeptide, a detectable label, a member of a specific binding pair. In one embodiment, the conjugation of the capturing and / or tracing and / or detecting antibodies to their conjugation partners is performed by chemical binding via the N-terminus and / or the ε-amino group (lysine), the ε-amino groups of different lysines, the carboxyl-, sulfhydryl-, hydroxyl- and / or phenolic functional groups of the amino acid backbone of the antibody, and / or the sugar alcohol groups of the carbohydrate structure of the antibody. In one embodiment, the capture antibody is conjugated to its conjugation partner via a binding pair. In a preferred embodiment, the capture antibody is conjugated to biotin and is immobilized to the solid support via avidin or streptavidin immobilized on the solid support. In one embodiment, the capture antibody is conjugated to its conjugation partner via a binding pair. In a preferred embodiment, the tracing antibody is conjugated to digoxigenin via a covalent bond as a detectable label.

[0144] Chromophores (fluorescent or luminescent groups and dyes), enzymes, NMR active groups or metal particles, haptens, such as digoxigenin are examples of "detectable labels". Detectable labels can also be photoactivatable crosslinking groups, such as azido or aziridine groups. Metal chelates detectable by electrochemiluminescence are also preferred signaling groups, particularly preferred are ruthenium chelates, such as ruthenium (bipyridyl) 32+ chelates. Suitable ruthenium labeling groups are described, for example, in EP 0 580 979, WO 90 / 05301, WO 90 / 11511 and WO 92 / 14138. For direct detection, the labeling group can be selected from any known detectable marker group, such as a dye, a luminescent labeling group, such as a chemiluminescent group, such as an acridinium ester or a dioxetane, or a fluorescent dye, such as fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and its derivatives. Further examples of labelling groups are luminescent metal complexes, such as ruthenium or europium complexes, enzymes (eg as used in ELISA or in CEDIA (Cloned Enzyme Donor Immunoassay, eg EP-A-0 061 888)) and radioactive isotopes.

[0145] Indirect detection systems include, for example, a detection reagent, such as a detection antibody, labeled with a first partner of a binding pair. Examples of suitable binding pairs are antigen / antibody, biotin or a biotin analog such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analog / complementary nucleic acid and receptor / ligand, such as steroid hormone receptor / steroid hormone. In a preferred embodiment, the first binding pair members include a hapten, an antigen and a hormone. In a preferred embodiment, the hapten is selected from digoxigenin, isohydroxydigoxigenin and biotin and analogs thereof. The second partner of such a binding pair, such as an antibody, streptavidin, etc. is typically labeled to allow direct detection, such as by a label as described above.

[0146] Immunoassays can generally be performed in three different formats. One is direct detection and one is indirect detection, or by sandwich assays. Direct detection immunoassays use a detection (or tracer) antibody that can be measured directly. An enzyme or other molecule allows the generation of a signal that will produce a color, fluorescence, or luminescence, allowing the signal to be visualized or measured (radioactive isotopes can also be used, although it is not commonly used nowadays). In an indirect assay, the primary antibody that binds to the analyte is used to provide a defined target for the secondary antibody (tracer antibody), which specifically binds to the target provided by the primary antibody (called the detector or tracer antibody). The secondary antibody produces a measurable signal. Sandwich assays use two antibodies, a capture antibody and a tracer (detector) antibody. The capture antibody is used to bind (immobilize) the analyte in solution or to bind to it in solution. This allows the analyte to be specifically removed from the sample. The tracer (detector) antibody is used in a second step to produce a signal (either directly or indirectly as described above). The sandwich format requires two antibodies, each with a different epitope on the target molecule. Furthermore, they must not interfere with each other, as both antibodies must bind to the target simultaneously.

[0147] Different principles for determining bispecific antibodies in immunoassays are known to the person skilled in the art:

[0148] 1) Capture and use

[0149] - one of the antigens;

[0150] -Anti-idiotypic antibodies directed against one of the binding sites;

[0151] 2) Detection and use

[0152] - corresponding other antigens;

[0153] - anti-idiotypic antibodies directed against the corresponding other binding sites;

[0154] These can be combined independently of each other.

[0155] Aspects of the alternative immunoassay according to the invention

[0156] The present invention is based, at least in part, on the discovery that the determination of multispecific therapeutic antibodies in serum samples may be hampered by interference from monospecific antibodies comprising one binding specificity of the therapeutic antibody. These antibodies may be present prior to administration of the multispecific therapeutic antibody as a result of administration of the monospecific antibodies.

[0157] Typically, in an immunoassay for determining a multispecific therapeutic antibody in a serum-containing sample, in a first step, extraction of the therapeutic antibody from the sample is achieved by a capture reagent bound to a solid phase having specificity for the multispecific therapeutic antibody, such as, for example, specifically binding to a binding specificity of the multispecific therapeutic antibody. In a second step, determination of the solid phase captured multispecific therapeutic antibody is achieved by a detection reagent, such as, for example, specifically binding to a binding specificity different from that of the multispecific therapeutic antibody used in the capture step. Figure 1 In the present invention, anti-idiotypic antibodies directed against the first and second binding specificities of the bispecific therapeutic antibody are exemplified as exemplary capture and detection agents.

[0158] If the sample contains, in addition to the multispecific therapeutic antibody, a monospecific antibody having at least one binding site with a binding specificity similar to one of the binding sites of the therapeutic antibody, interference resulting from this may occur via (nonspecific) binding to the solid phase and thus generate false positive signals, i.e. the specificity of the assay is biased based on the specificity of the capture reagent. Figure 2 This is particularly true if the presence of monospecific antibodies exceeds the presence of multispecific antibodies, for example, by 1,000 times or more in one embodiment, by 10,000 times or more in one embodiment, or by 100,000 times or more in one embodiment.

[0159] It has been found that if the formed capture antibody-therapeutic antibody complex is desorbed from the solid phase by incubation with a replacement antibody that replaces the capture antibody in the immobilized complex, this non-specific bias caused by the monospecific antibody can be reduced or even eliminated. The replacement antibody-therapeutic antibody complex thus produced is soluble and can be specifically detected after transfer to a new compartment. The capture of the desorbed complex for detection is preferably carried out via the second binding specificity of the therapeutic antibody, thereby reversing the bridging assay. The non-specifically bound monospecific antibody is not desorbed in this step. The interference of the monospecific antibody can thus be reduced or even eliminated.

[0160] In a preferred embodiment, a method for determining a multispecific therapeutic antibody, preferably a bispecific antibody, in a serum-containing sample is reported herein, the method comprising the following steps:

[0161] a) incubating a solid phase on which a capture agent that specifically binds to the therapeutic antibody has been immobilized with the sample to form an immobilized capture agent-therapeutic antibody-complex,

[0162] b) incubating the solid phase with a displacing agent that competes with the capture agent for binding to the therapeutic antibody and thereby separating the therapeutic antibody from the solid phase by forming a solution comprising the displacing agent-therapeutic antibody-complex,

[0163] c) determining said displacing agent-therapeutic antibody-complex in said solution obtained in step b) and thereby determining said therapeutic antibody in a serum-containing sample.

[0164] The capture agent and the displacing agent can be any compound that specifically binds to the therapeutic antibody, as long as the two agents compete with each other at the binding site of the therapeutic antibody, i.e., as long as the displacing agent displaces the capture agent when bound to the therapeutic antibody. Secondary parameters that affect the efficiency of displacement but not the occurrence of displacement are the amount / excess of the displacing antibody used and the incubation time.

[0165] Exemplary combinations of capture agents and displacing agents are:

[0166] - Capture agent: anti-idiotypic antibody 1; Displacer: anti-idiotypic antibody 2; anti-idiotypic antibody 1 and anti-idiotypic antibody 2 compete with each other for binding to the therapeutic antibody;

[0167] - Capture agent: an antigen of the therapeutic antibody; Displacer: anti-idiotypic antibody; the anti-idiotypic antibody competes with the antigen for binding to the therapeutic antibody;

[0168] - Capture agent: anti-idiotypic antibody; Displacer: antigen; anti-idiotypic antibody competes with antigen for binding to therapeutic antibody;

[0169] - Capture agent: antigen; Displacer: antigen

[0170] - Capture agent: anti-mutant Fc region antibody (eg, anti-P329G antibody or anti-L234A / L235A antibody); Displacer: anti-mutant Fc region antibody (eg, the same anti-PG antibody or the same anti-L234A / L235A antibody, respectively).

[0171] In a preferred embodiment, a method for determining a multispecific therapeutic antibody, preferably a bispecific antibody, in a serum-containing sample is reported herein, the method comprising the following steps:

[0172] a) incubating a solid phase on which a capture antibody that specifically binds to the therapeutic antibody has been immobilized with the sample to form an immobilized capture antibody-therapeutic antibody-complex,

[0173] b) incubating the solid phase with a displacement antibody that competes with the capture antibody for binding to the therapeutic antibody and thereby separating the therapeutic antibody from the solid phase by forming a solution comprising the displacement antibody-therapeutic antibody-complex,

[0174] c) determining said replacement antibody-therapeutic antibody-complex in said solution obtained in step b) and thereby determining said therapeutic antibody in the serum-containing sample.

[0175] The method according to the present invention is illustrated below with an anti-CD3 / CD20 bispecific antibody (CD20-TCB) in the form of TCB. This example is presented only as an example of the method according to the present invention and should not be construed as limiting. The true scope of the present invention is set forth in the appended claims.

[0176] CD20-TCB and (Anti-CD20 antibody) pretreatment represents an effective and safer approach to treating lymphoma patients and is currently being evaluated in a phase I multicenter study (NCT03075696) in patients with relapsed / refractory non-Hodgkin lymphoma (see Bacac, M. et al., Clin. Cancer. Res. 24 (2018) 1-13).

[0177] When using according to Figure 1 The prior art standard bridging immunoassay of the protocol described in (using anti-idiotypic anti-CD3 binding site antibodies as capture antibodies and anti-idiotypic anti-CD20 binding site antibodies as detection antibodies) obtained only a shallow curve when determining bispecific CD20-TCB (at a concentration of 1 ng / mL) in the presence of the corresponding monospecific anti-CD20 antibodies (at a concentration of 200 μg / mL) (see Example 1a, Figure 3 ).

[0178] In the absence of monospecific anti-CD20 antibodies, a steeper curve was obtained (see Example 1b, Figure 4 ).

[0179] The specific bridging immunoassay using anti-idiotypic anti-CD3 binding site antibody to capture the analyte was performed in the absence of CD20-TCB and the sample contained only monospecific anti-CD20 antibody. In this case, signals can be obtained in the presence and absence of anti-idiotypic anti-CD3 binding site antibody (see Example 1c).

[0180] Therefore, interference of monospecific anti-CD20 antibodies in the CD20-TCB bridging assay arises from nonspecific interactions with the solid phase.

[0181] CD20-TCB contains a specific mutation in the Fc region (P329G, according to Kabat numbering). Therefore, an alternative bridging assay using an anti-P329G-Fc region antibody as detection antibody was performed. Therein a correct bridging signal was obtained in the presence and absence of the corresponding anti-CD20 antibody. However, when using this assay setup, no information about the target binding capacity of the CD20 binding site of the CD20-TCB can be obtained, since this binding site no longer contributes to the generated signal (see Example 2, Figure 5 ).

[0182] Different approaches known in the art were tested to address non-specific binding of anti-CD20 antibodies so that the target binding capabilities of both binding sites of CD20-TCB could be addressed in a single assay.

[0183] Simple switching of capture and detection antibodies is not feasible, since the immobilized anti-idiotypic anti-CD20 binding site antibodies would be saturated with the monospecific anti-CD20 antibodies present in the sample and would thereby greatly reduce the assay sensitivity.

[0184] Next, the reduction of the density of streptavidin used to immobilize the capture antibody on the solid phase was tested. Non-specific binding can be reduced thereby, but the accuracy of the low quality control (LQC) and the lower limit of the quantitative quality control (LLQC) are still affected (see Example 3). This effect is also not robust, so for example high inter-assay variation occurs, resulting in unsuitable immunoassays (see Example 3).

[0185] Different buffers were tested. Compared with the assays performed in universal buffers, the use of 1% casein in PBS combined with medium density streptavidin solid phase reduces the interference of monospecific anti-CD20 antibodies. The appropriate assay range was determined, and the assay performance was evaluated by the accuracy assessment of quality control (QC) and the inter-assay precision. The results showed that the interference was reduced, but at the same time, QC and interference reduction were not robust, indicating high inter-assay variation (see Example 4). The specificity / selectivity assessment of the LLQC level also revealed that the method was not robust enough because the validation criteria for accuracy were not met (data not shown). Similarly, the use of medium streptavidin density, universal buffer and acidic washing conditions (pH 5.5) resulted in reduced interference, but at the same time, QC and interference reduction were not robust, indicating high inter-assay variation (see Example 5).

[0186] It has now been found that the above problems are solved by including an intermediate replacement / displacement step in the method. That is, first the anti-idiotypic anti-CD3 binding site antibody captures the CD20-TCB, and thereafter the formed complex is displaced from the solid surface for analysis. In more detail, the capture of CD20-TCB from the sample is achieved by incubation with an immobilized anti-idiotypic anti-CD3 binding site antibody on a first assay plate. After reaching equilibrium or after a specified time, the immobilized capture antibody in the complex is replaced with a soluble anti-idiotypic anti-CD3 binding site antibody (same or different from the capture antibody, such as binding to the same epitope or interfering spatially, as long as displacement can be performed). Thus, without being bound by this theory, only the bound CD20-TCB is released into the supernatant, while the non-specifically bound monospecific anti-CD20 antibody remains bound to the solid phase. Thereafter, the supernatant is transferred to a second assay plate, on which the anti-idiotypic anti-CD20 binding site antibody is immobilized as the capture antibody, i.e., reverse bridging.

[0187] Therefore, when using the immunoassay setup according to the present invention, the requirement of 1 ng / mL or lower assay sensitivity and identification of CD20-TCBs with targeted binding capacity is achieved in the presence of a large excess (about 1:250,000) of monospecific anti-CD20 antibodies. In addition, the immunoassay according to the present invention is feasible and robust to be performed within one working day.

[0188] Furthermore, the immunoassay setup according to the invention can be performed with a matrix content of up to 50% serum, thereby increasing sensitivity.

[0189] Figure 6 The concentration-response curves of the bridging immunoassay according to the invention comprising a displacement step are shown. Good responses can be obtained both in the presence and absence of the corresponding monospecific anti-CD20 antibody. Example 6 describes the corresponding instructions and data.

[0190] The table below provides a comparison of the different assay formats and their characteristic properties as described above (ID = idiotype).

[0191]

[0192] The assay for CD20-TCB according to the present invention was successfully characterized.

[0193]

[0194]

[0195] The following examples and drawings are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0196] Example 1

[0197] Bridging immunoassay using dual anti-idiotypic antibodies

[0198] General process:

[0199] Murine monoclonal antibodies were produced by Roche Diagnostics GmbH (Germany). Biotinylated M-4.25.93-IgG was used as anti-idiotypic anti-CD3 binding site antibody, and digoxigenylated M-6.15.528-IgG-Dig was used as anti-idiotypic anti-CD20 binding site antibody.

[0200] CD20-TCB calibrators or QCs (quality controls) and samples containing CD20-TCB and anti-CD20 antibodies were prepared in 100% pooled human serum obtained from Trina Bioreactives, Switzerland or 100% pooled cynomolgus monkey serum (Seralab, UK).

[0201] Antibody preparations and serum sample dilutions were made in Universal Buffer (Roche Diagnostics GmbH, Germany). Each wash consisted of 3 steps with 300 μL PBS, 0.05% Tween20 (Sigma Aldrich, Germany), incubation steps were performed at room temperature with shaking at 500 rpm and a working volume of 100 μL per well.

[0202] For capture, streptavidin plates (High Binding Capacity, Microcoat GmbH, Bernried, Germany) were coated with 0.2 μg / mL M-4.25.93-IgG-Bi for one hour, and unbound antibodies were removed by washing, followed by the addition of diluted (1:10) serum samples and incubation for one hour. After washing, the plates were then incubated for one hour by the sequential addition of M-6.15.528-IgG-Dig (0.2 μg / mL, 50 min) and covalently conjugated horseradish peroxidase. <dig>-Fab fragment (Roche Diagnostics GmbH, Germany; 40mU, 50 minutes) was used for analyte detection. Washing steps were applied between and after this step. ABTS (2'2'-azo-bis-3-ethyl-benzothiazoline-6-sulfonic acid; Roche Diagnostics GmbH, Germany) was added as a substrate for the colorimetric reaction, which was monitored by a photometric reading at 405nm (reference wavelength 490nm). Samples were analyzed in duplicate, and the average absorbance value was calculated and further processed. The concentration was determined by reverse calculation of the calibration curve using a nonlinear 4-parameter curve fitting function (Wiemer-Rodbard). The accuracy was calculated based on the nominal concentration in the spiked serum sample.

[0203] a) Sample containing CD20-TCB and 200 μg / mL anti-CD20 antibody:

[0204]

[0205] b) Samples containing only CD20-TCB:

[0206]

[0207] c) Samples containing only anti-CD20 antibody (reverse calculation on the CD20-TCB calibration curve in 10% cynomolgus monkey serum) Calculated value):

[0208]

[0209] Example 2

[0210] Bridging immunoassay with anti-idiotypic antibody capture and anti-P329G antibody detection

[0211] General process:

[0212] General: 4-step sandwich Elisa

[0213] Incubate at room temperature with shaking at 500 rpm

[0214] Working value: 100μL

[0215] Wash (hereinafter referred to as w): 3x 300 μL PBS, 0.05% Tween 20

[0216] Repeat n=2 and calculate the average absorbance value

[0217] Calibration curve fitting: nonlinear 4-parameter curve fitting function (Wiemer-Rodbard)

[0218] ·Measurement range: 1ng / mL-100ng / mL

[0219] Reagents:

[0220] Human pooled serum; Trina Bioreactives, Switzerland

[0221] Mouse monoclonal antibody; Roche Diagnostics GmbH, Germany

[0222] о Anti-idiotypic anti-CD3 binding site antibody, biotinylated: M-4.25.93-IgG-Bi

[0223] о Anti-P329G antibody, digoxigeninylated: M-1.7.24-IgG-Dig

[0224] Streptavidin plate (high binding capacity); Microcoat GmbH, Bernried, Germany Universal buffer (hereinafter referred to as UB); Roche Diagnostics GmbH, Germany Washing buffer; 1x PBS, 0.05% Tween (Sigma Aldrich, Germany)

[0225] Covalently bound to horseradish peroxidase <dig>-Fab fragment( <dig>-Fab-POD); Roche Diagnostics GmbH, Germany

[0226] ·2′2′-Azo-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS); Roche Diagnostics GmbH, Germany

[0227] Determination of behavior:

[0228] Coating: 0.2μg / mL M-4.25.93-IgG-Bi, 1 hour, w

[0229] Sample: 1 to 10 in UB, 1 hour, w

[0230] · Test 1: 0.2 μg / mL M-1.7.24-IgG-Dig, 50 min, w

[0231] ·Test 2: 50mU <dig>-Fab-POD, 50 minutes, w

[0232] Substrate reaction: ABTS, repeated absorbance measurement at 405 nm (reference wavelength 490 nm)

[0233] a) Sample containing CD20-TCB and 200 μg / mL anti-CD20 antibody:

[0234]

[0235]

[0236] b) Samples containing only CD20-TCB:

[0237]

[0238] Example 3

[0239] Bridging immunoassays using dual anti-idiotypic antibodies with reduced immobilization density

[0240] General process:

[0241] The same procedure as described in Example 1, except that streptavidin plates with medium binding capacity were used, Microcoat GmbH, Bernried, Germany

[0242] a) Sample containing CD20-TCB and 200 μg / mL anti-CD20 antibody:

[0243]

[0244] b) Samples containing only CD20-TCB:

[0245]

[0246] Example 4

[0247] Bridging immunoassay with immobilized double anti-idiotypic antibodies of reduced density using 1% casein in PBS as assay buffer

[0248] General process:

[0249] General: 4-step sandwich Elisa

[0250] Incubate at room temperature with shaking at 500 rpm

[0251] Working value: 100μL

[0252] Wash (hereinafter referred to as w): 3x 300 μL PBS, 0.05% Tween 20

[0253] Repeat n=2 and calculate the average absorbance value

[0254] Calibration curve fitting: nonlinear 4-parameter curve fitting function (Wiemer-Rodbard)

[0255] ·Measurement range: 1ng / mL-64ng / mL

[0256] Reagents:

[0257] Human pooled serum; Trina Bioreactives, Switzerland

[0258] Mouse monoclonal antibody; Roche Diagnostics GmbH, Germany

[0259] о Anti-idiotypic anti-CD3 binding site antibody, biotinylated: M-4.25.93-IgG-Bi

[0260] о Anti-idiotypic anti-CD20 binding site antibody, digoxigenylated: M-6.15.528-IgG-Dig

[0261] Streptavidin plates (medium binding capacity); Microcoat GmbH, Bernried, Germany

[0262] Casein (Hammersten); VWR Prolabo Chemicals, Germany Assay buffer (hereinafter AB): 1% casein in 1x PBS (Roche Diagnostics GmbH, Germany)

[0263] Washing buffer: 1x PBS, 0.05% Tween (Sigma Aldrich, Germany)

[0264] Covalently bound to horseradish peroxidase <dig>-Fab fragment( <dig>-Fab-POD); Roche Diagnostics GmbH, Germany

[0265] ·2′2′-Azo-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS); Roche Diagnostics GmbH, Germany

[0266] Determination of behavior:

[0267] Coating: 0.25 μg / mL M-4.25.93-IgG-Bi, 1 hour, w

[0268] Sample: 1 to 10 in AB, 1 hour, w

[0269] · Test 1: 0.25 μg / mL M-6.15.528-IgG-Dig, 60 min, w

[0270] ·Test 2: 40mU <dig>-Fab-POD, 60 minutes, w

[0271] Substrate reaction: ABTS, repeated absorbance measurements at 405 nm (reference wavelength 490 nm) a) Samples containing CD20-TCB and 200 μg / mL anti-CD20 antibody:

[0272]

[0273]

[0274]

[0275] b) Samples containing only CD20-TCB:

[0276]

[0277]

[0278] Example 5

[0279] Bridging immunoassay with dual anti-idiotypic antibodies of reduced immobilization density under acidic (pH 5.5) washing conditions

[0280] General process:

[0281] General: 4-step sandwich Elisa

[0282] Incubate at room temperature with shaking at 500 rpm

[0283] Working value: 100μL

[0284] Wash (hereinafter referred to as w): 3x 300 μL PBS, 0.05% Tween 20, pH 5.5

[0285] Repeat n=2 and calculate the average absorbance value

[0286] Calibration curve fitting: nonlinear 4-parameter curve fitting function (Wiemer-Rodbard)

[0287] ·Measurement range: 1ng / mL-64ng / mL

[0288] Reagents:

[0289] Human pooled serum; Trina Bioreactives, Switzerland

[0290] Mouse monoclonal antibody; Roche Diagnostics GmbH, Germany

[0291] о Anti-idiotypic anti-CD3 binding site antibody, biotinylated: M-4.25.93-IgG-Bi

[0292] о Anti-idiotypic anti-CD20 binding site antibody, digoxigenylated: M-6.15.528-IgG-Dig

[0293] Streptavidin plates (medium binding capacity); Microcoat GmbH, Bernried, Germany

[0294] Assay buffer: Universal Buffer (hereinafter referred to as UB); Roche Diagnostics GmbH, Germany

[0295] Wash buffer; 1x PBS, 0.05% Tween, pH 5.5 (Sigma Aldrich, Germany, pH adjusted with 2M HCl, Sigma Aldrich, Germany)

[0296] Covalently bound to horseradish peroxidase <dig>-Fab fragment( <dig>-Fab-POD); Roche Diagnostics GmbH, Germany

[0297] 3,3',5,5'-Tetramethylbenzidine (TMB); Roche Diagnostics GmbH, Germany

[0298] 1M H2SO4; Carl Roth, Germany

[0299] Determination of behavior:

[0300] Coating: 0.25 μg / mL M-4.25.93-IgG-Bi, 1 hour, w

[0301] Sample: 1 to 10 in UB, 1 hour, w

[0302] · Test 1: 0.25 μg / mL M-6.15.528-IgG-Dig, 60 min, w

[0303] ·Test 2: 10mU <dig>-Fab-POD, 60 minutes, w

[0304] Substrate reaction: TMB, stop with 50 μL 1M H2SO4

[0305] Photometric reading at 450nm (reference wavelength 690nm)

[0306] a) Sample containing CD20-TCB and 200 μg / mL anti-CD20 antibody:

[0307]

[0308]

[0309] b) Samples containing only CD20-TCB:

[0310]

[0311]

[0312] Example 6

[0313] Bridging immunoassay according to the present invention

[0314] General process:

[0315] Murine monoclonal antibodies were produced by Roche Diagnostics GmbH (Germany). Biotinylated M-4.35.77-IgG and digoxigenylated M-4.25.93-IgG were used as anti-idiotypic anti-CD3 binding site antibodies, and biotinylated M-6.15.528 was used as anti-idiotypic anti-CD20 binding site antibody.

[0316] CD20-TCB calibrators or QC (quality control) and samples containing CD20-TCB and anti-CD20 antibodies were prepared in 100% mixed human serum obtained from Trina Bioreactives, Switzerland. Antibody preparations and serum sample diluents were made in universal buffer (Roche Diagnostics GmbH, Germany). Each wash included 3 steps with 300 μL PBS, 0.05% Tween 20 (Sigma Aldrich, Germany). The incubation step was performed at room temperature with 500 rpm shaking. The process was divided into two parts and was performed on two streptavidin-coated microtiter plates (SA-MTP 1 and 2, high binding capacity, Microcoat GmbH, Bernried, Germany).

[0317] Part 1: For capture, SA-MTP 1 was coated with 135 μL / well of 0.21 μg / mL M-4.35.77-IgG-Bi for one hour, and unbound antibodies were removed by washing, followed by continuous addition of 67.5 μL / well universal buffer and 67.5 μL / well serum sample (generating 50% matrix) and incubation for 35 minutes. After washing, 135 μL of 1 μg / mL M-4.25.93-Dig (detection antibody) was added, and the plate was incubated for 2.5 hours without subsequent washing steps for desorption (replacement steps).

[0318] Part 2: 90 minutes after adding the detection antibody to SA-MTP1, SA-MTP2 was coated with 100 μL / well 0.25 μg / mL M-6.28.530-Bi for one hour and then washed. Thereafter, 100 μL of the formed complex (drug detection antibody) was transferred from SA-MTP1 to SA-MTP2 and incubated for 35 minutes. 100 μL / well 50 mU covalently bound to horseradish peroxidase was added. <dig>-Fab fragment (Roche Diagnostics GmbH, Germany) was used as the second detection reagent, and the plate was incubated for 45 minutes. A washing step was applied before and after this step. 100 μL / well ABTS (2′2′-azo-bis-3-ethyl-benzothiazoline-6-sulfonic acid; Roche Diagnostics GmbH, Germany) was used as the substrate for the chromogenic reaction, and the photometric reading was monitored at 405nm (reference wavelength 490nm). Samples were analyzed in duplicate, and the average absorbance value was calculated and further processed. The concentration was determined by reverse calculation of the calibration curve using a nonlinear 4-parameter curve fitting function (Wiemer-Rodbard). Accuracy was calculated based on the nominal concentration in the spiked serum sample.

[0319] a) Sample containing CD20-TCB and 200 μg / mL anti-CD20 antibody:

[0320]

[0321] b) Samples containing only CD20-TCB:

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Claims

1. A method for determining bispecific antibodies in a serum-containing sample, the method comprising the following steps: a) incubating a solid phase on which a capture antibody that specifically binds to the bispecific antibody has been immobilized with the serum-containing sample to form an immobilized capture antibody-bispecific antibody-complex, b) optionally washing the solid phase, wherein the capture antibody-bispecific antibody-complex remains bound to the solid phase, c) incubating the solid phase with a displacement antibody which competes with the capture antibody for binding to the bispecific antibody and thereby forms a solution comprising the displacement antibody-bispecific antibody-complex, d) determining the replacement antibody-bispecific antibody complex in the solution obtained in step c) and thereby determining the bispecific antibody in the serum-containing sample. 2 . The method according to claim 1 , wherein the replacement antibody is labeled and the replacement antibody-bispecific antibody-complex is determined by detecting the label.

3. The method according to any one of claims 1 to 2, wherein the replacement antibody is the capture antibody.

4. The method according to any one of claims 1 to 3, wherein the solid phase in step c) is incubated with the replacement antibody at a concentration of 6.67 nM or higher.

5. The method according to any one of claims 1 to 4, wherein the serum-containing sample comprises the bispecific antibody and a monospecific antibody comprising one binding site of the bispecific antibody, and the method is a method for reducing the interference of the monospecific antibody when determining the bispecific antibody.

6. The method according to any one of claims 1 to 5, wherein the bispecific antibody is an anti-CD3 / CD20 bispecific antibody.

7. The method of claim 6, wherein the bispecific anti-CD3 / CD20 antibody is RG6026.

8. The method according to any one of claims 5 to 7, wherein the monospecific antibody is an anti-CD20 antibody.

9. The method of claim 8, wherein the anti-CD20 antibody is obinutuzumab.

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