HK1230718A1 - Method for the detection of a binding partner of a multispecific binder - Google Patents

Description

Method for detecting binding partners of multispecific binders

The present application is a divisional application of PCT application PCT/EP2013/051604 entitled "method for detecting binding partners for multispecific binders" filed on 29.1.2013, which enters the national phase of china on 30.7.2014, 201380007278.5.

The present invention relates to a method for the detection/determination of a binding partner for a free (i.e. uncomplexed) multispecific binder in a sample, which binding partner may be specifically bound by the multispecific binder in the sample, wherein the binding partner bound to the multispecific binder is depleted from the sample prior to the detection of the free binding partner. Depleted multispecific binders may be used to detect/determine complexed binding partners.

Background

Standard solid phase immunoassays containing antibodies involve the formation of a complex between an antibody adsorbed/immobilized on a solid phase (capture antibody), an antigen and an antibody directed against another epitope of the antigen conjugated to an enzyme or a detectable label (tracer antibody). In the assay, a sandwich (sandwich) is formed: solid phase/capture antibody/antigen/tracer antibody. In reactions catalyzed by sandwich or the like, the activity of the antibody-conjugating enzyme is proportional to the antigen concentration in the incubation matrix. Anti-idiotype antibody assays are mentioned in e.g. US5,219,730; WO 87/002778; EP 0139389 and EP 0170302. Wadhwa, M. et al (J.Immunol. methods 278(2003)1-17) report strategies for detecting, measuring and characterizing undesirable antibodies induced by biological therapeutics. A method for producing anti-idiotype antibodies is reported in EP 1917854.

Chen, y. -p. et al (clin. vac. immunol.14(2007)720-725) reported rapid detection of hepatitis b virus surface antigen by a bispecific diabody-mediated agglutination assay directed simultaneously against human erythrocytes and hepatitis b virus surface antigen. Porter, r. et al reported an electroactive immunoassay system (EASI assay) using self-assembled monolayer modified electrodes (Biosensors bioetec.16 (2001) 9-12). Berkova, N.et al (Biotechnol. appl. biochem.23(1996) 163. 171) reported the development of an enzyme immunoassay for the measurement of human tumor necrosis factor-alpha (hTNF-. alpha.) using a bispecific antibody against hTNF-. alpha.and horseradish peroxidase. In EP 0962771, a detection device and method for the same purpose are also reported. Reinhartz, H.W. et al (Analyst 121 (1996)) 767-771 reported bispecific multivalent antibodies studied by real-time interaction analysis for the development of antigen-inhibitory enzyme-linked immunosorbent assays. Chemical generation of bispecific antibodies is reported by dopalapoudi, v.r. et al (proc.natl.acad.sci.107(2010) 22611-22616).

Summary of The Invention

Herein is reported a method for detecting the presence of, or for determining the amount of, a free (i.e. uncomplexed) binding partner in a sample, which binding partner may be specifically bound by a multispecific binder with at least one binding specificity, i.e. a first binding specificity.

It has been found to be advantageous to deplete the binding partner specifically bound by the multispecific binder, i.e. the binding partner-multispecific binder-complex, from the sample before determining the amount of free binding partner.

The method as reported herein achieves depletion of the multispecific binder by incubating a sample, which is either incubated with a binding partner (i.e. a second binding partner) which can be specifically bound by a different (i.e. a second) binding specificity of the multispecific binder, which does not bind to the binding partner to be determined (i.e. the first binding partner); the sample is either bound to a monospecific binder which specifically binds to one binding specificity of the multispecific binder, whereby the monospecific binder specifically binds to the binding specificity of the multispecific binder which does not bind to the binding partner to be determined (see figure 2).

One aspect as reported herein is an in vitro method for determining the presence and/or amount of a (first) antigen of a bispecific antibody in a sample, whereby the antigen to be detected can be specifically bound by a first binding specificity of the bispecific antibody, thereby complexing the antigen with the bispecific antibody (antigen-bispecific antibody-complex), comprising the steps of:

-incubating the sample comprising the antigen and the bispecific antibody with an anti-idiotype antibody specifically binding to a second binding specificity of the bispecific antibody different from the first binding specificity, wherein the anti-idiotype antibody is bound by an immobilization.

In one embodiment, the method comprises the steps of:

-incubating a sample comprising an antigen and a bispecific antibody with an anti-idiotype antibody which specifically binds to a second binding specificity of the bispecific antibody which is different from the first binding specificity, wherein the anti-idiotype antibody is bound by an immobilization, and

-detecting the antigen-bispecific antibody-anti-idiotype antibody complex, thereby determining the presence and/or amount of antigen of the bispecific antibody.

In one embodiment, the method comprises the steps of:

-incubating a sample comprising an antigen and a bispecific antibody with an anti-idiotype antibody which specifically binds to a second binding specificity of the bispecific antibody which is different from the first binding specificity, wherein the anti-idiotype antibody is bound by an immobilization, and

-incubating the complex formed in the first step with an antibody that specifically binds to an antigen at an epitope different from the epitope bound by the bispecific antibody, thereby determining the presence and/or amount of the antigen of the bispecific antibody in the sample.

In one embodiment, the method is for determining the presence and/or amount of an antigen of a bispecific antibody complexed with a bispecific antibody.

In one embodiment, the method comprises the steps of:

providing a sample comprising an antigen and a bispecific antibody, wherein at least 90% of the antigen is complexed with the bispecific antibody,

-incubating a sample comprising an antigen and a bispecific antibody with an anti-idiotype antibody which specifically binds to a second binding specificity of the bispecific antibody which is different from the first binding specificity, wherein the anti-idiotype antibody is bound by an immobilization, and

-incubating the complex formed in the first step with an antibody that specifically binds to an antigen at an epitope different from the epitope bound by the bispecific antibody, thereby determining the presence and/or amount of the antigen of the bispecific antibody in the sample.

In one embodiment, the method comprises the steps of:

incubating a sample comprising an antigen and a bispecific antibody with an amount of bispecific antibody to provide a sample in which at least 90% of the antigen is complexed by the bispecific antibody,

-incubating a sample comprising the antigen complexed by the bispecific antibody with an anti-idiotype antibody specifically binding to a second binding specificity of the bispecific antibody different from the first binding specificity, wherein the anti-idiotype antibody is bound by an immobilization, and

-incubating the complex formed in the preceding step with an antibody that specifically binds to an antigen at an epitope different from the epitope bound by the bispecific antibody, thereby determining the presence and/or amount of the antigen of the bispecific antibody in the sample.

In one embodiment, the amount of bispecific antibody is about 1 μ g/ml to 10 μ g/ml, preferably about 1.5 μ g/ml.

In one embodiment, the amount of bispecific antibody is 1mg/ml sample.

In one embodiment, at least 95% of the antigen is complexed with the bispecific antibody. In one embodiment, at least 98% of the antigen is complexed with the bispecific antibody.

One aspect as reported herein is an in vitro method for determining the amount of antibody-binding (first) antigen of a bispecific antibody in a sample, whereby the antigen can be specifically bound by a first binding specificity of the bispecific antibody, comprising the steps of:

incubating a first aliquot of the sample comprising the antigen and the bispecific antibody with an amount of bispecific antibody to provide a sample in which at least 90% of the antigen is complexed by the bispecific antibody,

-incubating a sample comprising the antigen complexed by the bispecific antibody with an anti-idiotype antibody specifically binding to a second binding specificity of the bispecific antibody different from the first binding specificity, wherein the anti-idiotype antibody is bound by an immobilization, and

incubating the complex formed in the preceding step with an antibody that specifically binds to an antigen at an epitope different from the epitope bound by the bispecific antibody, thereby determining the presence and/or amount of the antigen of the bispecific antibody in the sample, and determining the total amount of antigen present in the sample,

-incubating a second aliquot of the sample comprising the antigen and the bispecific antibody with an anti-idiotype antibody which specifically binds to a second binding specificity of the bispecific antibody which is different from the first binding specificity, wherein the anti-idiotype antibody is bound by an immobilisation, and

incubating the complex formed with an antibody that specifically binds to an antigen at an epitope different from the epitope bound by the bispecific antibody, thereby determining the amount of free antigen of the bispecific antibody present in the sample, and

-determining the amount of antibody-bound antigen of the bispecific antibody by the difference between the total amount of antigen present in the sample and the amount of free antigen present in the sample.

In one embodiment, the amount of bispecific antibody is about 1 μ g/ml to 10 μ g/ml, preferably about 1.5 μ g/ml.

In one embodiment, the amount of bispecific antibody is 1mg/ml sample.

One aspect as reported herein is a method for the in vitro determination of the presence and/or amount of a binding partner (antigen, target, analyte) which may be specifically bound by a first binding specificity of a multispecific binder, wherein the binding partner fraction which binds to the multispecific binder present in a sample is depleted by incubating the sample with a second binding partner which may be specifically bound by a second binding specificity of the multispecific binder, or a monospecific binder which specifically binds to a second binding specificity of the multispecific binder, prior to detecting the binding partner.

In one embodiment, the binding partner to be detected is an uncomplexed binding partner or a free binding partner.

Thus, one aspect as reported herein is an in vitro method for determining the presence and/or amount of a (first) binding partner of a multispecific binder, wherein said binding partner may be specifically bound by a first binding specificity of the multispecific binder, comprising the steps of:

-incubating the sample comprising the (first) binding partner and the multispecific binder with a second binding partner which can be specifically bound by a second binding specificity of the multispecific binder which is different from the first binding specificity.

In one embodiment, the method comprises the steps of:

-incubating a sample comprising a (first) binding partner and a multispecific binder with a monospecific binder which specifically binds to a second binding specificity of the multispecific binder which is different from the first binding specificity, and

-determining the amount of (free first) binding partner in the sample depleted of multispecific binder.

In one embodiment, the method comprises the steps of:

incubating a sample comprising a (first) binding partner and a multispecific binder with a monospecific binder which specifically binds to a second binding specificity of the multispecific binder which is different from the first binding specificity,

-depleting the monospecific binder-multispecific binder complex from the sample before determining the presence or amount of free binding partner, and

-determining the amount of (free first) binding partner in the sample depleted of multispecific binder.

The multispecific binder is removed/depleted from the sample by incubation with a second binding partner specifically bound by a second binding specificity of the multispecific binder. At the same time, the (first) binding partner-multispecific binder-complex is also removed from the sample.

In one embodiment, the multispecific binder is selected from an antibody, a fusion polypeptide comprising an antibody or antibody fragment and a non-antibody polypeptide, a fusion polypeptide comprising an antibody or antibody fragment and a soluble receptor, or a fusion polypeptide comprising an antibody or antibody fragment and a peptide binding molecule.

In one embodiment, the multispecific binder is an antibody. In one embodiment, the antibody is a bispecific antibody, or a trispecific antibody, or a tetraspecific antibody, or a pentaspecific antibody, or a hexaspecific antibody. In one embodiment, the antibody is a bispecific antibody.

In one embodiment, the monospecific conjugate is an anti-idiotype antibody.

In one embodiment, the binding specificity is a binding site or pair of an antibody heavy chain variable domain and an antibody light chain variable domain.

In one embodiment, the second binding partner or the monospecific binder is bound to a solid phase.

In one embodiment, the second binding partner is biotinylated, while the solid phase is coated with streptavidin. In one embodiment, the solid phase is streptavidin-coated paramagnetic beads or streptavidin-coated agarose beads.

One aspect as reported herein is a method for the immunological determination of the presence and/or amount of a binding partner for a multispecific binder in a sample using an immunoassay, wherein the multispecific binder is depleted from the sample prior to the determination of the binding partner.

In one embodiment of all aspects reported herein, the binding partner is a free binding partner, i.e. the binding partner is not bound or complexed by the multispecific binder.

In one embodiment, the second binding partner is a biotinylated second binding partner and is conjugated to the solid phase by streptavidin.

In one embodiment of the method as reported herein, the second binding partner is a mixture comprising at least two second binding partners that differ from the site of conjugation to the solid phase. In one embodiment, the site is an amino acid position of the amino acid sequence of the second binding partner.

In one embodiment, the first binding partner is a polypeptide.

In one embodiment, the second binding partner is a polypeptide.

In one embodiment, the conjugation of the polypeptide to its conjugation partner is performed by chemical binding via the N-terminus and/or-amino group (lysine), the-amino group of a different lysine, the carboxy-, thiol-, hydroxy-and/or phenol-functional group of the amino acid backbone of the polypeptide, and/or the sugar alcohol group of the carbohydrate structure of the polypeptide.

In one embodiment, the second binding partner is a mixture comprising a second binding partner conjugated to the solid phase via at least two different amino groups. Such coupling via different amino groups can be carried out as follows: a first step, acylation of a part of the amino group with a chemoprotectant, for example by citraconylation (cyclization). In a second step, conjugation is carried out through the remaining amino groups. Thereafter, citraconylation is removed and the binding partner is conjugated to the solid phase via the remaining free amino groups, i.e. the binding partner obtained is conjugated to the solid phase via an amino group which is not protected by citraconylation. Suitable chemoprotectants form bonds on the unprotected side chain amines, which are different from the N-terminal bonds and less stable. Many such chemoprotectants are known (see e.g. EP 0651761). In one embodiment, the chemoprotectant includes a cyclic dicarboxylic acid anhydride, such as maleic anhydride or citraconic anhydride.

In one embodiment, the second binding partner is conjugated to the solid phase by passive adsorption. Passive adsorption is described, for example, in Butler, J.E., in "Solid drugs in Immunoassay" (1996)205 and Diamandis, E.P., and Christopoulos, T.K, (eds.) in "Immunoassay" (1996) Academic Press (San Diego).

In one embodiment, the second binding partner is conjugated (immobilized) by a specific binding pair. In one embodiment, such binding pairs (first component/second component) are selected from streptavidin or avidin/biotin, antibodies/antigens (see, e.g., Hermanson, g.t. et al, Bioconjugate technologies, Academic Press (1996)), lectins/polysaccharides, steroid/steroid binding proteins, hormone/hormone receptors, enzymes/substrates, IgG/protein a and/or G, and the like. In one embodiment, the second binding partner is conjugated to biotin and the immobilization is performed by immobilized avidin or streptavidin.

One aspect as reported herein is a method for determining the presence and/or amount of a (first) antigen of a multispecific antibody in a sample, wherein the antigen to be detected can be specifically bound by the first binding specificity of the multispecific antibody, comprising the steps of:

-incubating the sample comprising the multispecific antibody, the (first) antigen to which the multispecific antibody is bound and the free (first) antigen with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity.

In one embodiment, the method comprises the steps of:

-incubating a sample comprising a multispecific antibody, a (first) antigen bound to the multispecific antibody and a free (first) antigen with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity, and

-determining the amount of (first) antigen in the sample depleted of multispecific antibodies.

In one embodiment, the method comprises the steps of:

incubating the sample comprising the (first) antigen and the multispecific antibody with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity,

-depleting the second antigen-multispecific antibody complex from the sample prior to determining the presence or amount of free antigen, and

-determining the amount of (first) antigen in the sample depleted of multispecific antibodies.

The multispecific antibody is removed from the sample by incubation with a second antigen that can be specifically bound by a second binding specificity of the multispecific antibody. At the same time, the (first) antigen-multispecific antibody-complex is also removed from the sample.

In one embodiment, the sample comprises a multispecific antibody, a free (first) antigen, and a multispecific antibody-antigen complex, the free (first) antigen of the multispecific antibody being detected.

In one embodiment, the second antigen is conjugated to a paramagnetic bead.

In one embodiment, the second antigen is conjugated to a solid phase.

In one embodiment, the second antigen is biotinylated and the solid phase is coated with streptavidin. In one embodiment, the solid phase is streptavidin-coated paramagnetic beads or streptavidin-coated agarose beads.

In one embodiment, the binding specificity is a binding site. In one embodiment, the binding site is a pair of an antibody heavy chain variable domain and an antibody light chain variable domain.

In one embodiment, the method comprises the steps of:

-incubating a sample comprising a multispecific antibody, a (first) antigen bound to the multispecific antibody and a free (first) antigen with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody different from the first binding specificity, forming a second antigen-multispecific antibody complex, and

-removing the second antigen-multispecific antibody complex from the sample.

In one embodiment, the second antigen-multispecific antibody complex is a mixture of the second antigen-multispecific antibody complex and the second antigen-multispecific antibody- (first) antigen complex.

In one embodiment, the method comprises the steps of:

-incubating a sample comprising a (first) antigen and a multispecific antibody with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity, to form a second antigen-multispecific antibody complex, and

-removing the second antigen-multispecific antibody complex from the sample, and

-determining the amount of (first) antigen in the sample depleted of multispecific antibodies.

In one embodiment, determining the amount of the (first) antigen comprises the steps of:

-incubating the sample depleted of multispecific antibody with a capture antibody which specifically binds to a (first) antigen, forming a capture antibody- (first) antigen complex, and

-correlating the amount of capture antibody- (first) antigen complex formed with the (first) antigen in the sample.

In one embodiment, determining the amount of the (first) antigen comprises the steps of:

incubating the sample depleted of multispecific antibody with a capture antibody which specifically binds to a (first) antigen forming a capture antibody- (first) antigen complex,

-incubating the capture antibody- (first) antigen complex with the tracer antibody, whereby the capture antibody and the tracer antibody bind to non-overlapping epitopes on the (first) antigen, and

-correlating the amount of capture antibody- (first) antigen-tracer antibody complex formed with the antigen in the sample.

In one embodiment, determining the amount of the (first) antigen comprises the steps of:

incubating the sample depleted of multispecific antibody with a capture antibody which specifically binds to a (first) antigen forming a capture antibody- (first) antigen complex,

incubating the capture antibody- (first) antigen complex with the tracer antibody, whereby the capture antibody and the tracer antibody bind to non-overlapping epitopes on the (first) antigen,

-incubating the capture antibody- (first) antigen-tracer antibody complex with a detection antibody comprising a detectable label, whereby the detection antibody specifically binds to the tracer antibody at an epitope outside the variable domain of the tracer antibody, and

-correlating the amount of formed capture antibody- (first) antigen-tracer antibody complex with the (first) antigen in the sample.

In one embodiment, the multispecific antibody is a bispecific antibody having a first binding specificity that specifically binds to a first antigen or a first epitope on an antigen and a second binding specificity that specifically binds to a second antigen or a second epitope on an antigen.

In one embodiment, the first antigen and the second antigen are the same antigen, the first binding specificity binds to a first epitope on the antigen, and the second binding specificity binds to a second epitope on the antigen, whereby the second epitope is an epitope that does not overlap with the first epitope, and binding to the first binding specificity does not interfere with binding to the second binding specificity.

In one embodiment, the method comprises the steps of:

-depleting the formed complex from the sample before determining the presence or amount of the (first) antigen.

One aspect as reported herein is an in vitro method for determining the presence and/or amount of a (first) antigen of a multispecific antibody in a sample, whereby the antigen to be detected can be specifically bound by a first binding specificity of the multispecific antibody, comprising the steps of:

-incubating the sample comprising the (first) antigen with a complex of a bispecific antibody and a second antigen or a complex of a bispecific antibody and an anti-idiotype antibody which specifically binds to a second binding specificity of the bispecific antibody which is different from the first binding specificity.

In one embodiment, the second antigen is a labeled second antigen. In one embodiment, the second antigen is immobilized on a solid phase by a specific binding pair. In one embodiment, the specific binding pair is biotin and streptavidin.

In one embodiment, the method comprises as a second step:

incubating the complex formed in the first step with an antibody that specifically binds to the first antigen at an epitope different from the epitope bound by the bispecific antibody.

One aspect as reported herein is an in vitro method for determining the presence and/or amount of a (first) antigen of a bispecific antibody in a sample, wherein the antigen to be detected can be specifically bound by the first binding specificity of the bispecific antibody, comprising the steps of:

-incubating the sample comprising the (first) antigen with the bispecific antibody and the second antigen or the bispecific antibody and the anti-idiotype antibody specifically binding to a second binding specificity of the bispecific antibody which is different from the first binding specificity, wherein the second antigen or the anti-idiotype antibody is bound by a solid phase.

In one embodiment, the method comprises as a second step:

incubating the complex formed in the first step with an antibody that specifically binds to the first antigen at an epitope different from the epitope bound by the bispecific antibody, whereby the amount of the (first) antigen of the bispecific antibody in the sample is determined.

One aspect as reported herein is an in vitro method for determining the presence and/or amount of a (first) antigen of a bispecific antibody complexed to a bispecific antibody (first antigen-bispecific antibody-complex) in a sample, wherein the antigen to be detected can be specifically bound by a first binding specificity of the bispecific antibody, comprising the steps of:

-incubating a sample comprising a (first) antigen and a bispecific antibody with an anti-idiotype antibody specifically binding to a second binding specificity of the bispecific antibody different from the first binding specificity, wherein the anti-idiotype antibody is bound by a solid phase.

In one embodiment, the method comprises as a second step:

incubating the complex formed in the first step with an antibody that specifically binds to the first antigen at an epitope different from the epitope bound by the bispecific antibody, whereby the amount of the (first) antigen of the bispecific antibody complexed to the bispecific antibody (first antigen-bispecific antibody-complex) in the sample is determined.

In one embodiment, the method comprises the steps of:

-depleting the formed complex from the sample before determining the presence or amount of the (first) antigen.

Detailed Description

Herein is reported an in vitro method for pre-treating a sample, detecting "free and/or total binding partners" of a multispecific binder, e.g. a bispecific antibody/drug, in pre-clinical and clinical samples.

It has been found to be advantageous to deplete the multispecific binder from the sample prior to detection of the free binding partner.

It has been found that it is advantageous to incubate the sample with a multispecific binder of the sample in order to convert almost all of the binding partner in the sample into a defined complex.

It has been found to be advantageous to use anti-idiotype antibodies to capture multispecific binders.

Herein is reported the use of a second binding partner specifically bindable by a second binding specificity of a therapeutic multispecific antibody for determining the level of a (free first) antigen that is capable of, but not yet bound by, the first binding specificity of the multispecific therapeutic antibody. The second antigen is used to deplete the multispecific antibody and multispecific antibody-antigen-complex to be detected from the sample.

Thus, herein is reported an in vitro method for determining the free (first) binding partner (antigen, target and analyte) of a multispecific binder, which free binding partner may be specifically bound by a first binding specificity of the multispecific binder, wherein prior to determining the free binding partner the multispecific binder is depleted from the sample by incubating the sample with a second binding partner capable of being specifically bound by a second binding specificity of the multispecific binder and subsequently depleting the multispecific binder and the second binding partner of the multispecific binder- (first) binding partner-complex from the sample, wherein the second binding specificity is different from the first binding specificity.

In the following, the methods reported herein are exemplified with such multispecific antibodies and antigens as embodiments of multispecific binders that specifically bind to multiple antigens or multiple epitopes on the same antigen as embodiments of (first) binding partners that are specifically bound by a first binding specificity of the multispecific antibody.

The term "antibody" is used broadly herein to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

In certain embodiments, the antibody is a multispecific antibody, e.g., a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one binding specificity is for a first antigen and the other binding specificity is for a second, different antigen. In certain embodiments, a bispecific antibody can bind 2 different epitopes of the same antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. In one embodiment, the antibody is a bispecific antibody that specifically binds to the first and second antigens. In one embodiment, the bispecific antibody has i) a first binding specificity that specifically binds to a first antigen or a first epitope on an antigen, and ii) a second binding specificity that specifically binds to a second antigen or a second epitope on the same antigen. In one embodiment, the second epitope on the same antigen is a non-overlapping epitope.

Multispecific antibodies are described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792 or WO 2010/145793.

An "antibody fragment" refers to a molecule other than an intact antibody, which molecule includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The "class" of an antibody refers to the type of constant domain or constant region that an antibody heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, several of which may be further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. And different classesThe heavy chain constant domains corresponding to immunoglobulin types are designated α, γ, and μ, respectively.

The term "free antigen" denotes an antigen that can be specifically bound by the binding specificity of an antibody, but which has not yet bound to this binding specificity. In one embodiment, the free antigen is an antigen that is not bound to an antibody or an antigen that is not complexed with an antibody.

The term "Fc-region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc-regions and variant Fc-regions. In one embodiment, the human IgG heavy chain Fc-region extends from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat, E.A. et al, Sequences of Proteins of immunological interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

"framework" or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. The FRs of a variable domain typically consist of 4 FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the following sequences of VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from an antibody from a non-human source using a human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically 2, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally can include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is highly variable in sequence and/or forms structurally defined loops ("hypervariable loops"). In general, a natural four-chain antibody comprises 6 HVRs; 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3). HVRs generally include amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia, C. and Lesk, A.M., J.mol.biol.196(1987) 901-917). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) occur at positions 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2 and 95-102 of H3 (Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, National Institutes of Health, Bethesda, MD (1991), NIHPubtilization 91-3242). In addition to CDR1 in VH, the CDRs generally include amino acid residues that form a hypervariable loop. CDRs also include "specificity determining residues" or "SDRs," residues that contact antigen. The SDR contained in the CDR region is called the abbreviation-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at positions 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (Almagro, J.C. and Fransson, J., Front.biosci.13(2008) 1619-. Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies comprised in the population are identical and/or bind the same epitope except for possible variant antibodies, e.g., containing naturally occurring mutations or mutations that arise during the course of producing a monoclonal antibody preparation, such variants typically being present in only minute amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies being described herein.

A "polypeptide" is a polymer composed of amino acids joined by peptide bonds, whether naturally or synthetically produced. Polypeptides of less than about 20 amino acid residues may be referred to as "peptides", while molecules consisting of 2 or more polypeptides, or molecules comprising one polypeptide of more than 100 amino acid residues, may be referred to as "proteins". The polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions or carboxylic acid esters. The non-amino acid components may be added by the cell expressing the polypeptide and may vary with the cell type. Herein, a polypeptide is defined in terms of the structure of the amino acid backbone of the polypeptide or the nucleic acid encoding it. Additives, such as carbohydrate groups, are generally not specifically indicated, but may be present.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs) (see, e.g., Kindt, t.j. et al, KubyImmunology, 6 th edition, w.h.freeman and co., n.y. (2007), page 91). A single VH or VL domain is sufficient to generate antigen-binding specificity. In addition, libraries of complementary VL or VH domains can be screened using the VH or VL domains, respectively, of antigen-binding antibodies to isolate antibodies that bind a particular antigen (see, e.g., Portolano, S. et al, J.Immunol.150(1993) 880-887; Clackson, T. et al, Nature 352(1991) 624-628).

The term "anti-idiotype antibody" denotes an antibody that specifically binds to the binding specificity (e.g. binding site) of a parent antibody, i.e. is directed against an antigen binding site of e.g. a parent antibody. In one embodiment, the anti-idiotype antibody specifically binds to one or more CDRs of a parent antibody. In one embodiment, the parent antibody is a therapeutic antibody. In one embodiment, the parent antibody is a multispecific antibody. In one embodiment, the parent antibody is a bispecific antibody.

Two epitopes are overlapping if a 50% or more, in one embodiment a 75% or more reduction in signal is detected by a Surface Plasmon Resonance (SPR) assay using an immobilized antibody and a soluble antigen, or vice versa, where the concentration of the epitope under investigation is 20-50nM and the concentration of the antibody required to detect the epitope overlap is 100 nM. Alternatively, a method may be used in which epitope overlap of two antibodies binding to the same antigen is determined with the aid of a competitive test system. For this purpose, cells expressing recombinant antigenic epitopes are used, for example with the aid of cell-based enzyme immunoassays (ELISA), to test whether antibodies requiring detection of epitope overlap compete with other antibodies for binding to the immobilized antigen. For this purpose, the immobilized antigen is incubated with an antibody in a labeled form and an excess of antibody that is required to determine epitope overlap. Epitope overlap can be verified simply by detecting the bound label. If, with respect to known antibodies, it was determined that at the same concentration, the signal reduction was greater than 70%, in one embodiment, a reduction of greater than 80%, or at higher concentrations (in one instance, 10)⁵A double excess of antibodies for which epitope overlap is to be determined), greater than 80% substitution, and in one embodiment greater than 90% substitution, then epitope consistency or overlap exists, and both antibodies bind the same or overlapping epitopes on the same antigen.

The principles of different immunoassays are described, for example, in Hage, d.s. (anal. chem.71(1999) 294R-304R). Lu, B. et al (Analyst 121(1996)29R-32R) reported directed immobilized antibodies for use in immunoassays. Avidin-biotin-mediated immunoassays are reported, for example, by Wilchek, M., and Bayer, E.A., in Methods enzymol.184(1990) 467-469.

Polypeptides and monoclonal antibodies and their constant domains contain a plurality of reactive amino acid side chains for coupling to binding partners such as surfaces, proteins, polymers (e.g., PEG, cellulose or polystyrene), enzymes or members of binding pairs. Chemically reactive groups of amino acids are, for example, amino groups (lysine, alpha-amino), mercapto groups (cystine, cysteine and methionine), carboxylic acid groups (aspartic acid, glutamic acid) and sugar alcohol groups. Such methods are described, for example, in "Bioconjugation" by Aslam M. and Dent, A. MacMillan Ref.Ltd.1999, pages 50-100.

One of the most common reactive groups of polypeptides and antibodies is the aliphatic-amine of the amino acid lysine. In general, almost all polypeptides and antibodies are rich in lysine. Above pH 8.0 (pK)_a9.18), lysinamines are fairly good nucleophilic groups and therefore react simply and completely with a variety of reagents to form stable bonds.amine-reactive reagents react primarily with lysine and the α -amino group of proteins.reactive esters, particularly N-hydroxy-succinimide (NHS) esters, are the most commonly used reagents to modify amine groups.the optimum pH for reaction in an aqueous environment is pH 8.0 to 9.0 isothiocyanates are amine modifying reagents to form thiourea bonds with proteinsGroup or aromatic amines, hydrazines and hydrazides react to form imine intermediates (Schiff bases). The Schiff base can be selectively reduced by mild or strong reducing agent (such as sodium borohydride or sodium cyanoborohydride) to derive stable alkylamine bond. Other reagents used to modify amines are anhydrides. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a bifunctional chelating agent containing 2 amine-reactive anhydride groups. It can react with the N-terminus and-amino group of an amino acid to form an amide bond. The anhydride ring is opened to create a multivalent metal-chelating arm that is capable of tightly binding to the metal in the coordination complex.

Another common reactive group in polypeptides and antibodies is a sulfhydryl residue from the sulfur-containing amino acid cystine and its reduction product cysteine (or half cystine). Cysteine contains a free sulfhydryl group, is more nucleophilic than amines, and is generally the most reactive functional group in proteins. Sulfhydryl groups are generally reactive at neutral pH and can therefore be selectively coupled to other molecules in the presence of amines. Since free sulfhydryl groups are relatively reactive, the groups of proteins bearing these groups are often present in the oxidized form of disulfide groups or disulfide bonds. In such proteins, reduction of disulfide bonds with reagents such as Dithiothreitol (DTT) is necessary to generate reactive free thiols. Thiol-reactive reagents are reagents on a polypeptide that will couple a thiol group to form a thioether-coupled product. These reagents react rapidly at mildly acidic to neutral pH and therefore can react selectively in the presence of amine groups. The literature reports the use of several thiolated crosslinkers, such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio) acetate (SATA) and sulfosuccinimidyl 6- [3- (2-pyridyldithio) propionamido ] hexanoate (Sulfo-LC-SPDP), for providing an efficient means of introducing multiple sulfhydryl groups through reactive amino groups. Haloacetyl derivatives (e.g., iodoacetamide) form thioether bonds and are also reagents for thiol modification. Other effective agents are maleimides. The reaction of maleimide with the thiol-reactive reagent is essentially the same as that with iodoacetamide. Maleimide reacts rapidly at weakly acidic to neutral pH.

Another common reactive group in polypeptides and antibodies is carboxylic acids. Polypeptides and antibodies contain a carboxylic acid group in the side chains of aspartic acid and glutamic acid at the C-terminal position. The relatively low reactivity of carboxylic acids in water often makes it difficult to selectively modify polypeptides and antibodies with such groups. When the modification is performed, the carboxylic acid group is typically converted to a reactive ester using a water-soluble carbodiimide and reacted with a nucleophile such as an amine, hydrazide, or hydrazine. The amine-containing reagent should be weakly basic to selectively react with the activated carboxylic acid in the presence of the more strongly basic-amino group of lysine to form a stable amide bond. When the pH is raised above 8.0, protein cross-linking can occur.

Sodium periodate can be used to oxidize the alcohol portion of the sugar in the carbohydrate moiety attached to the antibody to an aldehyde. Each aldehyde group can be reacted with an amine, hydrazide, or hydrazine as described for the carboxylic acid. Since the carbohydrate moiety is predominantly present in the crystallizable fragment (Fc) region of the antibody, conjugation can be achieved by site-directed modification of the carbohydrate away from the antigen-binding site. The Schiff base intermediate is formed and can be reduced to the alkylamine by reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strongly) water-soluble reducing agent.

The term "sample" includes, but is not limited to, any amount of material from a living or previous living. Such living 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. In one embodiment, such substances include, but are not limited to, whole blood, serum or plasma from an individual, which is the most commonly used sample source in clinical routine.

The term "solid phase" means a non-liquid substance, including particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glasses, and ceramics; gel substances such as silica, alumina and polymer gels; a capillary tube, which may be made of polymer, metal, glass and/or ceramic; zeolites and other porous materials; an electrode; a microtiter plate; a solid bar (strip); and a cuvette, test tube or other spectrophotometer specimen container. The solid phase component is distinguished from the surface of an inert solid in that the "solid phase" contains at least one moiety on its surface that is intended to interact with a substance in the sample. The solid phase may be a fixed component, such as a tube, strip, cuvette or microtiter plate, or may be a non-fixed component, such as beads and microparticles. A variety of microparticles that allow for non-covalent or covalent attachment of proteins and other substances may be used. Such particles include polymeric particles such as polystyrene and poly (methacrylate); gold particles, such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass, and metal oxide particles. See, e.g., Martin, C.R., et al, Analytical Chemistry-News & Features, 70(1998)322A-327A, or Butler, J.E., Methods 22(2000) 4-23.

In one embodiment, the detectable label is selected from the group consisting of chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups, metal particles or haptens (e.g., digoxigenin). The detectable label may also be a crosslinking group that can be photoactivated, for example, azido or 1H-aziridinyl. In yet another embodiment, the metal chelate which can be detected by electrochemiluminescence is a signaling group, with particular preference being given to ruthenium chelates, e.g.ruthenium (bipyridine)₃ ²⁺A chelate compound. Suitable ruthenium marker groups are described, for example, in EP 0580979, WO 90/05301, WO 90/11511 and WO 92/14138.

Herein is reported a method for determining the presence and/or amount of a (free first) antigen of a multispecific antibody in a sample, comprising a solid phase immobilized second antigen capable of being specifically bound by one binding specificity of the multispecific antibody, which is not the binding specificity of the multispecific antibody specifically binding to the (free first) antigen to be determined, for depleting the multispecific antibody in complexed or uncomplexed form from the sample prior to determining the amount of the (free first) antigen.

In one embodiment, the method comprises depleting the second antigen-multispecific antibody-complex from the sample prior to determining the presence or amount of free (first) antigen.

In one embodiment, the presence and/or amount of (free first) antigen in the multispecific antibody-depleted sample is determined by an antigen bridging immunoassay. In one embodiment, the immunoassay comprises a capture antibody and a tracer antibody, wherein the capture antibody is conjugated to a solid phase and the tracer antibody is conjugated to a detectable label.

One aspect as reported herein is an in vitro method for determining the presence and/or amount of a (free first) antigen of a multispecific antibody in a sample, wherein the antigen to be detected can be specifically bound by a first binding specificity of the multispecific antibody, comprising the steps of:

-incubating the sample comprising the (first) antigen and the multispecific antibody with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity, thereby removing the multispecific antibody from the sample.

It is known to those skilled in the art that due to thermodynamic equilibrium, a sample comprising an antigen and an antibody that can specifically bind to the antigen comprises a mixture of free antigen, antibody-bound antigen and free antibody.

In one embodiment, the method comprises the steps of:

-incubating a sample comprising a (first) antigen and a multispecific antibody with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity, to form a second antigen-multispecific antibody complex, and

-removing the second antigen-multispecific antibody complex from the sample.

In one embodiment, the method comprises the steps of:

incubating a sample comprising a (first) antigen and a multispecific antibody with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity, to form a second antigen-multispecific antibody complex,

-removing the second antigen-multispecific antibody complex from the sample, and

-determining the amount of (first) antigen in the sample depleted of multispecific antibodies.

In one embodiment, the method comprises the steps of:

-depleting the second antigen-multispecific antibody-complex from the sample prior to determining the presence or amount of free (first) antigen.

In one embodiment, the method comprises the steps of:

incubating the sample comprising the (first) antigen and the multispecific antibody with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity,

-depleting the second antigen-multispecific antibody complex from the sample prior to determining the presence or amount of free (first) antigen, and

-determining the amount of (first) antigen in the sample depleted of multispecific antibodies.

In one embodiment, the presence and/or amount of the (first) antigen is determined by an antigen bridging immunoassay.

In one embodiment, determining the presence and/or amount of the (first) antigen is determining the amount of free (first) antigen.

In one embodiment, determining the presence and/or amount of the (first) antigen comprises the steps of:

incubating the sample depleted of multispecific antibody with a capture antibody that specifically binds to a (first) antigen, forming a capture antibody-antigen complex, and

-correlating the amount of capture antibody- (first) antigen complex formed with the amount of antigen in the sample.

In one embodiment, determining the presence and/or amount of the (first) antigen comprises the steps of:

incubating the sample depleted of multispecific antibody with a capture antibody that specifically binds to a (first) antigen to form a capture antibody- (first) antigen complex,

-incubating the capture antibody- (first) antigen complex with the tracer antibody, wherein the capture antibody and the tracer antibody bind to non-overlapping epitopes on the (first) antigen, and

-correlating the amount of formed capture antibody- (first) antigen-tracer antibody complex with the (first) antigen in the sample.

In one embodiment, the tracer antibody comprises a detectable label.

In one embodiment, determining the presence and/or amount of the (first) antigen comprises the steps of:

incubating the sample depleted of multispecific antibody with a capture antibody that specifically binds to a (first) antigen to form a capture antibody- (first) antigen complex,

-incubating the capture antibody- (first) antigen complex with the tracer antibody, wherein the capture antibody and the tracer antibody bind non-overlapping epitopes on the (first) antigen,

-incubating the capture antibody- (first) antigen-tracer antibody complex with a detection antibody comprising a detectable label, wherein the detection antibody specifically binds to the tracer antibody at an epitope other than the variable domain of the tracer antibody, and

-correlating the amount of formed capture antibody- (first) antigen-tracer antibody complex with the (first) antigen in the sample.

In one embodiment, the capture antibody and the tracer antibody bind to non-overlapping epitopes located on the (first) antigen.

In one embodiment of the method as reported herein, the (first) antigen is a free (first) antigen.

In one embodiment, the second antigen and/or the capture antibody is conjugated to a solid phase.

The second antigen and/or capture antibody for use in the methods as reported herein may be conjugated to a solid phase. In one embodiment, conjugation is carried out by chemical binding via the N-terminus and/or-amino group (lysine), the-amino group of a different lysine, the carboxy-, thiol-, hydroxy-and/or phenol-functional group of the amino acid backbone of the antigen or antibody, and/or the sugar alcohol group of the carbohydrate structure of the antigen and/or antibody. In one embodiment, the second antigen and/or capture antibody is a mixture of at least two second antigens and/or antibodies conjugated to a solid phase, wherein the site of conjugation of the at least two second antigens and/or antibodies conjugated to the solid phase is different from the site of conjugation of the solid phase. For example, a mixture of at least two second antigens and/or two antibodies conjugated to a solid phase may comprise conjugation to the solid phase through an amino acid of the amino acid backbone, and conjugation to the solid phase through a sugar alcohol group of the carbohydrate structure. Furthermore, for example, a mixture of at least two second antigens and/or two antibodies conjugated to a solid phase may comprise second antigens and/or antibodies conjugated to the solid phase via different amino acid residues of their amino acid backbone. The expression "different amino acid residues" denotes two different types of amino acids, such as lysine and aspartic acid, or tyrosine and glutamic acid, or two amino acid residues in the amino acid backbone at different positions in the amino acid sequence of the second antigen and/or antibody. In the latter case, the amino acids may be of the same kind or of different kinds. The expression "different antibody sites" denotes a difference in the type of site, for example an amino acid or sugar alcohol group, or a difference in the number of amino acids of the amino acid backbone (for example at the position where the second antigen and/or antibody is conjugated to the solid phase). The same application can also be used for the tracer antibody used in the method as reported herein.

In one embodiment of the method, the immunoassay comprises a capture antibody, a tracer antibody and a detection antibody, wherein the capture antibody is a biotinylated antibody against an antigen conjugated to a solid phase by streptavidin, the tracer antibody is an antibody against an antigen conjugated to digoxigenin, and the detection antibody is an antibody against digoxigenin conjugated to horseradish peroxidase.

General method for depleting a complex consisting of bispecific antibodies specifically binding to antigen X and antigen Y from a sample comprising antigen X and/or antigen Y (for determining antigen X or antigen Y, respectively), comprising the following steps:

assembling a complex of bispecific antibody (anti-X/Y antibody) that specifically binds antigen X and antigen Y:

constant concentrations of antigen X were incubated with increasing amounts of bispecific monoclonal antibody having a first binding specificity specific for binding to antigen X and a second binding specificity specific for binding to antigen Y (anti-X/Y antibody) for 1 hour at room temperature. Thereafter, the sample was used as a positive control in the depletion step.

-a depletion step:

to deplete the antigen X bound to the anti-X/Y antibody, biotinylated antigen Y-BI was bound to approximately 10. mu.g/ml magnetic streptavidin coated beads (SA-beads). Each sample was washed with 600. mu.l of SA-beads and separated from the supernatant using a magnetic separator. Mu.l of the solution containing biotinylated antigen Y was mixed with SA-beads and incubated at room temperature for about 1 hour. Excess unbound antigen was removed by washing the beads 3 times with a magnetic separator. Then, the beads coated with antigen Y were incubated with about 250. mu.l of a sample containing a complex of an anti-X/Y antibody and antigen X. The mixture was incubated at room temperature with shaking for about 1 hour. After incubation, the sample is separated from the beads using a magnetic separator. Supernatants were collected for analysis of "free" antigen X in ELISA (see e.g. example 2). The remaining beads were transferred to an ELECSYS container and the antigen X bound to the beads (antigen X bound to bispecific antibody) was analyzed with an ELECSYS 2010 analyzer according to standard user-directed protocols.

To deplete antigen Y bound to anti-X/Y antibody, biotinylated antigen X (X-BI) was bound to approximately 10. mu.g/ml magnetic streptavidin coated beads (SA-beads). Each sample was washed with 600. mu.l of SA-beads and separated from the supernatant using a magnetic separator. Mu.l of the solution containing biotinylated antigen X was mixed with SA-beads and incubated at room temperature for about 1 hour. Excess unbound antigen X was removed by washing the beads 3 times with a magnetic separator. Antigen X beads were then incubated with approximately 250. mu.l of sample containing complexes of anti-X/Y antibodies with antigen Y. The mixture was incubated at room temperature with shaking for about 1 hour. After incubation, the sample is separated from the beads using a magnetic separator. Supernatants were collected for analysis of "free" antigen Y in ELISA (see e.g. example 2). The remaining beads were transferred to an ELECSYS container and the antigen Y bound to the beads (antigen Y bound to bispecific antibody) was analyzed with an ELECSYS 2010 analyzer according to standard user-directed protocols.

To determine the pharmacokinetic properties of the multispecific antibody in vivo, the distribution or amount of free first antigen, free second antigen, free multispecific antibody, and multispecific antibody-first and/or second antigen-complex may be determined.

One aspect as reported herein is an in vitro method suitable for determining the presence and/or amount of a free (first) antigen of a bispecific antibody, wherein the (first) antigen can be specifically bound by a first binding specificity of the bispecific antibody, comprising the steps of:

-incubating a sample comprising a (first) antigen and a bispecific antibody with an anti-idiotype antibody which specifically binds to a second binding specificity in the bispecific antibody which is different from the first binding specificity.

In one embodiment, the method comprises the steps of:

incubating a sample comprising a (first) antigen and a bispecific antibody with an anti-idiotype antibody which specifically binds to a second binding specificity of the bispecific antibody which is different from the first binding specificity,

-removing the anti-idiotype antibody-bispecific antibody complex from the sample, and

-determining the amount of antigen in the sample depleted of bispecific antibody.

The anti-idiotype antibody may be bound to a solid phase.

Detection of the bispecific antibody can be carried out by immunological determination using a bridging assay comprising a capture molecule, a tracer molecule and a detector molecule.

The capture molecules may be bound to a solid phase. The capture molecule may generally be any binding partner of the bispecific antibody (e.g., an antigen), a generic complexing agent for the bispecific antibody (e.g., an Fc-receptor or anti-Fc-region antibody in the case of a full-length antibody), or an anti-idiotypic antibody that specifically binds to one binding specificity of the bispecific antibody.

The tracer molecule may be any binding partner of a multispecific binder (e.g. one antigen of a bispecific antibody, but if one antigen is used as capture molecule, a different antigen is used as tracer molecule), a generic complexing agent for a bispecific antibody (e.g. an Fc-receptor in the case of a full-length antibody, provided that the molecule is not yet used as capture molecule, or an anti-Fc-region antibody in the case of a full-length antibody, provided that the antibody binds a different epitope if the same type of antibody is also used as capture molecule), or a first partner of a binding pair (if the bispecific antibody is derived with a second partner of a binding pair) (provided that a different binding pair is used than that used to immobilize the capture molecule), or an anti-idiotypic antibody that specifically binds the binding specificity of a bispecific antibody (provided that it binds with a different binding specificity than the anti-idiotypic antibody used as capture molecule) .

One aspect as reported herein is an in vitro method for determining the presence and/or amount of a (first) antigen of a bispecific antibody complexed to a bispecific antibody (first antigen-bispecific antibody-complex) in a sample, wherein the antigen to be detected can be specifically bound by a first binding specificity of the bispecific antibody, comprising the steps of:

-incubating a sample comprising a (first) antigen and a bispecific antibody with an anti-idiotype antibody specifically binding to a second binding specificity of the bispecific antibody different from the first binding specificity, wherein the anti-idiotype antibody is bound by a solid phase.

In one embodiment, the anti-idiotype antibody-bispecific antibody- (first) antigen-complex is formed in a first step of the method.

In one embodiment, the method comprises as a second step:

incubating the complex formed in the first step with an antibody that specifically binds to the (first) antigen at an epitope different from the epitope bound by the bispecific antibody, thereby determining the presence and/or amount of the (first) antigen of the bispecific antibody complexed to the bispecific antibody ((first) antigen-bispecific antibody-complex) in the sample.

Determination of total, antibody-bound and free antigens helps to monitor the treatment with therapeutic antibodies. For example, in the case of bispecific antibodies that specifically bind ANG2 and VEGF, the mechanism of action is to block both antigens from binding to their respective receptors. In the absence of free ligand, the signaling pathway is blocked. Thus, the possibility of determining the fraction of free antigen and antigen bound to antibody has an impact on the treatment, in particular for determining the dose and dosing frequency. Total antigen represents the sum of free and bound (antibody) antigen.

In the course of treating a patient, an antigen and a therapeutic antibody are present in parallel in the patient and form a complex thereof. Thus, there is a balance between antigen bound antibody and free antigen in vivo. In vitro, the distribution of the equilibrium can be influenced by, for example, diluting the sample or by the selected antibody for detection or capture. In particular for free antigens, there may be a potential difference between the amount present "in vivo" and the amount determined "in vitro". In addition to the pretreatment method, the antigen bound to the antibody and the total antigen can be determined analytically and then further overcome based on the above determination of the free antigen. Typically, the assay format for determining the antigen bound to the antibody and the total antigen is set differently, e.g., the type of assay may be different between the assay for determining the antigen bound to the antibody and the assay for determining the total antigen, the antibody used for capture and detection may be different, and the order and timing of the incubation steps may be different.

In contrast, an assay is described in example 9 and figure 13, which can be used to determine total antigen and antigen bound antibody. This unique feature is conferred by the bispecific nature of the (therapeutic) antibody and utilizes anti-idiotypic antibodies directed against the second binding specificity.

Bound target is determined directly in an in vivo sample, such as a plasma sample.

Briefly, free antigen present in a sample is converted to antibody-bound antigen by adding excess bispecific antibody in vitro. Thus, the total antigen is determined by performing exactly the same assay again for the above-described bound target. The difference between the in vitro assays with or without bispecific antibody addition reflects the amount of transformed target, i.e., the free target originally present.

The following examples and figures are provided to aid the understanding of the present invention, but the appended claims set forth the true scope of the invention. It is understood that modifications may be made to the process described without departing from the spirit of the invention.

Description of the drawings

FIG. 1 equilibrium between drug-bound target (antigen bound to bispecific antibody) and free target (free antigen)

FIG. 2 depletes the c-MET binding to bispecific anti-c-MET/HER 3 antibodies by using HER 3; biotinylated HER3 immobilized on streptavidin coated magnetic beads; after incubating these magnetic beads with a sample (e.g., a serum sample), bispecific anti-c-MET/HER 3 antibodies are bound and depleted by immobilized HER 3; co-depleting c-MET bound to the bispecific antibody; free c-MET (not bound to bispecific antibody) remains in the supernatant of the sample.

FIG. 3 Sandwich ELISA for detection of c-MET: biotinylated anti-c-MET antibody was bound to streptavidin coated microtiter plates; the immobilized anti-c-MET antibody specifically binds to free c-MET, while the second, DIG-labeled anti-c-MET antibody allows detection of bound c-MET; the assay was used to detect "free" c-MET in the supernatant of depleted samples.

FIG. 4(A) measured signal levels of c-MET in buffer before and after immunodepletion: preparing a sample containing 100ng/ml c-MET and increasing amounts of bispecific anti-c-MET/HER 3 antibody; depleting the complex of bsmAb and bound c-MET with biotinylated HER3 bound to magnetic beads; the graph shows the c-MET concentrations before and after depletion as determined by ELISA.

FIG. 4(B) measured signal levels of c-MET in serum before and after immunodepletion: preparing a sample containing 100ng/ml c-MET and increasing amounts of bispecific anti-c-MET/HER 3 antibody; depleting the complex of bsmAb and bound c-MET with biotinylated HER3 bound to magnetic beads; the graph shows the c-MET concentrations before and after depletion as determined by ELISA.

Fig. 5(a) ELISA for detection of antigen of bispecific antibody with the aid of other antigens: biotinylated HER3 was bound to streptavidin-coated microtiter plates and used to immobilize bispecific anti-c-MET/HER 3 antibodies; c-MET binds to an immobilized bispecific anti-c-MET/HER 3 antibody; a second anti-c-MET antibody (DIG-labeled) together with a polyclonal HRP-labeled anti-DIG antibody allows detection of bound c-MET.

FIG. 5(B) ELISA for detection of antigen of bispecific antibody by means of anti-idiotype antibody against other binding specificities of the bispecific antibody: biotinylated anti-idiotypic antibodies directed against the binding specificity, which specifically bind to HER3(idmAb < HER3> -BI), were bound to streptavidin-coated microtiter plates and used to immobilize bispecific anti-c-MET/HER 3 antibodies; c-MET binds to an immobilized bispecific anti-c-MET/HER 3 antibody; a second anti-c-MET antibody (DIG-labeled) together with a polyclonal HRP-labeled anti-DIG antibody allows detection of bound c-MET.

Figure 6 calibration curve of ELISA for detection of antigen of bispecific antibody with the aid of other antigens.

FIG. 7 Sandwich ELISA for VEGF detection with anti-ANG 2/VEGF antibody (bispecific antibody-bound VEGF): biotinylated anti-idiotype antibodies specific for ANG2 binding of bispecific antibodies were conjugated to streptavidin coated microtiter plates. The immobilized anti-idiotypic anti-ANG 2 antibody forms a complex with an anti-ANG 2/VEGF antibody. A second digoxigenin-labeled anti-VEGF antibody was used to detect VEGF bound to the bispecific antibody.

FIG. 8 calibration curve of ELISA for detection of VEGF complexes with anti-ANG 2/VEGF antibody. To serum containing 500. mu.g/ml anti-ANG 2/VEGF antibody was added 0ng/ml to 50ng/ml serial dilutions of VEGF and incubated for 1 hour at room temperature. Samples were analyzed as described in example 6.

Figure 9 sandwich ELISA for detection of ANG2 complex with anti-ANG 2/VEGF antibody (ANG 2 bound by bispecific antibody): biotinylated anti-idiotype antibodies directed against the VEGF binding specificity of the bispecific antibody bind to streptavidin coated microtiter plates. The immobilized anti-idiotypic anti-VEGF antibody forms a complex with the anti-ANG 2/VEGF antibody. A second digoxigenin-labeled anti-ANG 2 antibody was used to detect bound ANG 2.

Fig. 10 calibration curve for ELISA to detect the complex of ANG2 and anti-ANG 2/VEGF antibody. To the serum containing 5 μ g/ml anti-ANG 2/VEGF antibody was added 0ng/ml to 5000ng/ml serial dilutions of ANG2 and incubated for 1 hour at room temperature. Samples were analyzed as described in example 7.

Figure 11 sandwich ELISA for detection of VEGF complexes with anti-ANG 2/VEGF antibody (VEGF bound by bispecific antibody): biotinylated anti-VEGF antibody was bound to streptavidin coated microtiter plates. The immobilized anti-VEGF antibody forms a complex with the anti-ANG 2/VEGF antibody-VEGF complex. Digoxigenin-labeled anti-idiotype anti-ANG 2 antibody was used to detect the complex bound to the antibody.

FIG. 12 calibration curve for ELISA to detect complexes of VEGF with anti-ANG 2/VEGF antibody. To the serum containing anti-ANG 2/VEGF antibody was added 0ng/ml to 10ng/ml serial dilutions of VEGF and incubated for 1 hour at room temperature.

Figure 13 sandwich ELISA for detection of ANG2 complex with anti-ANG 2/VEGF antibody (bispecific antibody bound ANG 2): free ANG2 was converted to antibody-bound ANG2 by incubating the samples with a bispecific anti-ANG 2/VEGF antibody. Biotinylated anti-idiotype antibodies directed against the VEGF binding specificity of the bispecific antibody bind to streptavidin coated microtiter plates. The immobilized anti-idiotypic anti-VEGF antibody forms a complex with the ANG 2-anti-ANG 2/VEGF antibody complex. A second digoxigenin-labeled anti-ANG 2 antibody that specifically binds to an epitope on ANG2 different from the anti-ANG 2/VEGF antibody was used to detect total ANG 2.

Example 1

In the case of bispecific drug molecules, the drug-binding target (antibody-binding antigen) is depleted

A) Complex assembling bispecific anti-c-MET/HER 3 antibodies and c-MET

A constant concentration of c-MET was incubated with increasing amounts of bispecific antibody having a first binding specificity that specifically binds c-MET and a second binding specificity that specifically binds HER3 (bispecific anti-c-MET/HER 3 antibody) for 1 hour at room temperature. These samples were then used as positive controls in the depletion step.

B) Depletion step

To deplete the c-MET bound to the bispecific anti-c-MET/HER 3 antibody, biotinylated HER3(HER3-BI) was bound to 10 μ g/ml magnetic streptavidin coated beads (SA beads). Each sample was washed with 600. mu.l of SA-beads and separated from the supernatant using a magnetic separator. Approximately 600. mu.l of a solution containing HER3-BI was mixed with SA-Beads and incubated for 1 hour at room temperature. Excess unbound HER3-BI was removed by washing the beads 3 times with a magnetic separator. The antigen coated beads were then incubated with 250. mu.l of a sample containing a complex of a bispecific anti-c-MET/HER 3 antibody and c-MET. The samples were incubated at room temperature with shaking for 1 hour. After incubation, the sample is separated from the beads using a magnetic separator. Supernatants were collected for analysis of "free" c-MET in ELISA (see example 2).

Example 2

ELISA for the detection of c-MET

Biotinylated monoclonal antibodies against c-MET were coated onto streptavidin microtiter plates in the first step. The supernatant sample of the depletion step (see example 1) was diluted 10-fold and added to the wells of a microtiter plate coated with anti-c-MET antibody. Free c-MET contained in the sample is bound by anti-c-MET antibodies coated in the wells of the microtiter plates. After 1 hour incubation at room temperature, the plates were washed 3 times and the samples were removed. Then, a monoclonal DIG-labeled anti-c-MET antibody with a different specificity (i.e., epitope) from the coating antibody was added to the wells, and incubated at room temperature for 1 hour. After another washing step, polyclonal HRP-labeled anti-DIG antibody was added to the plate and incubated for an additional 1 hour. The development reaction was triggered using ABTS substrate solution (see figure 3).

Example 3

Drug-bound c-MET depleted in human serum and buffer

According to example 1, bispecific anti-c-MET/HER 3 antibodies were diluted to concentrations of 20/10/5/1/0.5/0.1 and 0 μ g/ml, respectively, and incubated with a constant concentration of 100ng/ml c-MET. The dilution was carried out in two different matrices:

PBS/BSA buffer

Human pooled serum (Trina, NHS Base matrix)

The samples were incubated at room temperature with shaking for 1 hour. The sample was then depleted as described in example 1.

Complexes of c-MET with bispecific anti-c-MET/HER 3 antibodies were captured using HER 3-BI.

After depletion, supernatants were measured in a c-MET ELISA as described in example 2.

As shown in figure 4a, c-MET bound to bispecific anti-c-MET/HER 3 antibody was removed by immunodepletion. In the presence of 5. mu.g/ml or more of bispecific antibody, the c-MET signal after depletion was similar to the assay background signal.

Similar behavior was observed in the serum samples shown in figure 4 b.

Example 4

ELISA for detection of antigen of bispecific antibody by means of other antigen

A) Detecting the amount of (total) c-MET in a sample

Biotinylated HER3 was bound to the streptavidin microtiter plate in the first step. In parallel, the bispecific anti-c-MET/HER 3 antibody was pre-incubated with the sample/standard for 1 hour. During the pre-incubation, c-MET in the sample binds to bifunctional anti-c-MET/HER 3 antibodies. After washing the streptavidin-coated plates, a pre-incubation mixture of c-MET and anti-c-MET/HER 3 antibody was added to the plates and incubated for 1 hour at room temperature. After another washing step to remove unbound components from the sample, digoxigenin-labeled anti-c-MET antibody (binding to a different epitope on c-MET than the bifunctional anti-c-MET/HER 3 antibody) was added and incubated for 1 hour. After another washing step, polyclonal horseradish peroxidase (HRP) -labeled anti-DIG antibody was added to the plate and incubated for 1 hour. The development reaction was triggered using ABTS substrate solution (see figure 5 a).

B) Detection of complexes of bispecific anti-c-MET/HER 3 antibodies and c-MET (Presence) in a sample

Biotinylated HER3 was bound to the streptavidin microtiter plate in the first step. After washing the plates, samples and standards were added to the plates and incubated for 1 hour at room temperature. Complexes of bispecific anti-c-MET/HER 3 antibodies and c-MET bind to immobilized HER 3-BI. After another washing step, digoxigenin-labeled anti-c-MET antibody (specifically binding to a different epitope on c-MET than the bifunctional anti-c-MET/HER 3 antibody) was added and incubated for 1 hour. After another washing step, polyclonal HRP-labeled anti-DIG antibody was added to the plate and incubated for 1 hour. The development reaction was triggered using ABTS substrate solution (see fig. 5 (a)).

Example 5

ELISA for detection of a first antigen of a bispecific antibody by means of an anti-idiotype antibody directed against a second binding specificity of the bispecific antibody

a) Detecting the amount of (total) c-MET in a sample

Biotinylated anti-idiotype antibodies directed against the binding specificity of specifically binding to HER3 (anti-idiotype anti-HER 3 antibody-BI) were bound to streptavidin microtiter plates in the first step. In parallel, the bispecific anti-c-MET/HER 3 antibody was pre-incubated with the sample or standard for 1 hour. In the pre-incubation step, c-MET in the sample is specifically bound by a bispecific anti-c-MET/HER 3 antibody. After washing the streptavidin-coated plates, a pre-incubation mixture of c-MET and bispecific anti-c-MET/HER 3 antibody was added to the plates and incubated for 1 hour at room temperature. After another washing step to remove unbound components, digoxigenin-labeled anti-c-MET antibodies (specifically binding to a different epitope on c-MET than the bispecific anti-c-MET/HER 3 antibody) were added and incubated for 1 hour. After another washing step, polyclonal HRP-labeled anti-DIG antibody was added to the plate and incubated for an additional 1 hour. The development reaction was triggered using ABTS substrate solution (see fig. 5 (B)).

b) Detection of complexes of c-MET with (pre-existing) anti-c-MET/HER 3 antibodies in a sample

Biotinylated anti-idiotype antibodies directed against the binding specificity of specifically binding to HER3 (anti-idiotype anti-HER 3 antibody-BI) were bound to the streptavidin coated microtiter plate in the first step. After washing the plates, the samples and standards were added to the plates for 1 hour at room temperature. The complex of anti-c-MET/HER 3 antibody and c-MET was captured by an immobilized anti-idiotype antibody. After another washing step, digoxigenin-labeled anti-c-MET antibodies (specifically binding to a different epitope on c-MET than the bispecific anti-c-MET/HER 3 antibody) were added and incubated for 1 hour. After another washing step, polyclonal HRP-labeled anti-DIG antibody was added to the plate and incubated for 1 hour. The development reaction was triggered using ABTS substrate solution (see fig. 5 (B)).

Example 6

ELISA for detection of complexes of VEGF with anti-ANG 2/VEGF bispecific antibodies

Antibodies to the anti-ANG 2 antibody of biotinylated monoclonal anti-idiotype, which specifically bind to the ANG2 binding specificity of the anti-ANG 2/VEGF antibody, were coated on streptavidin-coated microtiter plates (MTP). Samples with unknown amounts of VEGF-anti-ANG 2/VEGF antibody complexes were diluted 10-fold and added to wells of MTP coated with anti-idiotype anti-ANG 2 antibody antibodies. The bispecific antibody that specifically binds ANG2 and VEGF is complexed by an immobilized anti-idiotypic antibody directed against the ANG2 binding specific CDRs of the bispecific antibody. Complexes of bispecific antibodies and VEGF were also bound. After incubation for 1 hour at room temperature, the sample/supernatant was removed, after which the plates were washed 3 more times. Thereafter, a monoclonal digoxigenin-labeled anti-VEGF antibody (which binds to an epitope on VEGF different from the bispecific anti-ANG 2/VEGF antibody to be detected) was added to the wells and incubated at room temperature for 1 hour. After the washing step, polyclonal horseradish peroxidase (HRP) -labeled anti-digoxigenin antibody (anti-DIG antibody) was added to the plate and incubated for 1 hour. After removal of the supernatant and washing, ABTS substrate solution was added for the color reaction (see fig. 7).

Example 7

ELISA for detection of complexes of ANG2 with bispecific anti-ANG 2/VEGF antibodies

Biotinylated monoclonal anti-idiotype antibodies that specifically bind to the VEGF binding specificity of the anti-ANG 2/VEGF antibodies were coated on streptavidin coated microtiter plates (MTP). Samples with unknown amounts of ANG2 complex with anti-ANG 2/VEGF antibody were diluted 10-fold and added to the wells of MTP coated with anti-idiotypic anti-VEGF antibody. Bispecific antibodies that specifically bind ANG2 and VEGF were complexed by immobilized anti-idiotypic antibodies directed against the CDRs of the VEGF binding specificity of the bispecific anti-ANG 2/VEGF antibody. Also binding to a complex of bispecific antibody and ANG 2. After 1 hour incubation at room temperature, the sample/supernatant was removed and the plate washed 3 more times. Thereafter, a monoclonal digoxigenin-labeled anti-ANG 2 antibody (which specifically binds to an epitope different from the ANG2 binding specificity of the bispecific anti-ANG 2/VEGF antibody) was added to the wells and incubated at room temperature for 1 hour. After the washing step, polyclonal HRP-labeled anti-digoxigenin antibody was added to the plate and incubated for 1 hour. After removal of the supernatant and washing, ABTS substrate solution was added for the color reaction (see fig. 9).

Example 8

ELISA for detection of complexes of VEGF with bispecific anti-ANG 2/VEGF antibodies

Biotinylated monoclonal antibodies against VEGF were coated on streptavidin coated microtiter plates (MTP). After washing, samples with unknown amounts of VEGF complexed with anti-ANG 2/VEGF antibody were diluted 10-fold and added to the anti-VEGF antibody coated MTP wells. The immobilized antibody against VEGF binds VEGF at a different binding site than the bispecific anti-ANG 2/VEGF antibody. Complexes of VEGF and anti-ANG 2/VEGF antibody bind the immobilized anti-VEGF antibody. After 1 hour incubation at room temperature, the sample/supernatant was removed and the plate washed 3 more times. Thereafter, digoxin-labeled monoclonal anti-idiotypic antibodies (which specifically bind to the ANG2 binding specificity of the anti-ANG 2/VEGF antibody) were added to the wells and incubated for 1 hour at room temperature. After the washing step, polyclonal HRP-labeled anti-digoxigenin antibody was added to the plate and incubated for 1 hour. After removal of the supernatant and washing, ABTS substrate solution was added for the color reaction (see fig. 11). Fig. 12 shows the corresponding calibration curve.

Example 9

ELISA for detection of Total ANG2 by conversion of free ANG2 to antibody-bound ANG2 and incubation with bispecific anti-ANG 2/VEGF antibody

Biotinylated monoclonal anti-idiotype antibodies that specifically bind to the VEGF binding specificity of the anti-ANG 2/VEGF antibodies were bound to streptavidin coated microtiter plates (MTPs). A first aliquot of sample with an unknown amount of ANG2 was incubated with 1.5 μ g/mL of bispecific anti-ANG 2/VEGF antibody for 1 hour to convert free ANG2 to ANG2 that bound anti-ANG 2/VEGF antibody. The second (i.e., non-incubated) aliquot and the antibody-incubated aliquot were diluted 10-fold and added to the wells of the MTP coated with anti-idiotype antibodies that specifically bind to the VEGF-binding specificity of the bispecific antibody. The bispecific antibody is bound by an immobilized anti-idiotype antibody. Likewise, complexed ANG2 was bound by a bispecific antibody. After incubation at room temperature for 1 hour, the supernatant (sample) was removed and the plates were washed 3 more times. Thereafter, a monoclonal digoxigenin-labeled anti-ANG 2 antibody (which specifically binds to an epitope different from the ANG2 binding specificity of the bispecific anti-ANG 2/VEGF antibody) was added to the wells and incubated at room temperature for 1 hour. After the washing step, polyclonal HRP-labeled anti-digoxigenin antibody was added to the plate and incubated for 1 hour. After removal of the supernatant and washing, ABTS substrate solution was added for the color reaction (see fig. 13). From the difference between the results obtained from the first aliquot and the results obtained from the second aliquot, the amount of free ANG2 was calculated. Thus, using this assay, the amount of antibody-bound ANG2 and free ANG2 was determined.

Claims (6)

1. Method for the in vitro determination of the presence and/or amount of binding partners (antigens, targets, analytes) which can be specifically bound by a first binding specificity of a multispecific binder, wherein, prior to the detection of a binding partner, the part of the binding partner present in a sample which binds to the multispecific binder is depleted by incubating the sample with a second binding partner, which can be specifically bound by a second binding specificity of the multispecific binder, or a monospecific binder which specifically binds to a second binding specificity of the multispecific binder.

2. An in vitro method for determining the presence and/or amount of a (first) binding partner of a multispecific binder, which binding partner may be specifically bound by a first binding specificity of the multispecific binder, comprising the steps of:

-incubating the sample comprising the (first) binding partner and the multispecific binder with a second binding partner which can be specifically bound by a second binding specificity of the multispecific binder which is different from the first binding specificity.

3. Method for the immunological determination of the presence and/or amount of a binding partner for a multispecific binder in a sample using an immunoassay, wherein the multispecific binder is depleted from the sample prior to the determination of the binding partner.

4. Method for determining the presence and/or amount of a (first) antigen of a multispecific antibody in a sample, wherein the antigen to be detected can be specifically bound by a first binding specificity of the multispecific antibody, comprising the steps of:

-incubating the sample comprising the multispecific antibody, the (first) antigen to which the multispecific antibody is bound and the free (first) antigen with a second antigen which can be specifically bound by a second binding specificity of the multispecific antibody which is different from the first binding specificity.

5. An in vitro method for determining the presence and/or amount of a (first) antigen of a multispecific antibody in a sample, whereby the antigen to be detected can be specifically bound by a first binding specificity of the multispecific antibody, comprising the steps of:

-incubating the sample comprising the (first) antigen with a complex of a bispecific antibody and a second antigen or a complex of a bispecific antibody and an anti-idiotype antibody which specifically binds to a second binding specificity of the bispecific antibody which is different from the first binding specificity.

6. An in vitro method for determining the presence and/or amount of a (first) antigen of a bispecific antibody in a sample, wherein the antigen to be detected can be specifically bound by a first binding specificity of the bispecific antibody, comprising the steps of:

-incubating the sample comprising the (first) antigen with the bispecific antibody and the second antigen or the bispecific antibody and the anti-idiotype antibody specifically binding to a second binding specificity of the bispecific antibody which is different from the first binding specificity, wherein the second antigen or the anti-idiotype antibody is bound by a solid phase.