ADA response specificity assay

Abstract

This article reports a method for determining the epitope of an antibody that specifically binds to a therapeutic antibody, the method comprising the steps of: a) culturing a sample comprising serum and the antibody that specifically binds to the therapeutic antibody separately with: i) at least one Fab fragment of the therapeutic antibody, and ii) at least one Fab fragment of the therapeutic antibody, wherein the HVRs that form the complementary site have been replaced by germline sequences, and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to at least one Fab fragment in either i) or ii), and b) determining that the epitope of the antibody that specifically binds to the therapeutic antibody is in at least one HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii).

Description

[0001] The present invention belongs to the field of assays for determining and quantifying antidrug responses to therapeutic antibodies (drugs). Using the assays reported herein, it is possible to determine the spatial binding regions of antidrug antibodies (ADA) in a sample, especially a plasma sample. Background Art

[0002] Many biotherapeutic drugs under development today are designed to achieve multiple functions and complex mechanisms of action. This can be accomplished through engineering incorporating multiple functional domains and / or specific amino acid sequences.

[0003] However, all therapeutic proteins have the potential to induce antidrug antibodies (ADA) in recipient animals (preclinical) or human (clinical) organisms. Clinically relevant ADA can affect the efficacy and / or safety of biotherapeutic agents. Immunogenicity assessment strategies evaluate binding and neutralizing ADA and use a risk-based approach to determine the need for additional characterization (e.g., epitope, titer, etc.).

[0004] ADA characterization can influence the interpretation of the risk profile of a given therapeutic agent and provide a deep understanding of risk mitigation and management opportunities.

[0005] This is of particular relevance for the treatment of chronic diseases such as AMD and DME, as the treatment is intended to be applied for months or even years. If an immune response occurs, it is important to understand which region or domain of the molecule the response is directed against. In this way, information is collected regarding whether, for example, engineered parts of the molecule that can be reengineered (e.g., removing potential T cell epitopes) are immunogenic or whether the immunogenic response is directed against the antigen-binding portion (HVR) of the biotherapeutic agent, i.e., the fundamental intrinsic property of the molecule. Appropriate risk mitigation strategies can then be developed.

[0006] Established methods for immunogenicity response or anti-drug antibody (ADA) characterization are the classical domain competition assay and domain detection assay (DCA, DDA; see, e.g., Hoofring, S.A. et al., Bioanal. 5 (2013) 1041-1055; Gorovits, B. et al., J. Immunol. Meth. 408 (2014) 1-12; Stubenrauch, K. et al., J. Pharm. Biomed. Anal. 114 (2015) 296-304). Here, the drug candidate is cleaved into individual domains or specific sub-fragments are recombinantly expressed to allow for the development of domain-specific ADA assays. These widely used methods are of particular value for large protein molecules (such as IgG or IgG-like forms and fusion proteins). For smaller biotherapeutics, such as single-chain Fv fragments (scFv), Fab fragments or bispecific Fabs, the above methods have limitations because these molecules are generally not easily cleaved into sub-domains or these sub-domains have a stability liability. Therefore, alternative, more generally applicable methods are needed.

[0007] The disadvantages of competition / inhibition-based ADA detection methods, especially considering mixed affinity and mixed epitope ADA responses, have been reported by Hoofring et al. (Bioanal. 5 (2013) 1041-1055) and Stubenrauch et al. (J. Pharm. Biomed. Anal. 10 (2015) 296-304).

[0008] WO 2018 / 178307 reports an improved immunogenicity assay that is capable of differentiating between neutralizing and non-neutralizing anti-drug antibodies. More specifically, it reports an assay / method that is carried out in the presence of a sufficient amount, and preferably an excess, of an inactive variant of an immunoglobulin single variable domain (ISV)-based drug, such that most, and preferably all, non-neutralizing ADAs, especially those that bind to the ISV framework sequence present in the ISV-based drug, bind to the inactive variant in the assay reaction mixture rather than to the ISV-based drug, and thus essentially only the neutralizing ADAs against the ISV-based drug are detected or measured (even when the sample also contains non-neutralizing ADAs against the ISV-based drug). In the inactive variant, only individual residues have been changed, but there is no complete CDR sequence.

[0009] Stubenrauch et al. reported on the epitope characterization of ADA responses against targeted immunocytokines (J. Pharmaceut. Biochem. Anal. 114 (2015) 296-304).

[0010] Stubenrauch et al. reported on the evaluation of a biosensor immunoassay for the simultaneous characterization of the isotype and binding regions of human anti-Tocilizumab antibodies using replacement standards (Anal. Biochem. 390 (2009) 189-196).

[0011] Zhu et al. reported on the construction of a large initial human phage display Fab library by one-step cloning (Methods. Mol. Biol. 1045 (2009) 129-142). SUMMARY OF THE INVENTION

[0012] Many biologic agents that provide tailored properties and functions for a wide range of indications are currently under development. During the preclinical development of therapeutic antibodies, it is particularly important to evaluate the immune response against the compound and its potential impact on pharmacokinetic characteristics, target binding properties, safety, and efficacy.

[0013] The present invention relates to an analytical method that allows the immunogenicity of different domains of a therapeutic antibody (such as a bispecific Fab) in experimental animals such as cynomolgus monkeys to be classified. In the method according to the invention, a molecular engineering method is combined with a domain detection assay. The present invention is at least partially based on the discovery that by replacing a single antigen-binding region, i.e., the complementarity-determining region (CDR), in the Fab with a human germline sequence, it is possible to distinguish anti-drug antibodies (ADA) against either of the two target-binding CDRs and the constant part of the bispecific Fab. The method according to the invention has advantageous properties especially for smaller biotherapeutic agents, such as Fab or scFv fragments. The method allows for a reliable characterization of the ADA response using readily generated tool variant antibodies and a better understanding of preclinical immunogenicity.

[0014] Without being bound by this theory, considering the known methods available, the method according to the invention provides improvements, especially in terms of simpler feasibility and higher reliability regarding false-negative results. The second advantage is at least partially based on the direct capture of variants of the therapeutic antibody in which at least one complete hypervariable region (HVR) has been replaced by a complete non-binding HVR, thereby preventing the binding of the corresponding anti-HVR anti-drug antibody, i.e., at the wrong position. Methods using variants in which only some residues or only individual residues in the HVR are replaced to generate non-target-binding therapeutic antibodies and thus in which, although target binding is eliminated, part of the CDR remains, are more prone to false-negative results. This is because neutralizing antibodies that specifically bind to the remaining residues of the therapeutic antibody CDR may still interfere.

[0015] One aspect of the present invention is a bioanalytical method for characterizing the immunogenic response after application of a therapeutic antibody to an experimental animal such as a cynomolgus monkey.

[0016] The present invention is at least in part based on the discovery that by combining molecular engineering methods with an effective and easily set up domain detection assay, the spatial location of the antidrug response to a therapeutic antibody can be determined and quantified in a simple and reliable manner. It has been found that by replacing individual antigen-binding regions with germline sequences, the assay is able to distinguish between antidrug antibodies (ADA) against the target-binding paratope and the constant part of the therapeutic antibody. It has further been found that in combination with timely and comprehensive reagent development, the assay according to the present invention is particularly advantageous for smaller therapeutic antibodies such as scFvs and Fabs. Using the assay according to the present invention, a reliable characterization of the ADA response and a better understanding of preclinical immunogenicity can be achieved. The method according to the present invention can be applied to any therapeutic antibody or antibody format to obtain ADA epitope information, provided that the paratope is formed by a VH / VL pair, thereby reducing the effort required to generate reference antibodies with the removed paratope compared to methods known in the art.

[0017] More specifically, a novel bioanalytical method is reported herein for characterizing the immunogenic response to a therapeutic antibody (especially a bispecific antibody Fab fragment) by reverting the corresponding antigen-binding site / paratope to germline sequences, thereby eliminating the binding specificity for, e.g., antigen 1, antigen 2, or both antigens. The domain detection assay according to the present invention is based on these variants of the therapeutic antibody. By using these variants, it is possible to distinguish between antidrug antibodies (ADA) against the first specificity and the second specificity, both specificities, and the constant part of the molecule. In addition, an improvement lies in reducing the effort required to generate paratope variants of the therapeutic antibody.

[0018] One aspect of the present invention is a method for determining the epitope of an antibody that specifically binds to a therapeutic antibody (antidrug antibody), the method comprising the steps of:

[0019] a) culturing a sample comprising serum and an antibody that specifically binds to a therapeutic antibody separately with

[0020] i) at least one Fab fragment of the therapeutic antibody, and

[0021] ii) at least one Fab fragment of the therapeutic antibody, wherein at least one (complete)

[0022] HVR has been replaced by a (complete) non-binding HVR,

[0023] Detecting the binding or non - binding of an antibody that specifically binds to a therapeutic antibody to at least one Fab fragment in any one of i) to ii)

[0024] And

[0025] b) Determining the epitope of an antibody that specifically binds to a therapeutic antibody

[0026] - In at least one (complete) HVR that has been replaced in ii), provided that

[0027] Binding is detected in i)

[0028] And

[0029] Non - binding is detected in ii)

[0030] - In the HVR framework junction of at least one (complete) HVR that has been replaced in ii), or in the framework region, or in the constant domain, provided that

[0031] Binding is detected in i) and ii)

[0032] In one embodiment of the method according to the invention, step a) further comprises

[0033] iii) At least one Fab fragment of a therapeutic antibody, wherein all HVRs have been (completely) replaced by non - binding HVRs

[0034] And detecting the binding or non - binding of an antibody that specifically binds to a therapeutic antibody to at least one Fab fragment in any one of i) to iii)

[0035] And step b) is

[0036] Determining the epitope of an antibody that specifically binds to a therapeutic antibody

[0037] - In at least one (complete) HVR that has been replaced in ii), provided that

[0038] Binding is detected in i)

[0039] And

[0040] Non - binding is detected in ii) and iii)

[0041] - In the HVR framework region junction of at least one (complete) HVR that has been replaced in ii), provided that

[0042] Binding is detected in i) and ii)

[0043] And

[0044] Non-binding was detected in (iii).

[0045] - in the framework region and / or constant domain, provided that

[0046] Binding was detected in (i) to (iii).

[0047] In one embodiment of the method according to the invention, step a) further comprises

[0048] iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one (complete) HVR different from the HVR of (ii) has been replaced by a (complete) non-binding HVR,

[0049] and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to at least one Fab fragment in any one of (i), (ii) and (iv) and (iii) if present,

[0050] and step b) is

[0051] Determining the epitope of the antibody specifically binding to the therapeutic antibody

[0052] - in at least one (complete) HVR that has been replaced in (ii) or (iv), provided that

[0053] Binding was detected in (i),

[0054] and

[0055] Non-binding was detected in one of (ii) or (iv) and (iii) if present,

[0056] - in the framework region junction of at least one HVR that has been replaced in (ii) or (iv), provided that

[0057] Binding was detected in (i) and in one of (ii) or (iv),

[0058] and

[0059] Non-binding was detected in (iii) if present,

[0060] - optionally in the framework region and / or constant domain, provided that

[0061] Binding was detected in (i), (ii), (iv) and (iii) if present.

[0062] In one embodiment of the method according to the invention, step a) further comprises

[0063] v) at least one Fab fragment of a non-binding antibody from the germline, which is different from the Fab fragment of the framework region of the therapeutic antibody,

[0064] and step b) is

[0065] determining the epitope of the antibody that specifically binds to the therapeutic antibody

[0066] - in at least one (complete) HVR that has been replaced in ii) or iv) if present, provided that

[0067] binding is detected in i)

[0068] and

[0069] non - binding is detected in ii) and v), iii) if present, and iv) if present

[0070] - in the framework region junction of at least one (complete) HVR that has been replaced in ii) or iv) if present, provided that

[0071] binding is detected in i) and in one of ii) or iv) if present

[0072] and

[0073] non - binding is detected in v) and iii) if present

[0074] - in the framework region and / or constant domain, provided that

[0075] binding is detected in i) and ii) and iii) if present and iv) if present

[0076] and

[0077] non - binding is detected in v)

[0078] In one embodiment of the method according to the invention, the (complete) non - binding HVR is a (complete) germline HVR obtained from the same germline as the framework region of the therapeutic antibody.

[0079] In one embodiment of the method according to the invention, the therapeutic antibody is a monospecific Fab or a bispecific Fab.

[0080] In one embodiment of the method according to the invention

[0081] - the therapeutic antibody is a bispecific Fab, wherein the first complementary site is formed by HVR H - 1, H - 3 and L - 2, and the second complementary site is formed by HVR H - 2, L - 1 and L - 3

[0082] - in step a), step ii) is

[0083] A therapeutic antibody in which the (complete) HVR H-1, H-3 and L-2 have been replaced by (complete) non-binding HVRs

[0084] - Step a) further comprises

[0085] vi) A therapeutic antibody in which the (complete) HVR H-2, L-1 and L-3 have been replaced by (complete) non-binding HVRs

[0086] - Step b) is

[0087] Determining the epitope of an antibody that specifically binds to the therapeutic antibody

[0088] - In the first complementarity-determining region of the bispecific Fab, provided that

[0089] Binding is detected in i) and vi),

[0090] And

[0091] Non-binding is detected in ii), iii) if present and v) if present

[0092] - In the second complementarity-determining region of the bispecific Fab, provided that

[0093] Binding is detected in i) and ii),

[0094] And

[0095] Non-binding is detected in vi), iii) if present and v) if present

[0096] - In the HVR framework region junction of the first complementarity-determining region, provided that

[0097] Binding is detected in i) and vi)

[0098] And

[0099] Non-binding is detected in ii), iii) if present and v) if present

[0100] - In the HVR framework region junction of the second complementarity-determining region, provided that

[0101] Binding is detected in i) and ii)

[0102] And

[0103] Non-binding is detected in vi), iii) if present and v) if present

[0104] - In the framework region and / or constant domain, provided that

[0105] Binding is detected in i), ii), iv), and if present, iii).

[0106] and

[0107] Non - binding is detected in v) if present.

[0108] In all aspects and embodiments of the present invention, prior to step a), i.e., prior to incubation with at least a Fab of a therapeutic antibody or a modified variant of its complementarity determining region, a soluble variant of the therapeutic antibody or its fragment is not added to the sample or an aliquot of the sample.

[0109] In all aspects and embodiments, the therapeutic antibody comprises a VH / VL pair that forms a therapeutic target - binding complementarity determining region.

[0110] In all aspects and embodiments, the complementarity determining region of the therapeutic antibody that binds to the therapeutic target is not a single - domain antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 Gene usage frequencies observed for VH domains of human (n = 9340) Ig sequences published in the IMGT / LIGM - DB database. According to the IMGT / High - V - QUEST statistical analysis report, each mapped IGHV gene usage is calculated as the percentage of the total unique population of productive and in - frame sequences (adapted from B. Shi et al., Ther. Biol. Med. Model. 11(2014)30, Supplementary Figure 1 A).

[0112] Figure 2 Germlining scheme for the heavy - chain variable domain. The corresponding segments of the heavy - variable domain denoted by "F", "C", "-", and "J" are used as query sequences in the alignment with the human IMGT germline repertoire to identify the most identical germline sequences. "-" represents the gaps used in the alignment, and the corresponding sequences of the identified germline are used to replace the corresponding HVR sequences of the antibody.

[0113] Figure 3 Germlining scheme for the light - chain variable domain. The corresponding segments of the light - variable domain denoted by "F", "C", "-", and "J" are used as query sequences in the alignment with the human IMGT germline repertoire to identify the most identical germline sequences. "-" represents the gaps used in the alignment, and the corresponding sequences of the identified germline are used to replace the corresponding HVR sequences of the antibody.

[0114] Figure 4 Schematic representation of bispecific Fab biotherapeutics, engineered Fab variants, and control molecules generated for the detection of domain - specific anti - drug antibodies (ADA). Constructs 2 to 4 are modified versions of the original Duta original drug molecule.

[0115] Figure 5 : Principle of domain detection assay: Different domains ( Figure 4 as described in) are directly coated onto NuncMaxiSorp TM plates and used to capture domain-specific ADA. The bound ADA is detected by Dig-labeled anti-cynomolgus IgG. A signal is generated using an anti-Dig antibody conjugated to horseradish peroxidase (HRP), resulting in substrate / ABTS color conversion.

[0116] Figure 6 : Schematic diagram of an exemplary domain detection assay according to the invention as used in Example 5. Five different Fab variants representing different domains are immobilized on the surface. ADAs with different specificities can attach to different domains. Further identification of the ADA is accomplished by anti-cynomolgus antibodies.

[0117] Figure 7 : Expected positive pattern of an exemplary assay setup of the method according to the invention as used in Example 5.

[0118] Figure 8 : ADA response "type 8" determined using the assay according to the invention.

[0119] Figure 9 : ADA response "type 9" determined using the assay according to the invention.

[0120] Figure 10 : ADA response "type 10" determined using the assay according to the invention.

[0121] Figure 11 : ADA response "type 11" determined using the assay according to the invention. Detailed Description

[0122] Definitions

[0123] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art. Generally, terms and techniques related to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0124] Unless otherwise defined herein, the term "comprising" shall include the term "consisting of".

[0125] As used herein in connection with a particular value (e.g., temperature, concentration, time, etc.), the term "about" shall mean a variation of + / - 1% of the particular value to which the term "about" refers.

[0126] The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0127] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments.

[0128] 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. In certain aspects, the antibody is of the IgG1 isotype. In certain aspects, the antibody is of the IgG1 isotype having P329G, L234A, and L235A mutations to reduce Fc region effector function. In other aspects, the antibody is of the IgG2 isotype. In certain aspects, the antibody is of the IgG4 isotype having an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated alpha, delta, epsilon, gamma, and mu, respectively. The light chain of an antibody can be assigned to one of two types based on the amino acid sequence of its constant domain, and these two types are called kappa (κ) and lambda (λ).

[0129] "Framework" or "FR" refers to the variable domain residues other than the complementarity-determining regions (CDRs). The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0130] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that contains an Fc region as defined herein.

[0131] The terms "variable region" or "variable domain" refer to the domains of an antibody heavy or light chain that participate in binding of the antibody to antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind a particular antigen can be isolated using, respectively, the VH or VL domains from an antibody that binds that antigen to screen a library for complementary VL or VH domains.

[0132] As used herein, the terms "hypervariable region" or "HVR" refer to each of the regions within the variable domain of an antibody that are highly variable in sequence and that define antigen-binding specificity, such as "complementarity determining regions" ("CDRs"). These regions form the paratope or binding site.

[0133] Typically, an antibody includes six antigen-binding specificity-determining regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary antigen-binding specificity-determining regions herein include:

[0134] (a) hypervariable loops (HVRs) at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987));

[0135] (b) complementarity determining regions CDRs at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0136] (a + b) is present at the HVRs (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987) + Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 26 - 35 (H1), 50 - 65 (H2) and 95 - 102 (H3);

[0137] and

[0138] (c) is present at the antigen - contact points (MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996)) at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2) and 93 - 101 (H3).

[0139] Unless otherwise specified, HVRs are determined according to the method described by Kabat et al. (ibid.). Those skilled in the art will understand that antigen - binding specific - determining region names can also be determined according to the methods described by Chothia (ibid.), McCallum (ibid.) or any other scientifically - accepted naming system.

[0140] As used interchangeably herein, "complementary site" or "antigen - binding site" refers to the part of an antibody that recognizes and binds an antigen. The complementary site is formed by several individual amino acid residues from the variable domains of the antibody heavy and light chains, which are spatially adjacent in the tertiary structure of the Fv region. The complementary site of an endogenous human antibody is formed by 6 CDR (complementary - determining region) loops, which recognize the complementary epitope on its antigen surface. In the case of antibodies specific for small antigens (e.g., small molecules), only a few amino acids from some of the CDRs, but not all, are involved in antigen recognition. The CDR amino acid residues involved in direct antigen contact are called specific - determining regions (SDRs). By analyzing the 3 - dimensional structure of the antigen - antibody complex amino acid residues, the directly - contacting antigens can be identified based on their distances. The FR (framework) residues intervening between the CDRs can also participate in antigen recognition, but to a lesser extent (the surface of such regions may account for up to 15% of the antigen - antibody contact surface) (see, e.g., Altshuler, E.P., Chemie 50 (2010) 203 - 258; Bujotzek, A. et al., mAbs 8 (2016) 288 - 305).

[0141] In one embodiment, the therapeutic antibody in the method according to the present invention is a bispecific Fab. In one embodiment, the bispecific Fab comprises two "non-overlapping" complementarity-determining regions (CDRs) in a homologous VH / VL pair. "Non-overlapping" means that the amino acids included in one of the two CDRs are not included in the other CDR.

[0142] "Bispecific Fab" is a bispecific antibody as disclosed in WO 2012 / 163520. In the bispecific Fab, a single VH domain and VL domain pair specifically binds to two different epitopes, wherein one CDR comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3, and the other CDR comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2. The bispecific Fab comprises two non-overlapping CDRs within a homologous VH / VL pair and can bind to two different epitopes simultaneously. The bispecific Fab and methods for its production by screening libraries comprising monospecific Fab fragments are disclosed in WO 2012 / 163520.

[0143] In one embodiment, the therapeutic antibody in the method according to the present invention is a bispecific Fab. In one embodiment, the bispecific Fab specifically binds to a first antigen (antigen 1) and a second antigen (antigen 2). In one embodiment, the bispecific Fab specifically binds to the first antigen (antigen 1) through its first CDR and to the second antigen (antigen 2) through its second CDR. In one embodiment, the bispecific Fab comprises a first CDR and a second CDR within a homologous pair of variable light chain domain (VL domain) and variable heavy chain domain (VH domain), wherein the first CDR comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, and wherein the second CDR comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the antibody.

[0144] In one embodiment, the bispecific Fab binds to a first antigen and a second antigen and comprises a first CDR and a second CDR within a homologous pair of VL domain and VH domain, wherein the variable light chain domain and the variable light chain domain pair can bind to the first antigen and the second antigen simultaneously.

[0145] In one embodiment, the bispecific Fab binds to a first antigen and a second antigen and comprises a first CDR and a second CDR within a homologous pair of VL domain and VH domain, wherein the amino acids included in the first CDR are not included in the second CDR.

[0146] As used herein, the term "sample" refers to any biological matrix from which an ADA response can be determined. Exemplary but non-limiting samples are serum, plasma, aqueous humor, vitreous humor, retinal tissue lysate, and tumor tissue. In a preferred embodiment, the sample is plasma.

[0147] The term "epitope" refers to a site on a protein or non-protein antigen to which an antigen binds. Epitopes can be formed either by a continuous stretch of amino acids (linear epitopes) or by amino acids that are not contiguous (conformational epitopes), such as due to antigen folding, i.e., spatial proximity due to the tertiary folding of a protein antigen. Linear epitopes are generally still bound by antibodies after the protein antigen has been exposed to a denaturing agent, while conformational epitopes are generally disrupted after treatment with a denaturing agent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8 - 10 amino acids in a unique three-dimensional conformation.

[0148] As used herein, the term "anti-drug antibody" refers to an antibody produced by the innate immune system of the recipient of a therapeutic antibody against the therapeutic antibody after administration of the therapeutic antibody.

[0149] As used herein, the term "immunogenicity" refers to the likelihood of a therapeutic antibody to induce an immune response in a human or animal. During drug development, immunogenicity is primarily evaluated by measuring binding and neutralizing anti-drug antibodies.

[0150] Unbound (germline) CDR refers to a CDR obtained from a human germline amino acid sequence to which the framework region of a therapeutic antibody has the highest homology and which does not specifically bind to the target of the therapeutic antibody either alone or in combination with other CDRs of the therapeutic antibody.

[0151] A method according to the present invention

[0152] 1. A method for determining the (spatial location of) epitope of an antibody that specifically binds to a therapeutic antibody (anti-drug antibody), the method comprising the following steps:

[0153] a) Culturing a sample comprising serum and an antibody that specifically binds to a therapeutic antibody (the sample being obtained from an (experimental) animal that has been administered the therapeutic antibody) separately with

[0154] i) at least one Fab fragment of the therapeutic antibody, and

[0155] ii) at least one Fab fragment of the therapeutic antibody, wherein at least one CDR has been replaced by an unbound (germline) CDR, and

[0156] Detecting the binding or non - binding of an antibody that specifically binds to a therapeutic antibody to at least one Fab fragment in any one of i) to ii)

[0157] And

[0158] b) Determining the epitope (spatial location) of an antibody that specifically binds to a therapeutic antibody

[0159] - In at least one CDR that has been replaced in ii), provided that

[0160] Binding is detected in i)

[0161] And

[0162] Non - binding is detected in ii)

[0163] - In the CDR framework junction, or in the framework region, or in the constant domain of at least one CDR that has been replaced in ii), provided that

[0164] Binding is detected in i) and ii)

[0165] 2. The method according to item 1, wherein

[0166] Step a) further comprises

[0167] iii) At least one Fab fragment of a therapeutic antibody, wherein all CDRs have been replaced by non - binding (germline) CDRs

[0168] And step b) is

[0169] Determining the epitope (spatial location) of an antibody that specifically binds to a therapeutic antibody

[0170] - In at least one CDR that has been replaced in ii), provided that

[0171] Binding is detected in i)

[0172] And

[0173] Non - binding is detected in ii) and iii)

[0174] - In the CDR framework region junction of at least one CDR that has been replaced in ii), provided that

[0175] Binding is detected in i) and ii)

[0176] And

[0177] Non - binding is detected in iii)

[0178] - In the framework region and / or constant domain, provided that

[0179] Binding was detected in i) to iii).

[0180] 3. The method according to any one of items 1 to 2, wherein

[0181] Step a) further comprises

[0182] iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one CDR different from the CDR of ii) has been replaced by a non-binding (germline) CDR,

[0183] and step b) is

[0184] Determining the spatial location of the epitope of the antibody that specifically binds to the therapeutic antibody

[0185] - in at least one CDR that has been replaced in ii) or iv), provided that

[0186] Binding was detected in i)

[0187] and

[0188] Non-binding was detected in one of ii) or iv) and optionally in iii),

[0189] - in the framework region junction of the CDR of at least one CDR that has been replaced in ii) or iv), provided that

[0190] Binding was detected in i) and in one of ii) or iv)

[0191] and

[0192] Non-binding was detected in iii),

[0193] - optionally in the framework region and / or constant domain, provided that

[0194] Binding was detected in i) to iv).

[0195] 4. The method according to any one of items 1 to 3, wherein

[0196] Step a) further comprises

[0197] v) at least one Fab fragment of a non-binding (germline) antibody from the germline, which is different from the Fab fragment of the framework region of the therapeutic antibody,

[0198] and step b) is

[0199] Determining the spatial location of the epitope of the antibody that specifically binds to the therapeutic antibody

[0200] - in at least one CDR that has been replaced in ii) or optionally in iv), provided that

[0201] binding is detected in i)

[0202] and

[0203] non - binding is detected in ii) and v), optionally in iii), optionally in iv),

[0204] - in the framework region junction of a CDR of at least one CDR that has been replaced in ii) or optionally in iv), provided that

[0205] binding is detected in i) and ii), or optionally in iv)

[0206] and

[0207] non - binding is detected in v) and optionally in iii),

[0208] - optionally in the framework region and / or constant domain, provided that

[0209] binding is detected in i) and ii), and optionally in iii), and optionally in iv)

[0210] and

[0211] non - binding is detected in v).

[0212] 5. The method according to any one of items 1 to 4, wherein each HVR is a complete HVR determined according to the combined Kabat - Chothia definition.

[0213] 6. The method according to any one of items 1 to 5, wherein the HVR is determined according to the combined Kabat - Chothia definition, and HVR - L1 contains residues 24 - 34, HVR - L2 contains residues 50 - 59, HVR - L3 contains residues 89 - 97, HVR - H1 contains residues 26 - 35, HVR - H2 contains residues 50 - 65, and HVR - H3 contains residues 95 - 102.

[0214] Exemplary embodiments and experimental results

[0215] It is expressly stated that the following is presented only as an example of the method according to the present invention. It should not be construed as limiting. The scope of the present invention is set forth in the claims.

[0216] The method according to the present invention will be illustrated by using bispecific Fabs as an example of therapeutic antibodies, which have been designed and optimized to simultaneously recognize two separate targets. The main intended applications of these molecules are ophthalmic indications such as age-related macular degeneration (AMD) or diabetic macular edema (DME), where the simultaneous inhibition of two soluble factors (e.g., angiogenesis-promoting factors and inflammatory mediators) is considered to have beneficial / synergistic / superior effects compared to single standard monotherapies alone. Bispecific Fabs show a major advantage especially for ophthalmic applications because they combine two target sites within a relatively small molecular size (about 50 kDa), resulting in a favorable drug:target ratio. In addition, they are highly concentrated and can be fully engineered for the required specificity, affinity, stability, and PK properties.

[0217] The method according to the present invention was applied to analyze the immunogenic responses in a preclinical non-human primate study involving 8 cynomolgus monkeys, which were administered therapeutic bispecific Fab fragments (see the Examples section for details). An ADA response was observed after intravitreal application of the molecule. Using the method according to the present invention, the immunogenic responses could be deeply characterized to identify the major immunogenic parts of the therapeutic antibody.

[0218] Design of Sequence Variants

[0219] Typically, the non-naturally occurring CDR and framework region sequences that are expected to be involved in binding to antigen 1 or antigen 2 are analyzed separately and replaced with germline sequences of the human species that provide the closest sequence match. It must be noted that for this replacement, not every non-contiguous part of the complementarity-determining region must be replaced with a fragment from the same, i.e., single germline gene. More typically, the replacement of different non-contiguous fragments is carried out with sequences derived from different germline sequences. Optionally, the central and usually buried residues in the variable domain can be retained to maintain the stable core packing of the VH-VL pair when the binding surface is exchanged and to optimally preserve the shape of the Fab fragment.

[0220] Typically, to achieve the back-to-germline modification (germlining) of the drug antibody under discussion, the variable domain is divided into different parts because the framework region and the variable region must be germlined.

[0221] For the germlining of frameworks, the N-terminal half of FR1 or the C-terminal half of FR3 of the drug antibody to be germlined is aligned with the human germline repertoire using standard tools such as, for example, Blast. FR1 and FR3 and the corresponding other parts of FR2 are obtained by germlining the corresponding CDRs. The sequence with the highest number of identical residues at the same positions is selected as the replacement sequence. If two or more similar identical germline sequences are identified, the sequence with a higher usage frequency in the natural human antibody repertoire is selected. As a basis for the usage frequency, the list provided by B. Shi et al. (Ther. Biol. Med. Model. 11 (2014) 30, supplement Figure 1 A; as Figure 1 reproduced).

[0222] For the germlining of HVRs, sequences corresponding to the sequences of the halves of the frameworks adjacent (front) to the N-terminus and adjacent (rear) to the C-terminus of the drug antibody, together with the gaps of the HVRs, are aligned with the human germline repertoire using standard tools such as, for example, Blast. The HVRs are determined according to the combined Kabat-Chothia definition, i.e., HVR-L1 is residues 24-34, HVR-L2 is residues 50-59, HVR-L3 is residues 89-97, HVR-H1 is residues 26-35, HVR-H2 is residues 50-65, and HVR-H3 is residues 95-102. The sequence with the highest number of identical residues outside the HVRs at the same positions and with the same HVR length is selected. If two or more germline sequences are identified, the sequence with a higher usage frequency in the natural human antibody repertoire is selected. As a basis for the usage frequency, the list provided by B. Shi et al. (Ther. Biol. Med. Model. 11 (2014) 30, supplement Figure 1 A; as Figure 1 reproduced). Although the sequences of the corresponding HVRs are not used for alignment to identify the corresponding most identical germline framework sequences, the corresponding HVR sequences in the identified germline sequences are used to replace the HVRs of the antibody to be germlined.

[0223] The heavy chain HVR-H3 is the result of VDJ rearrangement that occurs during antibody maturation from 1) a portion of V element, 2) D element, and 3) a portion of J element. More specifically, the heavy chain HVR-H3 is generated by combining residues from variable (VH) gene segments, diversity (D) gene segments, and joining (J) gene segments. Although this diversity has been generated using different structural units, additional diversity can be generated during the joining process by adding short palindromic nucleotides to the ends of coding sequences, or by deleting a variable number of nucleotides from the ends of coding segments, or by inserting a variable number of non-templated nucleotides at the VH-D and D-J junctions by terminal deoxynucleotidyl transferase or by a hypermutation mechanism that introduces point mutations to alter amino acid codons (see Rosner, K. et al., Immunology 103 (2001) 179-187).

[0224] In Figure 2 A and Figure 3 A general scheme of germlining is outlined.

[0225] Thus, an easy-to-use method of complementarity germlining is provided herein as an advantage over current methods.

[0226] The exemplary bispecific Fab used in this example includes a single VH domain and VL domain pair that specifically binds to two different epitopes. One complementarity-determining region (CDR) includes amino acid residues from HVR-H2, HVR-L1, and HVR-L3, while the other CDR includes amino residues from HVR-H1, HVR-H3, and HVR-L2.

[0227] Thus, in this exemplary case, for the first CDR as Figure 2 C and Figure 3 E and for the second CDR as Figure 2 E and Figure 3 C, the HVRs and FRs shown are only partially germlined.

[0228] Thus, to create a Germ1 control construct in which only the antigen 1-binding CDR is retained and the antigen 2-binding CDR is removed (i.e., germlined), the following modifications were made:

[0229] - The N-terminal portion of HC-FR1 was replaced with the germline sequence VH3-21 (IMGT),

[0230] - The C-terminal portion of HC-FR1, HVR1, and the N-terminal portion of HC-FR2 were replaced with the germline sequence VH3-23 (IMGT),

[0231] -HC-FR3 is replaced by the germline sequence VH3-23 (IMGT),

[0232] -Depending on the practice used, such as the replacement of potential immunogenic residue 96D by non-immunogenic A in alanine scanning,

[0233] -HC-HVR3 residues 98-102 and FR4 are replaced by IGHJ4 (IMGT),

[0234] -LC-HVR2 is replaced by IGKV1-33 (IMGT),

[0235] Thus, to create a Germ2 control construct in which only the antigen 2-binding complementarity-determining regions are retained and the antigen 1-binding complementarity-determining regions are removed (i.e., germlined), the following modifications were made:

[0236] -The C-terminal part of HC-FR2, HVR2, and the N-terminal part of HC-FR3 are replaced by the germline sequence VH3-23 (IMGT),

[0237] -The N-terminal part of LC-FR1 is replaced by the germline sequence IGKV1-6 (IMGT),

[0238] -The C-terminal part of LC-FR1, HVR1, and the N-terminal part of LC-FR2 are replaced by the germline sequence VGKV1-27 (IMGT),

[0239] -LC-FR3 is replaced by the germline sequence IGKV1d-43 (IMGT),

[0240] -LC-HVR3 residues 97 and FR4 are replaced by IGKJ2 (IMGT).

[0241] To create a GermGerm control construct, all of the above-described replacements for the Germ1 control and Germ2 control constructs were combined, thereby eliminating both the antigen 1-binding complementarity-determining regions and the antigen 2-binding complementarity-determining regions from the bispecific Fab.

[0242] In Figure 4 are schematically shown all of the constructs generated in this study and applied to the characterization of the immunogenic response.

[0243] The domain detection assay (DDA) according to the present invention employs the same principle as the conventional bridging ADA assay. The difference of the present invention lies in using a defined drug domain as the capture reagent. Thus, the domain-specific ADA fractions can be selectively determined.

[0244] In a preferred embodiment of the method according to the present invention, the specific domain is directly immobilized on a solid surface (see Figure 5)。Therefore, an exemplary material is Nunc MaxiSorp TM MTP. In a preferred embodiment, digoxin-labeled anti-cynomolgus IgG is used to detect domain-specific ADA responses.

[0245] In addition to the ADA domain detection assay according to the present invention, several other assay settings are also applied to sample analysis and evaluation of data reliability.

[0246] In the domain competition assay (DCA) setting, Figure 4 and Figure 5 variants of biotin and digoxin for different domains as described therein are used to form drug / drug variant:ADA complexes, which can be immobilized on streptavidin-coated MTPs and detected by anti-Dig-HRP antibodies. Unlabeled versions of the corresponding constructs are used as specific competition reagents. Compared to the domain detection assay according to the present invention, this assay produces comparable results; however, it is much more laborious in terms of reagent production and execution. Due to its indirect working mode (signal reduction corresponding to the detection of domain-specific ADA), it also has lower sensitivity. In terms of workload and result quality, DCA has been shown to be inferior to the DDA according to the present invention for ADA characterization.

[0247] A further assay applied to ADA characterization is the ADA bridging assay. In this assay, in the presence of anti-drug antibodies in the sample, biotin-labeled and digoxin-labeled versions of the drug candidate corresponding to different control constructs are used to generate a signal. To confirm the specificity of the ADA, a large excess of the unlabeled drug corresponding to the control construct is incorporated. A significant signal quenching ability of the unlabeled molecule is considered positive for the presence of specific ADA. In this example, this principle acts on the original bispecific Fab as well as two partially germline variants (one with binding specific activity and the other with non-functional binding), but not on the GermGerm variant and other control constructs, indicating no ADA specific to the constant part. This finding is regarded as a flaw / weakness in the design of the ADA bridging assay, which may not fully address alternative and lower affinity ADAs against the constant antibody part (see Figure 4 ).

[0248] The third assay applied to the ADA characterization of cynomolgus monkey study samples is the immune complex assay (see, e.g., Wessels, U. et al., Bioanal. 10 (2018) 803 - 814), which uses a combination of anti - human Fab antibody as the capture and anti - cynomolgus monkey IgG antibody as the detection reagent to detect ADA:drug complexes in biological samples. Since this assay is not very tolerant to the drug, even with trace amounts of residual drug (Fab - naive) still present in the sample, it will produce a positive signal, making it unable to distinguish between different constructs.

[0249] The domain - detection assay according to the invention, i.e., the combination of the assay and the corresponding molecular engineering method (generation of germline - like variants of the original drug compound), lacks all the limitations outlined above, since the different domains are directly immobilized on a solid surface, such as, for example, MTP, and are recognized by the corresponding ADA. This makes the assay very specific and sensitive. The assay also shows a high degree of drug tolerance, since the concentration of the capture antibody is at least 200 - fold higher compared to the level of residual drug present in the sample on day 35 after dosing. This was also confirmed by titration experiments, showing that the specific ADA recognition signal remained highly positive when the drug was diluted out of the sample.

[0250] In terms of biological relevance and further development, the finding that the immune response is distinguished by the antigen - binding and constant parts with a lower prevalence for (the vast majority of) bispecific Fab molecules supports the hypothesis of a complex polyclonal anti - drug antibody response against fully human drug candidates in the context of non - human primates. The finding of ADA against the constant part of the bispecific Fab molecule also cross - reacts with unbound human Fab, and ranibizumab is particularly noteworthy with respect to the intended human application, since ranibizumab is a drug for intravitreal application and is well - tolerated in humans. Thus, the results obtained do not provide a hint of the intrinsic immunogenic properties of the molecules in the bispecific Fab modality.

[0251] Summary and Outlook

[0252] A novel and creative concept is reported herein to facilitate the characterization of the immune response against biotherapeutic drug candidates in a pre - clinical setting. This is achieved by combining a molecular engineering method with a highly efficient and easy - to - perform domain - detection assay setup.

[0253] By replacing the individual HVR and framework region sequences of biotherapeutic drug candidates with human germline sequences, a novel and creative assay tool has been generated, which enables the development of a highly specific and sensitive ADA domain - characterization assay according to the invention. This method according to the invention can be used to evaluate the immunogenic response of samples from pre - clinical non - human animals, such as, for example, primate studies.

[0254] This assay can distinguish anti-drug antibodies against all major domains (i.e., different constant parts or complementarity-determining regions) of biotherapeutics and also provides a profound understanding of the cross-reactivity behavior of anti-drug antibodies by using different control molecules including commercial Fab molecules approved for human therapy.

[0255] The described method is applicable to all types of therapeutic antibodies, especially to protein biotherapeutics of smaller size, such as scFv or Fab, because there is no need to enzymatically cleave the molecule into sub-fragments prior to ADA characterization. In terms of data quality, robustness, and ease of use, this assay has proven to be superior to other bioanalytical methods.

[0256] From a project perspective, as part of the developability activities, early inclusion of germline variants of clinical lead candidates can add significant value in terms of immunogenicity understanding and risk mitigation, because molecules with inherent immunological risks can be identified early and excluded from the development of more promising candidates.

[0257] ***

[0258] The following examples, sequences, and figures are provided to assist in understanding the present invention, the true scope of which is set forth in the appended claims. It should be understood that the procedures described may be modified without departing from the spirit of the present invention.

[0259] Examples

[0260] Chemicals, Reagents and Equipment

[0261] The therapeutic antibody and specific assay reagents were all provided by Roche Diagnostics GmbH, Penzberg, Germany, and were aliquoted and stored at -80 °C until use. The bispecific therapeutic Fab ("Fab - original", the drug) consisted of monoclonal Fabs against two different antigens obtainable from recombinant expression. It was used after purification and analytical characterization. In the bridging ADA assay, the biotinylated (Fab - original - Bi) and digoxigenylated (Fab - original - Dig) versions of the bispecific construct "Fab - original" were used as the capture and detection reagents, respectively.

[0262] For the domain detection assay (DDA) and domain competition assay (DCA), the following unlabeled reagents were used for capture: "Fab - original" (bispecific therapeutic Fab), "Germ1 - control" (bispecific therapeutic Fab in which the first complementarity-determining region (CDR) is intact and the second CDR has reverted to the germline sequence), and "Germ2 - control" (bispecific therapeutic Fab in which the second CDR is intact and the first CDR has reverted to the germline sequence), "GermGerm - control" (bispecific therapeutic Fab in which both CDRs have reverted to the corresponding germline sequences), ranibizumab (a commercial human Fab biotherapeutic agent approved for intraocular use), non - binding human Fab (DP47). Digoxigeninylated monoclonal anti - cynomolgus IgG antibody was used as the detection reagent. Polyclonal sheep antibody Fab against digoxigenin conjugated to horseradish peroxidase (HRP) (pAb - Dig - S - Fab - HRP) was used as the second detection reagent in all ADA assays.

[0263] Positive control, PC = generated in vivo or by phage display

[0264] Capture control, CC = PC substitute when PC is not available

[0265] The following compounds were used as positive controls (PC), e.g., as (functional (cloned, generated, immobilized) capture controls (CC): Total ADA screening assay: Monoclonal mouse antibody against human IgGκ (mAb - anti - hu - κ, Roche Diagnostics GmbH, Mannheim, Germany); DDA: Digoxigeninylated recombinant antigen 1 and digoxigeninylated recombinant antigen 2 were used as capture controls (detected by anti - Cκ), and plasma samples from pre - dosing cynomolgus CTAD (CTAD = citrate - theophylline - adenine - dipyridamole; anticoagulant) from each animal were used as negative controls.

[0266] Pooled cynomolgus CTAD plasma was prepared from 40 individual drug - naïve female and male animals. Plasma samples were obtained from Sera Laboratory International, Ltd., Haywards Heath, UK. Wash buffer (phosphate - buffered saline (PBS) / 0.05% Tween 20 / 0.002% Bronidox) and 2,2'-azino - bis - 3 - ethylbenzothiazoline - 6 - sulfonic acid (ABTS) substrate were provided by Roche Diagnostics GmbH, Mannheim, Germany. Ready - to - use low cross - reactivity buffer was obtained from Candor Bioscience GmbH, Wangen, Germany and was used as diluent and assay buffer in the bridging ADA assay (ELISA). All chemicals were of analytical grade.

[0267] Three dose groups of four animals (adult monkeys; cynomolgus monkeys), each dose group was treated with 0 (placebo), 5, and 10 mg / eye for up to 43 days. A second treatment was administered on day 29. From this study, plasma samples from 39 animals dosed with the bispecific Fab original construct were used for ADA testing. Among them, 31 samples were found to be positive in the total ADA screening assay (samples tested at a dilution of 1:20). Finally, 16 samples from 8 different animals (pre-dose samples from each animal as specific negative controls and samples on day 35 after treatment) were selected for further evaluation by domain-specific methods due to significant signal intensities (>1.0 AU) reflecting high ADA concentrations.

[0268] Streptavidin-coated microtiter plates (SA-MTP) for bridging ADA assays were obtained from Microcoat Biotechnologie GmbH, Bernried, Germany. Uncoated NuncMaxiSorp TM microtiter plates for DDA were obtained from Thermo Fisher Scientific, Germany.

[0269] Example 1

[0270] Bridging ADA assay (ADA screening assay) for detecting overall ADA responses

[0271] The bridging ADA assay was applied to analyze plasma samples obtained from the cynomolgus monkey study (see above) to detect against bispecific <ag1 ag2>ADA response of Fab (bispecific Fab against antigen 1 / antigen 2). Samples were tested at a dilution of 1:20. The bridging ADA assay was used as a reference for the domain-specific method.

[0272] The bridging ADA assay is a sandwich enzyme-linked immunosorbent assay (ELISA). Generally, the antibody preparation is made in a dilution buffer (low cross-reactivity buffer, Candor Biosciences GmbH, Wangen, Germany). The reagents and samples are incubated with shaking at 500 rpm at room temperature. The washing step consists of three cycles which are application of 300 μL of wash buffer followed by a final aspiration step. Generally, 15 μL of sample is added to 285 μL of incubation mixture in a polypropylene pre-incubation plate which contains 0.5 μg / mL each of Duta-Original-Bi and Duta-Original-Dig. PC (mAb-anti-hu-κ M-1.7.10) is processed in parallel. After 1 hour of incubation, 100 μL of each sample mixture in duplicate is transferred to SA-MTP for further incubation for 1 hour. After washing with PBS / Tween to remove unbound material and a final aspiration, 100 μL of polyclonal anti-Dig-S-Fab-HRP conjugate (12.5 mU / mL) is added. After 1 hour of incubation, the SA-MTP is washed 3 times followed by a final aspiration. Then the substrate solution ABTS is added and the HRP catalyzes a colorimetric reaction and the reading is at 405 nm wavelength (reference wavelength: 490 nm). Only samples containing the bridging complex (i.e., antibodies that bind to both Duta-Original-Bi and Duta-Original-Dig) can generate a signal. The signal intensity is proportional to the amount of ADA present. For each sample, the absorbance values are determined in two wells. If the precision of the mean ≤ 20% CV, the absorbance values are averaged and accepted. The screening cut-off point (CP) was evaluated using the 95th percentile applicable to 40 drug-naïve donor samples according to the method of Shankar et al. By calculating the plate-specific CP (pooled plasma blank multiplied by a normalization factor), negative samples (< plate-specific CP) and positive samples (> plate-specific CP) were identified. In the context of this article, it should be mentioned that drug tolerance is not considered a critical parameter. The drug was administered intravitreally and the study samples in this formula were sampled at time points of low drug levels, i.e., before subsequent dosing. Since the Fab original level in the samples was B.L.Q., it was considered unnecessary to evaluate drug tolerance.

[0273] Example 2

[0274] Generation of bispecific and variant Fab fragments

[0275] Synthetic genes encoding the bispecific construct Duta-Original and the monospecific, partially germline-constructs Germ1-Control and Germ2-Control, as well as the fully germline, non-binding construct GermGerm-Control were purchased from Geneart.

[0276] The synthetic antibody genes were cloned into a vector, a dicistronic vector for periplasmic expression of Fab fragments in E. coli, carrying an expression cassette including the LacZ promoter, LC ribosome binding site, LC signal peptide, Vk variable domain, Ck constant domain, HC ribosome binding site, HC signal peptide, VH variable domain, IgG1 CH1 constant domain, and the upper hinge of IgG1.

[0277] Plasmids encoding the Fab fragments were transformed into TG1 E. coli cells (Zymo Research), and single colonies were pre-cultured in TB medium at 37 °C, which was supplemented with 2% glucose to inhibit antibody expression. At the end of the lag phase, the pre-culture was diluted into TB expression medium supplemented with glucose at a final concentration of 0.05%, and the expression culture was induced to a final concentration of 1 mM by supplementing IPTG at the end of the logarithmic phase. The Fab fragments were expressed at 30 °C for 16 h, and the culture supernatant was clarified by centrifugation.

[0278] To ensure that only full-length Fab fragments with intact heavy and light chain constant domains were used in the ADA assay, the Fab fragments were purified by double affinity in two steps, first using CaptureSelect IgG-CH1 resin and then using CaptureSelect κXL resin, both purchased from GE Healthcare. The protein concentration of the purified Fab fragments was determined by spectrophotometry using the absorbance at 280 nm.

[0279] Example 3

[0280] Domain Detection Assay (DDA) for detecting domain-specific ADA responses

[0281] The Domain Detection Assay (DDA) represents a four-step sandwich enzyme-linked immunosorbent assay (ELISA).

[0282] 3.33 μg / mL each and 150 μL of Fab-Original, Germ1-Control, Germ2-Control, GermGerm-Control, ranibizumab, and non-binding human Fab per well were used as capture reagents in Nunc MaxiSorp TM Incubate on MTP for 1 hour. After washing the plate 3 times with PBS / Tween to remove unbound materials, block the plate with 150 μL of assay buffer for 30 minutes to avoid non-specific binding of samples in the next step. After washing the plate, dilute 3 μL of each sample with 297 μL of assay buffer and incubate 100 μL of each sample in duplicate for 1 hour. Process the capture controls (recombinant antigen 1-Dig and recombinant antigen 2-Dig) in parallel. The pre-dose samples from each animal represent the negative controls. Digoxigeninylated monoclonal anti-cynomolgus IgG is used as the detection reagent (100 μL / well). The wells with capture controls are filled with 100 μL of assay buffer. The polyclonal sheep antibody Fab fragment against Dig conjugated with horseradish peroxidase (HRP) (pAb-Dig-S-Fab-HRP) is used as the second detection reagent in all ADA assays (100 μL / well). Add ABTS to each well and monitor the subsequent chromogenic reaction by photometric reading at 405 nm (reference wavelength 490 nm).

[0283] To distinguish positive samples from negative samples, a cut-off point (CP) determination was performed on a set of 8 pre-dose samples from cynomolgus monkeys studied for each of the 5 DDAs.

[0284] Table: Capture control assays for 5 different DDAs with expected positive / negative.

[0285]

[0286] All samples were diluted 3 times (1 to 100 / 1 to 1000 / 1 to 10000) for measurement. Samples were diluted in low cross-reactivity buffer TM and measured in parallel in all 5 assays.

[0287] Example 4

[0288] Capture control assay (CCA)

[0289] To test the reproducibility of the coating process for 5 different DDAs, three different binding control assays were established. First, Nunc MaxiSorp TM plates were coated as described in the DDA. Subsequently, digoxigenin-labeled anti-human κ light chain antibody or digoxigenin-labeled antigen 1 or antigen 2 was incubated at a concentration of 50 ng / mL. With HRP (pAb <dig>The polyclonal sheep antibody Fab fragment against digoxin conjugated with S-Fab-HRP was used as the second detection reagent. ABTS was added to each well, and the subsequent chromogenic reaction was monitored by photometric readings at 405 nm (reference wavelength 490 nm). These assays were used to examine the reproducibility of the coating and the functionality of the coated protein. In addition, during sample measurement, a capture control 1 assay was performed on the measurement plate as a quasi-positive control ("substitute PC") to ensure the comparability of individual measurements on different plates. The signal of this CCA was monitored and the assay was stopped at a signal intensity of 1.8 - 2.2 OD 405 nm and the signal intensity of

[0290] Example 5

[0291] Characterization of the domain detection assay (DDA) according to the present invention

[0292] The samples used were collected from a preclinical tolerance study in cynomolgus monkeys with bispecific anti-antigen 1 / antigen 2 Fab as the drug. Based on ADA positivity in the initial bridging assay used for ADA screening (Wessels, U. et al., Bioanal. 10 (2018) 803 - 814), 16 plasma samples from 8 different animals treated with bispecific anti-antigen 1 / antigen 2 Fab were selected for ADA characterization using the method according to the present invention. For each animal, pre-dose samples and samples taken 35 days after dosing, which served as specific negative controls, were tested. The pre-dose cynomolgus monkey citrate-theophylline-adenine-dipyridamole (CTAD) plasma samples from each animal were used as negative controls for the DDA.

[0293] Determination of Qualitative

[0294] The example of the method according to the present invention described in this example was used to characterize the ADA response and was based on the results of the analysis of 5 individual DDAs that were related to each other (see Figure 6 and Figure 7 , shows the settings used in this example and the expected positive pattern). This requires generating 5 different positive controls. This is a common challenge with DDA and is not limited to the assays described in this disclosure (Gorovits, B. et al., J. Immunol. Meth. 408 (2014) 1 - 12; Hock, M. B. et al., AAPS J. 17 (2015) 35 - 43). For optimal qualification of these assays, specific positive controls must be generated, which can be achieved by immunizing mice with the full compound and a good screening concept. Another possibility is to use antibodies already available against individual domains (Stubenrauch, K. et al., J. Pharm. Biomed. Anal. 114 (2015) 296 - 304). Due to the nature of the Fab therapeutics used in this example, the second approach is not feasible and, unfortunately, specific immunization is very time - consuming and laborious. In the absence of positive controls, the standard methods for qualifying these assays are not feasible. Therefore, a combination of capture control assays and assay - specific cut - point determination is used as a practical method for generating reliable data.

[0295] Capture Consistency

[0296] Data from the capture control assays allows for the determination of precision values. These precision values reflect the reproducibility of the coating. The CCA based on anti - κ IgG antibody is a measure of the efficiency of Fab surface coating. This value was determined for all 5 individual DDAs. The CCA based on AG1 and AG2 also shows whether the CDRs of the bound Fab fragments are available for the antigen or potential ADA. The precision data shows that in 3 different in - house assay runs, the variation in all 3 CCAs was very small, only 1% to 3%.

[0297] Table: Precision data for all 3 capture control assays in five domain detection assays.

[0298]

[0299] It can be seen that the amount of coated antibody and their ability to bind antigen are constant. This robustness of the coating is a prerequisite for data comparability. The data also shows that all the constructs used either have or no longer have the required function.

[0300] DDA Cleavage Point Determination

[0301] Specific cleavage points were calculated for all 5 DDAs to assess ADA positivity by measuring the corresponding pre-dose samples (n = 8 animals). To calculate the cleavage points, the 99th percentile was used. The following table lists all the measured multiplication normalization factors and the corresponding cleavage points.

[0302]

[0303] The assay range was determined by using digoxin-labeled antigens (Antigen 1 and Antigen 2). The assay was carried out at an antigen 1-Dig or antigen 2-Dig concentration of 50 ng / ml until a signal of 1.8 - 2.2 OD 405 nm was reached. This signal range was used as the standard to stop the reaction in the measurement of real samples. Real samples were diluted 1:100 in the assay buffer. In the case of reaching a signal higher than 2.2 OD 405nm the samples were further diluted in steps of 1:10.

[0304] For each individual construct, cleavage point determination was performed using the corresponding pre-dose samples.

[0305] Table: Cleavage point determination: Obtain the signal of the pre-dose samples in the DDA assay. As shown below, the 99th percentile was used to calculate the assay-specific cleavage point.

[0306]

[0307] Drug Tolerance of DDA

[0308] The samples used were from a later time point (day 35 after drug administration). Therefore, the measured residual drug levels were very low, and it was expected that the residual drugs would not affect these assays. To discuss this theoretically, it has to be said that in this assay, the labeled drug was coated at a very high concentration, which directly competes with the residual drugs in the samples. Therefore, the theoretical drug tolerance should be at least as good as that of the standard bridging assay. If necessary, this can be improved in a way similar to the bridging ADA assay. For example, the samples can be pretreated with acid to dissolve the existing drug-ADA complexes (see, for example, Kavita, U. et al., J. Immunol. Meth. 448 (2017) 91 - 104).

[0309] This assay has already shown a high degree of drug tolerance because the mass concentration of the capture antibody / domain is several orders of magnitude higher than the residual drug levels present in the samples on day 35 after dosing. This was also confirmed by titration experiments, indicating that when the drug was diluted out of the samples, the specific ADA recognition signal remained highly positive.

[0310] Sample Analysis Using Domain Detection Assay

[0311] The numerical data for all samples diluted 3 times used can be found in the table below.

[0312] Table: Results of the numerical ADA domain detection assay. Figures 8 to 11 The values used for illustration in the figure are in bold.

[0313]

[0314] As Figures 8 to 11 shown, different dilutions were selected to compare the data with each other. The linear range of the chromogenic ABTS substrate is limited, and the samples had to be diluted to different extents to make the signal heights clearly distinguishable. For 6 out of 8 animals, dilutions from 1 to 1000 were used. In animal 3, the immune response was weak, and dilutions from 1 to 100 were selected, while in animal 1, dilutions from 1 to 10000 were selected because the immune response was very strong.

[0315] Four ADA response patterns (hereinafter referred to as 8, 9, 10, and 11, consistent with the Figures 8 to 11 corresponding results therein) can be distinguished, which represent all 8 ADA-positive animals studied.

[0316] Pattern 8: Animals 1 and 2 revealed a mixed ADA response against different parts of the molecule (CDR and framework). A response against the fully bound competent <ag1 ag2>Bispecific F(ab), Germ <ag1>- Control and Germ <ag2>- Strong responses to the control. Mild responses were detected against the fully germline control construct (GermGerm-control) and ranibizumab, indicating the presence of ADA against the molecular constant region. Most of the ADA seemed to target both CDRs in similar proportions.

[0317] Pattern 9: Animals 3 and 4 showed recognition only of the CDR regions, without ADA against the constant region. In Animal 3, the signals against the two CDRs here were lower than in Pattern A, indicating a milder ADA response, while Animal 4 revealed a signal intensity similar to Pattern A. In both of these animals, there also seemed to be a similar number of ADA against the two CDRs.

[0318] Pattern 10: Animals 5 and 6 were similar to Pattern 8, indicating a mixed response against both the CDR and the constant region. However, compared to Pattern 8, the proportion of ADA against the molecular constant region was much lower.

[0319] Pattern 11: Animals 7 and 8 did not show ADA against the constant region and showed only ADA against the CDR. Different from Pattern 9 and all other animals, we saw a different distribution of ADA between the two CDRs of Antigen 1 and Antigen 2. There seemed to be more ADA against the AG1 binding site.

[0320] Overall, all pre-dose samples were ADA negative in all five DDAs. Therefore, we concluded that all immunogenic responses observed in the cynomolgus monkey study samples after dosing from Day 35 were treatment-related ADA responses.

[0321] The signals of the pre-dose samples were also well comparable to the minor variations in all 5 DDAs. Therefore, these values were used to calculate the DDA specific to the study cut-off point (see above: DDA cut-off point determination). For this procedure, the number of baseline samples used was relevant, and it was recommended (Shankar, G. et al., J. Pharm. Biomed. Anal. 48 (2008) 1267 - 1281; Amaravadi, L. et al., Bioanal. 7 (2015) 3107 - 3124) to use at least 50 pre-dose samples. In many pre-clinical studies such as this, the number of animals was significantly reduced, making this infeasible. However, considering the exploratory nature of this measurement, this method was chosen.

[0322] In summary, using the method according to the present invention, it was found that all eight animals showed ADA against CDRS, and in only two of the eight animals, these responses were slightly different. Due to this mixed immune response, it is unlikely that one of the two CDR regions of the bispecific molecule is mainly responsible for the observed immune response. No individual domain was observed to exhibit strongly different immunogenicity.

[0323] Another issue to be addressed is whether the engineered bispecific F(ab) fragment used here (which has a slightly different structure compared to the wild-type Fab fragment) has a higher immunogenicity risk. Four of the eight animals showed antibodies against the constant region. No higher signal of the samples was found in the DDA with the Germ-Germ variant compared to the DDA with ranibizumab. This would be evidence of an immune response against a new epitope present only on the Germ-Germ variant and not on ranibizumab, which is an ocular therapeutic agent known for its low immunogenicity, as shown in several clinical studies (Figurska, M. et al., Klin. Oczna. 112 (2010) 147-150).

[0324] Based on the above findings, all immunogenic responses from the above cynomolgus monkey study were treatment-related ADA responses. The dual / bispecific Fab specificity of treatment-induced ADA was confirmed. Overall, the immunogenic responses were against the HVR and the constant Fab portion. In 50% of the animals, a pure "monospecific" response against the HVR was observed, while in the other half, a mixed response against both the HVR and the framework was seen.

[0325] No ADA response against the constant framework was observed in the absence of HVR contribution. Notably, the drug antibodies against the constant portion of the bispecific Fab cross-react with typical human Fab molecules such as the non-binding (DP47) control molecule and ranibizumab. This is an important result for potential human applications, as there is no hint of the intrinsic immunogenic properties of the molecule in the bispecific Fab. < / dig>

Claims

1. A method for determining the epitope of an anti-drug antibody that specifically binds to a therapeutic antibody, wherein the therapeutic antibody specifically binds to a therapeutic target, the method comprising the steps of: a) incubating aliquots of a sample comprising the anti-drug antibody separately with: i) at least a Fab fragment of the therapeutic antibody, and ii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs of the complementarity-determining regions (CDRs) against the therapeutic target have been replaced by germline or non-binding HVRs, and detecting the binding or non-binding of the anti-drug antibody to the at least Fab fragment in any one of i) to ii), and b) determining the epitope of the anti-drug antibody - in the complementarity-determining regions against the therapeutic target, provided that binding is detected in i) and non-binding is detected in ii).

2. The method according to claim 1, wherein the non-binding or germline HVRs are obtained from the same germline as the framework regions of the therapeutic antibody.

3. The method according to any one of claims 1 to 2, wherein the therapeutic antibody is a monospecific or bispecific Fab.

4. The method according to claim 3, wherein the bispecific Fab comprises two non-overlapping complementarity-determining regions in one cognate VH / VL pair.

5. The method according to claim 3, wherein the bispecific Fab specifically binds to a first antigen and a second antigen.

6. The method according to claim 3, wherein the bispecific Fab specifically binds to the first antigen through its first complementarity-determining region and specifically binds to the second antigen through its second complementarity-determining region.

7. The method according to claim 3, wherein the bispecific Fab comprises a first complementarity-determining region and a second complementarity-determining region in one cognate pair of variable light chain domain and variable heavy chain domain, wherein the first complementarity-determining region comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the therapeutic antibody, and wherein the second complementarity-determining region comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the therapeutic antibody.

8. The method according to any one of claims 4 to 7, wherein the complementarity-determining regions are formed by the VH / VL pair.

9. The method according to any one of claims 1 to 2, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one of the HVRs that have been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii), - in the HVR-framework region junction of at least one of the HVRs that have been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii); - in the framework region and / or constant domain, provided that Binding was detected in i) to iii).

10. The method according to claim 4, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one HVR that has been replaced in ii), provided that binding was detected in i) and non-binding was detected in ii) and iii), - in the HVR-framework region junction of at least one HVR that has been replaced in ii), provided that binding was detected in i) and ii) and non-binding was detected in iii); - in the framework region and / or constant domain, provided that binding was detected in i) to iii).

11. The method according to claim 5, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one HVR that has been replaced in ii), provided that binding was detected in i) and non-binding was detected in ii) and iii), - in the HVR-framework region junction of at least one HVR that has been replaced in ii), provided that binding was detected in i) and ii) and non-binding was detected in iii); - in the framework region and / or constant domain, provided that binding was detected in i) to iii).

12. The method according to claim 6, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one HVR that has been replaced in ii), provided that binding was detected in i) and non-binding was detected in ii) and iii), - in the HVR-framework region junction of at least one HVR that has been replaced in ii), provided that binding was detected in i) and ii) and non-binding was detected in iii); - in the framework region and / or constant domain, provided that binding was detected in i) to iii).

13. The method according to claim 7, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i) to iii) and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii) - at the HVR-framework region junction of at least one HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii) - in the framework region and / or constant domain, provided that binding is detected in i) to iii) 14. The method according to claim 8, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been replaced by non-binding HVRs and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i) to iii) and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii) - at the HVR-framework region junction of at least one HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii) - in the framework region and / or constant domain, provided that binding is detected in i) to iii) 15. The method according to any one of claims 1 to 2, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been completely replaced by non-binding HVRs and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i) to iii) and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii) - at the HVR-framework region junction of at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii) - in the framework region and / or constant domain, provided that binding is detected in i) to iii) 16. The method according to claim 4, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all the HVRs have been completely replaced by non-binding HVRs and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i) to iii) and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii), - at the HVR-framework region junction of at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii); - in the framework region and / or constant domain, provided that binding is detected in i) to iii).

17. The method according to claim 5, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all HVRs have been completely replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii), - at the HVR-framework region junction of at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii); - in the framework region and / or constant domain, provided that binding is detected in i) to iii).

18. The method according to claim 6, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all HVRs have been completely replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii), - at the HVR-framework region junction of at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii); - in the framework region and / or constant domain, provided that binding is detected in i) to iii).

19. The method according to claim 7, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all HVRs have been completely replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii), - in the HVR-framework region junction of at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii); - in the framework region and / or constant domain, provided that binding is detected in i) to iii).

20. The method according to claim 8, wherein step a) further comprises iii) at least a Fab fragment of the therapeutic antibody, wherein all HVRs have been completely replaced by non-binding HVRs; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i) to iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and non-binding is detected in ii) and iii), - in the HVR-framework region junction of at least one complete HVR that has been replaced in ii), provided that binding is detected in i) and ii) and non-binding is detected in iii); - in the framework region and / or constant domain, provided that binding is detected in i) to iii).

21. The method according to claim 9, wherein step a) further comprises iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one HVR different from the HVR of ii) has been replaced by a non-binding HVR; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i), ii) and iv) and if present iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one HVR that has been replaced in ii) or iv), provided that binding is detected in i) and non-binding is detected in one of ii) or iv) and in if present iii); - in the HVR-framework region junction of at least one HVR that has been replaced in ii) or iv), provided that binding is detected in i) and in one of ii) or iv) and non-binding is detected in if present iii); - optionally in the framework region and / or constant domain, provided that binding is detected in i), ii), iv) and if present iii).

22. The method according to claim 10, wherein step a) further comprises iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one HVR different from the HVR of ii) has been replaced by a non-binding HVR; and detecting the binding or non-binding of the antibody specifically binding to the therapeutic antibody to the at least Fab fragment in any one of i), ii) and iv) and if present iii), and step (b) is determining the epitope of the antibody specifically binding to the therapeutic antibody - in at least one HVR that has been replaced in ii) or iv), provided that Binding is detected in (i) and non - binding is detected in one of (ii) or (iv) and, if present, in (iii); - in the HVR - framework region junction of at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and in one of (ii) or (iv) and non - binding is detected in (iii), if present; - optionally in the framework region and / or constant domain, provided that binding is detected in (i), (ii), (iv) and, if present, (iii).

23. The method according to claim 11, wherein step (a) further comprises iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one HVR different from the HVR of (ii) has been replaced by a non - binding HVR; and detecting the binding or non - binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of (i), (ii) and (iv) and, if present, (iii), and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and non - binding is detected in one of (ii) or (iv) and, if present, in (iii); - in the HVR - framework region junction of at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and in one of (ii) or (iv) andnon - binding is detected in (iii), if present; - optionally in the framework region and / or constant domain, provided that binding is detected in (i), (ii), (iv) and, if present, (iii).

24. The method according to claim 12, wherein step (a) further comprises iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one HVR different from the HVR of (ii) has been replaced by a non - binding HVR; and detecting the binding or non - binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of (i), (ii) and (iv) and, if present, (iii), and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and non - binding is detected in one of (ii) or (iv) and, if present, in (iii); - in the HVR - framework region junction of at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and in one of (ii) or (iv) andnon - binding is detected in (iii), if present; - optionally in the framework region and / or constant domain, provided that binding is detected in (i), (ii), (iv) and, if present, (iii).

25. The method according to claim 13, wherein step (a) further comprises ​ ​ iv) one or more additional at least Fab fragments of the therapeutic antibody, wherein at least one HVR different from the HVRs of ii) has been replaced by a non-binding HVR; and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i), ii), iv), and iii) if present; and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or iv), provided that binding is detected in i) and non-binding is detected in one of ii) or iv) and in iii) if present; - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or iv), provided that binding is detected in i) and in one of ii) or iv) and non-binding is detected in iii) if present; - optionally in the framework region and / or constant domain, provided that binding is detected in i), ii), iv), and iii) if present.

26. The method according to claim 14, wherein step a) further comprises iv) one or more additional at least Fab fragments of the therapeutic antibody, wherein at least one HVR different from the HVRs of ii) has been replaced by a non-binding HVR; and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i), ii), iv), and iii) if present; and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or iv), provided that binding is detected in i) and non-binding is detected in one of ii) or iv) and in iii) if present; - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or iv), provided that binding is detected in i) and in one of ii) or iv) and non-binding is detected in iii) if present; - optionally in the framework region and / or constant domain, provided that binding is detected in i), ii), iv), and iii) if present.

27. The method according to claim 15, wherein step a) further comprises iv) one or more additional at least Fab fragments of the therapeutic antibody, wherein at least one complete HVR different from the HVRs of ii) has been replaced by a complete non-binding HVR; and detecting the binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i), ii), iv), and iii) if present; and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii) or iv), provided that binding is detected in i) and non-binding is detected in one of ii) or iv) and in iii) if present; - at the HVR-framework region junctions of at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and one of (ii) or (iv); and non-binding is detected in (iii) if present; - optionally in the framework region and / or constant domain, provided that binding is detected in (i), (ii), (iv) and (iii) if present.

28. The method according to claim 16, wherein step (a) further comprises iv) one or more additional at least Fab fragments of the therapeutic antibody, wherein at least one complete HVR different from the HVR of (ii) has been replaced by a complete non-binding HVR; and detecting binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of (i), (ii) and (iv) and (iii) if present, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and non-binding is detected in one of (ii) or (iv) and in (iii) if present; - at the HVR-framework region junctions of at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and one of (ii) or (iv); and non-binding is detected in (iii) if present; - optionally in the framework region and / or constant domain, provided that binding is detected in (i), (ii), (iv) and (iii) if present.

29. The method according to claim 17, wherein step (a) further comprises iv) one or more additional at least Fab fragments of the therapeutic antibody, wherein at least one complete HVR different from the HVR of (ii) has been replaced by a complete non-binding HVR; and detecting binding or non-binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of (i), (ii) and (iv) and (iii) if present, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and non-binding is detected in one of (ii) or (iv) and in (iii) if present; - at the HVR-framework region junctions of at least one HVR that has been replaced in (ii) or (iv), provided that binding is detected in (i) and one of (ii) or (iv); and non-binding is detected in (iii) if present; - optionally in the framework region and / or constant domain, provided that binding is detected in (i), (ii), (iv) and (iii) if present.

30. The method according to claim 18, wherein step (a) further comprises iv) one or more additional at least Fab fragments of the therapeutic antibody, wherein at least one complete HVR different from the HVR of (ii) has been replaced by a complete non-binding HVR; And detecting the binding or non - binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i), ii), iv), and iii) if present. And step (b) is Determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii) or iv), provided that Binding is detected in i) And Non - binding is detected in one of ii) or iv) and in iii) if present; - at the HVR - framework region junction of at least one HVR that has been replaced in ii) or iv), provided that Binding is detected in i) and in one of ii) or iv) And Non - binding is detected in iii) if present; - optionally in the framework region and / or constant domain, provided that Binding is detected in i), ii), iv), and iii) if present.

31. The method according to claim 19, wherein step a) further comprises iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one complete HVR different from the HVR of ii) has been replaced by a complete non - binding HVR; And detecting the binding or non - binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i), ii), iv), and iii) if present, And step (b) is Determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii) or iv), provided that Binding is detected in i) And Non - binding is detected in one of ii) or iv) and in iii) if present; - at the HVR - framework region junction of at least one HVR that has been replaced in ii) or iv), provided that Binding is detected in i) and in one of ii) or iv) And Non - binding is detected in iii) if present; - optionally in the framework region and / or constant domain, provided that Binding is detected in i), ii), iv), and iii) if present.

32. The method according to claim 20, wherein step a) further comprises iv) one or more other at least Fab fragments of the therapeutic antibody, wherein at least one complete HVR different from the HVR of ii) has been replaced by a complete non - binding HVR; And detecting the binding or non - binding of the antibody that specifically binds to the therapeutic antibody to the at least Fab fragment in any one of i), ii), iv), and iii) if present, And step (b) is Determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii) or iv), provided that Binding is detected in i) And Non - binding is detected in one of ii) or iv) and in iii) if present; - at the HVR - framework region junction of at least one HVR that has been replaced in ii) or iv), provided that Binding is detected in i) and in one of ii) or iv) And Detect non-binding in iii) if present; - Optionally in the framework region and / or constant domain, provided that binding is detected in i), ii), iv) and iii) if present.

33. The method according to claim 21, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or iv) if present, provided that binding is detected in i) and non-binding is detected in ii) and v), iii) if present and iv) if present; - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or iv) if present, provided that binding is detected in i) and in one of ii) or iv) if present and non-binding is detected in v) and iii) if present; - in the framework region and / or constant domain, provided that binding is detected in i) and ii) and iii) if present and iv) if present and non-binding is detected in v).

34. The method according to claim 22, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or iv) if present, provided that binding is detected in i) and non-binding is detected in ii) and v), iii) if present and iv) if present; - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or iv) if present, provided that binding is detected in i) and in one of ii) or iv) if present and non-binding is detected in v) and iii) if present; - in the framework region and / or constant domain, provided that binding is detected in i) and ii) and iii) if present and iv) if present and non-binding is detected in v).

35. The method according to claim 23, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or iv) if present, provided that binding is detected in i) and non-binding is detected in ii) and v), iii) if present and iv) if present; - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or iv) if present, provided that binding is detected in i) and in one of ii) or iv) if present and Non-binding is detected in v) and, if present, iii); - in the framework region and / or the constant domain, provided that binding is detected in i) and ii) and, if present, iii) and, if present, iv); and non-binding is detected in v).

36. The method according to claim 24, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i); and non-binding is detected in ii) and v), and, if present, iii) and, if present, iv); - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and in one of ii) or, if present, iv); and non-binding is detected in v) and, if present, iii); - in the framework region and / or the constant domain, provided that binding is detected in i) and ii) and, if present, iii) and, if present, iv); and non-binding is detected in v).

37. The method according to claim 25, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i); and non-binding is detected in ii) and v), and, if present, iii) and, if present, iv); - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and in one of ii) or, if present, iv); and non-binding is detected in v) and, if present, iii); - in the framework region and / or the constant domain, provided that binding is detected in i) and ii) and, if present, iii) and, if present, iv); and non-binding is detected in v).

38. The method according to claim 26, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i); and non-binding is detected in ii) and v), and, if present, iii) and, if present, iv); - at the HVR-framework region junction of at least one HVR that has been replaced in ii) or, if present, iv), provided that Binding is detected in one of i) and ii) or iv) if present and Non-binding is detected in v) and iii) if present; - In the framework region and / or constant domain, provided that Binding is detected in i), ii), iii) if present, and iv) if present and Non-binding is detected in v).

39. The method according to claim 27, wherein step a) further comprises v) At least a Fab fragment of a non-binding antibody from a germline different from the germline of the framework region of the therapeutic antibody, and step (b) is Determining the epitope of the antibody that specifically binds to the therapeutic antibody - In at least one complete HVR that has been replaced in ii) or iv) if present, provided that Binding is detected in i) and Non-binding is detected in ii), v), iii) if present, and iv) if present; - At the HVR-framework region junction of at least one complete HVR that has been replaced in ii) or iv) if present, provided that Binding is detected in one of i) and ii) or iv) if present and Non-binding is detected in v) and iii) if present; - In the framework region and / or constant domain, provided that Binding is detected in i), ii), iii) if present, and iv) if present and Non-binding is detected in v).

40. The method according to claim 28, wherein step a) further comprises v) At least a Fab fragment of a non-binding antibody from a germline different from the germline of the framework region of the therapeutic antibody, and step (b) is Determining the epitope of the antibody that specifically binds to the therapeutic antibody - In at least one complete HVR that has been replaced in ii) or iv) if present, provided that Binding is detected in i) and Non-binding is detected in ii), v), iii) if present, and iv) if present; - At the HVR-framework region junction of at least one complete HVR that has been replaced in ii) or iv) if present, provided that Binding is detected in one of i) and ii) or iv) if present and Non-binding is detected in v) and iii) if present; - In the framework region and / or constant domain, provided that Binding is detected in i), ii), iii) if present, and iv) if present and Non-binding is detected in v).

41. The method according to claim 29, wherein step a) further comprises v) At least a Fab fragment of a non-binding antibody from a germline different from the germline of the framework region of the therapeutic antibody, and step (b) is Determining the epitope of the antibody that specifically binds to the therapeutic antibody - In at least one complete HVR that has been replaced in ii) or iv) if present, provided that Binding is detected in i) and Non-binding is detected in ii), v), iii) if present, and iv) if present; - at the HVR-framework region junctions of at least one complete HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and in one of ii) or, if present, iv) and non-binding is detected in v) and, if present, iii); - in the framework region and / or constant domain, provided that binding is detected in i) and ii) and, if present, iii) and, if present, iv) and non-binding is detected in v).

42. The method according to claim 30, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and non-binding is detected in ii) and v) and, if present, iii) and, if present, iv); - at the HVR-framework region junctions of at least one complete HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and in one of ii) or, if present, iv) and non-binding is detected in v) and, if present, iii); - in the framework region and / or constant domain, provided that binding is detected in i) and ii) and, if present, iii) and, if present, iv) and non-binding is detected in v).

43. The method according to claim 31, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and non-binding is detected in ii) and v) and, if present, iii) and, if present, iv); - at the HVR-framework region junctions of at least one complete HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and in one of ii) or, if present, iv) and non-binding is detected in v) and, if present, iii); - in the framework region and / or constant domain, provided that binding is detected in i) and ii) and, if present, iii) and, if present, iv) and non-binding is detected in v).

44. The method according to claim 32, wherein step a) further comprises v) at least a Fab fragment of a non-binding antibody from a germline that is different from the germline of the framework region of the therapeutic antibody, and step (b) is determining the epitope of the antibody that specifically binds to the therapeutic antibody - in at least one complete HVR that has been replaced in ii) or, if present, iv), provided that binding is detected in i) and Non-binding is detected in ii) and v), in iii) if present, and in iv) if present; - in the HVR-framework region junctions of at least one complete HVR that has been replaced in ii) or in iv) if present, provided that binding is detected in i) and in one of ii) or in iv) if present and non-binding is detected in v) and in iii) if present; - in the framework region and / or constant domain, provided that binding is detected in i) and ii) and in iii) if present and in iv) if present and non-binding is detected in v).

45. The method according to claim 15, wherein the complete non-binding HVR is a complete germline HVR obtained from the same germline as the framework region of the therapeutic antibody.

46. The method according to any one of claims 16 to 20, wherein the complete non-binding HVR is a complete germline HVR obtained from the same germline as the framework region of the therapeutic antibody.

47. The method according to any one of claims 27 to 32, wherein the complete non-binding HVR is a complete germline HVR obtained from the same germline as the framework region of the therapeutic antibody.

48. The method according to any one of claims 39 to 44, wherein the complete non-binding HVR is a complete germline HVR obtained from the same germline as the framework region of the therapeutic antibody.

49. The method according to any one of claims 1 to 2, wherein - the therapeutic antibody is a bispecific Fab, wherein the first complementarity-determining region is formed by HVR H-1, H-3 and L-2, and the second complementarity-determining region is formed by HVR H-2, L-1 and L-3, - in step a), step ii) is the therapeutic antibody, wherein HVR H-1, H-3 and L-2 have been replaced by non-binding HVRs, - step a) further comprises vi) the therapeutic antibody, wherein HVR H-2, L-1 and L-3 have been replaced by non-binding HVRs, - step b) is determining the epitope of the antidrug antibody - in the first complementarity-determining region of the bispecific Fab, provided that binding is detected in i) and vi), and non-binding is detected in ii), - in the second complementarity-determining region of the bispecific Fab, provided that binding is detected in i) and ii), and non-binding is detected in vi), - in the HVR-framework region junctions of the first complementarity-determining region, provided that binding is detected in i) and vi) and non-binding is detected in ii), - in the HVR-framework region junctions of the second complementarity-determining region, provided that binding is detected in i) and ii) and non-binding is detected in vi).

50. The method according to any one of claims 1 to 2, wherein no soluble variant of the therapeutic antibody or its fragment is added to the sample or an aliquot of the sample before incubation with at least the Fab immobilized of the therapeutic antibody or its complementarity-determining region-modified variant in step (a).

51. The method according to claim 21, wherein a soluble variant of the therapeutic antibody or a fragment thereof without the therapeutic antibody is added to the sample or an aliquot of the sample before incubation with at least the Fab immobilized of the therapeutic antibody or a complementarity-determining region-modified variant thereof in step (a).

52. The method according to claim 22, wherein a soluble variant of the therapeutic antibody or a fragment thereof without the therapeutic antibody is added to the sample or an aliquot of the sample before incubation with at least the Fab immobilized of the therapeutic antibody or a complementarity-determining region-modified variant thereof in step (a).

53. The method according to claim 23, wherein a soluble variant of the therapeutic antibody or a fragment thereof without the therapeutic antibody is added to the sample or an aliquot of the sample before incubation with at least the Fab immobilized of the therapeutic antibody or a complementarity-determining region-modified variant thereof in step (a).

54. The method according to claim 24, wherein a soluble variant of the therapeutic antibody or a fragment thereof without the therapeutic antibody is added to the sample or an aliquot of the sample before incubation with at least the Fab immobilized of the therapeutic antibody or a complementarity-determining region-modified variant thereof in step (a).

55. The method according to claim 25, wherein a soluble variant of the therapeutic antibody or a fragment thereof without the therapeutic antibody is added to the sample or an aliquot of the sample before incubation with at least the Fab immobilized of the therapeutic antibody or a complementarity-determining region-modified variant thereof in step (a).

56. The method according to claim 26, wherein a soluble variant of the therapeutic antibody or a fragment thereof without the therapeutic antibody is added to the sample or an aliquot of the sample before incubation with at least the Fab immobilized of the therapeutic antibody or a complementarity-determining region-modified variant thereof in step (a).

57. The method according to any one of claims 1 to 2, wherein the complementarity-determining region of the therapeutic antibody that binds to the therapeutic target is not a single-domain antibody.

58. The method according to any one of claims 1 to 2, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

59. The method according to claim 50, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

60. The method according to claim 51, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

61. The method according to claim 52, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

62. The method according to claim 53, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

63. The method according to claim 54, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

64. The method according to claim 55, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

65. The method according to claim 56, wherein at least the Fab fragment of step a) is directly immobilized on a solid surface.

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