Method for the determination of anti-drug antibodies in miniature pig samples

Abstract

The present invention relates to an in vitro method for determining the presence or absence of anti-drug antibodies (ADA) or drug / ADA immune complexes in a miniature pig sample. The present invention also relates to antibodies that specifically bind to miniature pig IgG and can be used in the method of the present invention.

Description

Technical Field

[0001] The present invention relates to an in vitro method for determining anti-drug antibodies in miniature pig samples and antibodies that specifically bind to miniature pig IgG. Background of the Invention

[0003] Clinical development of new therapeutic antibodies requires assessment of their potential immunogenicity by appropriate assays (Kaliyaperumal, A. and Jing, S., Curr. Pharm. Biotechnol. 10 (2009) 352-358). Anti-drug antibody (ADA) testing generally involves a two-tier approach: (1) an assay to detect ADA and (2) an assay to characterize ADA. ADA detection assays include screening and specificity validation (confirmatory) assays. Microtiter plate-based enzyme-linked immunosorbent assay (ELISA) is the most widely used format for screening ADA due to its high throughput efficiency, simplicity, and high sensitivity (Geng, D. et al., J. Pharm. Biomed. Anal. 39 (2005) 364-375). ADA ELISAs are most often designed in a bridge format that provides high selectivity, detection of all isotypes, and pan-species ADA detection capabilities (Mire-Sluis, AR et al., J. Immunol. Methods 289 (2004) 1-16). WO 2008031532A1 discloses an ADA ELISA for detecting ADA in samples derived from monkey species. SUMMARY OF THE INVENTION

[0005] The present invention relates to an in vitro method for determining the presence or absence of anti-drug antibodies (ADA) in a miniature pig sample, the method comprising:

[0006] contacting the sample with an excess of drug, wherein the ADA binds to the drug to form a drug / ADA complex,

[0007] contacting the drug / ADA complex with an antibody that specifically binds to minipig IgG, wherein the antibody that specifically binds to minipig IgG does not specifically bind to human IgG,

[0008] Binding of the drug / ADA complex by antibodies that specifically bind to minipig IgG is determined to determine the presence of ADA in the sample.

[0009] In another aspect, the present invention relates to an in vitro method for determining the presence or absence of an immune complex comprising a drug and an antibody that specifically binds to the drug (drug / ADA complex) in a miniature pig sample, the method comprising:

[0010] contacting the minipig sample with an immobilized antibody that specifically binds to the drug to immobilize the drug / ADA complex present in the minipig sample,

[0011] contacting the drug / ADA complex with an antibody that specifically binds to minipig IgG, wherein the antibody that specifically binds to minipig IgG does not specifically bind to human IgG, and

[0012] Binding of the drug / ADA complex by antibodies that specifically bind to minipig IgG is determined to determine the presence of ADA in the sample.

[0013] In one embodiment of the invention, the antibody that specifically binds to minipig IgG does not specifically bind to human IgG. In one embodiment of the invention, the antibody that specifically binds to minipig IgG does not specifically bind to mouse IgG.

[0014] In one embodiment of the present invention, the antibody that specifically binds to minipig IgG specifically binds to the Fc domain of minipig IgG. In one embodiment, the antibody that specifically binds to the Fc domain of minipig IgG comprises the heavy chain CDR1 of SEQ ID NO: 3, the heavy chain CDR2 of SEQ ID NO: 4, and the heavy chain CDR3 of SEQ ID NO: 5, the light chain CDR1 of SEQ ID NO: 6, the light chain CDR2 of SEQ ID NO: 7, and the light chain CDR3 of SEQ ID NO: 8 (which correspond to the six CDRs of antibody "2.8.14" as disclosed herein).

[0015] In one embodiment of the present invention, the antibody that specifically binds to minipig IgG specifically binds to the Fab domain of minipig IgG. In one embodiment, the antibody that specifically binds to the Fab domain of minipig IgG comprises the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12, and the heavy chain CDR3 of SEQ ID NO: 13, the light chain CDR1 of SEQ ID NO: 14, the light chain CDR2 of SEQ ID NO: 15, and the light chain CDR3 of SEQ ID NO: 16 (which correspond to the six CDRs of antibody "1.2.6" as disclosed herein). In one embodiment, the antibody that specifically binds to the Fab domain of minipig IgG comprises the heavy chain CDR1 of SEQ ID NO: 19, the heavy chain CDR2 of SEQ ID NO: 20, and the heavy chain CDR3 of SEQ ID NO: 21, the light chain CDR1 of SEQ ID NO: 22, the light chain CDR2 of SEQ ID NO: 23, and the light chain CDR3 of SEQ ID NO: 24 (which correspond to the six CDRs of antibody "2.3.8" as disclosed herein). In one embodiment, the antibody that specifically binds to the Fab domain of minipig IgG comprises the heavy chain CDR1 of SEQ ID NO:27, the heavy chain CDR2 of SEQ ID NO:28, and the heavy chain CDR3 of SEQ ID NO:29, the light chain CDR1 of SEQ ID NO:30, the light chain CDR2 of SEQ ID NO:31, and the light chain CDR3 of SEQ ID NO:32 (which correspond to the six CDRs of antibody "2.11.16" as disclosed herein).

[0016] In one embodiment of the invention the drug is a drug antibody (DA).

[0017] In one embodiment of the present invention, the method is a sandwich assay, wherein in step a), the drug / ADA complex is contacted with an antibody that specifically binds to human IgG, wherein the antibody that specifically binds to human IgG is immobilized on a support, thereby immobilizing the drug / ADA complex on the support, and wherein according to step b), the immobilized drug / ADA complex is subsequently contacted with an antibody that specifically binds to minipig IgG.

[0018] Another aspect of the present invention is an antibody that specifically binds to miniature pig IgG, said antibody comprising CDR1 of the heavy chain of SEQ ID NO:3, CDR2 of the heavy chain of SEQ ID NO:4, and CDR3 of the heavy chain of SEQ ID NO:5, CDR1 of the light chain of SEQ ID NO:6, CDR2 of the light chain of SEQ ID NO:7, and CDR3 of the light chain of SEQ ID NO:8 (which correspond to the six CDRs of antibody "2.8.14" as disclosed herein).

[0019] Another aspect of the present invention is an antibody that specifically binds to miniature pig IgG, said antibody comprising CDR1 of the heavy chain of SEQ ID NO: 11, CDR2 of the heavy chain of SEQ ID NO: 12, and CDR3 of the heavy chain of SEQ ID NO: 13, CDR1 of the light chain of SEQ ID NO: 14, CDR2 of the light chain of SEQ ID NO: 15, and CDR3 of the light chain of SEQ ID NO: 16 (which correspond to the six CDRs of antibody "1.2.6" as disclosed herein).

[0020] Another aspect of the present invention is an antibody that specifically binds to miniature pig IgG, said antibody comprising CDR1 of the heavy chain of SEQ ID NO: 19, CDR2 of the heavy chain of SEQ ID NO: 20, and CDR3 of the heavy chain of SEQ ID NO: 21, CDR1 of the light chain of SEQ ID NO: 22, CDR2 of the light chain of SEQ ID NO: 23, and CDR3 of the light chain of SEQ ID NO: 24 (which correspond to the six CDRs of antibody "2.3.8" as disclosed herein).

[0021] Another aspect of the present invention is an antibody that specifically binds to miniature pig IgG, said antibody comprising CDR1 of the heavy chain of SEQ ID NO:27, CDR2 of the heavy chain of SEQ ID NO:28, and CDR3 of the heavy chain of SEQ ID NO:29, CDR1 of the light chain of SEQ ID NO:30, CDR2 of the light chain of SEQ ID NO:31, and CDR3 of the light chain of SEQ ID NO:32 (which correspond to the six CDRs of antibody "2.11.16" as disclosed herein).

[0022] Another aspect of the invention is an antibody of the invention that specifically binds to minipig IgG for use in a method of determining the presence or absence of anti-drug antibodies (ADA) in a minipig sample.

[0023] The methods and antibodies of the present invention allow reliable detection of ADA and drug / ADA complexes in miniature pig samples (e.g., plasma, vitreous humor, and aqueous humor). The antibodies of the present invention show no relevant cross-reactivity with IgG from humans or mice. The antibodies of the present invention are particularly suitable for use in the methods of the present invention because they show a low signal-to-noise ratio. The methods of the present invention allow reliable detection in the presence of high drug doses (e.g., in vitreous humor samples). Therefore, these methods are, for example, useful for evaluating ADA formed in response to ophthalmic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Binding of monoclonal anti-minipig IgG 2.8.14 and commercially available HRP-conjugated polyclonal anti-pig IgG (pAb) and digoxigenated monoclonal anti-pig IgG (mAb) from Abd-Serotec to IgG from minipigs, humans, baboons, rhesus monkeys, cynomolgus monkeys, marmosets, dogs, mice, rats, hamsters, guinea pigs and rabbits (assessed as described in Example 3).

[0026] Figure 2 : Binding of monoclonal anti-minipig IgG 2.8.14 and 2.3.8 and commercially available polyclonal anti-pig IgG antibodies to different human IgG1 class drug antibodies (assessed as described in Example 4).

[0027] Figure 3 : Schematic diagram of an exemplary method for detecting drug / ADA complexes from miniature pig samples (IC assay without pre-incubation) [method of the present invention]

[0028] Figure 4 : Use of the anti-minipig IgG monoclonal antibody of the present invention versus the commercially available anti-pig IgG antibody in the method of the present invention (IC assay without pre-incubation with drug) for detecting drug / ADA complexes in minipig samples. For a description of the experiment, see Example 5.

[0029] Figure 5 : Schematic diagram of an example of a method for detecting ADA from miniature pig samples (IC assay with pre-incubation) [method of the present invention]

[0030] Figure 6 : Application of the anti-minipig IgG monoclonal antibody of the present invention versus commercially available anti-pig IgG antibody in the method of the present invention (IC assay with pre-incubation with drug) for detecting ADA in minipig samples. For a description of the experiment, see Example 6.

[0031] Figure 7 : Detection of ADA from minipig samples using the method of the present invention (IC assay with pre-incubation with drug) compared to detection by the bridging assay as described in Example 7.

[0032] Detailed description of the invention

[0033] 1. Definition

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires, singular terms should include plural terms and plural terms should include the singular. The disclosed methods and techniques are generally carried out according to conventional methods well known in the art. In general, the nomenclature used in connection with the present invention and the techniques of biochemistry, enzymology, molecule and cell biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known in the art and commonly used.

[0035] Unless otherwise indicated herein, the term "comprising of" shall include the term "consisting of."

[0036] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0037] The terms "anti-minipig IgG antibody" and "antibody that binds to minipig IgG" refer to an antibody that can bind to minipig IgG with sufficient affinity so that the antibody can be used as a diagnostic agent targeting minipig IgG. In certain embodiments, the antibody that binds to minipig IgG has an affinity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (Kd) of the

[0038] An antibody is said to "specifically bind" to its target when it has a Kd of 1 μM or less. Thus, an antibody that does not specifically bind to its target is understood herein to have a Kd of more than 1 μM.

[0039] As used herein, the term "drug" refers to a therapeutically active compound. As used herein, the term "drug antibody" refers to an antibody that can be administered to an individual to treat a disease, for example, a therapeutic monoclonal antibody. Drug antibodies (therapeutic monoclonal antibodies) are widely used to treat a variety of diseases, such as oncological diseases (for example, hematological tumors and solid malignancies, including non-Hodgkin's lymphoma, breast cancer and colorectal cancer), immunological diseases, central nervous system diseases, vascular diseases or infectious diseases. Such antibodies are described, for example, by Levene, AP et al., Journal of the Royal Society of Medicine 98 (2005) 145-152. Such antibodies are, for example, antibodies to CD20, CD22, HLA-DR, CD33, CD52, EGFR, G250, GD3, HER2, PSMA, CD56, VEGF, VEGF2, CEA, Lewis Y antigen, IL-6 receptor, or IGF-1 receptor. Therapeutic antibodies are also described by Groner, B. et al., Curr. Mol. Meth. 4 (2004) 539-547; Harris, M., Lancet Oncol. 5 (2004) 292-302.

[0040] As used herein, "anti-drug antibodies" or "ADA" refer to antibodies that specifically bind to a drug (e.g., a drug antibody). As an immunogenic response of a patient, ADA may be formed during antibody therapy (see Pan, Y. et al., FASEB J.9 (1995) 43-49). If the drug is a drug antibody, ADA may specifically bind to any region of the drug antibody, for example, a variable domain, a constant domain, or a sugar structure of the drug antibody. However, in many cases, ADA can bind to one or more complementary determining regions of a drug antibody.

[0041] As used herein, "variable domain" or "variable region" refers to each pair of light and heavy chains that are directly involved in the binding of an antibody to an antigen. The variable domain of a light chain is abbreviated as "VL" and the variable domain of a light chain is abbreviated as "VH". The variable domains of human light and human heavy chains have the same overall structure. Each variable domain contains four framework (FR) regions whose sequences are widely conserved. The FRs are connected by three "hypervariable regions" (or "complementarity determining regions", CDRs). The CDRs on each chain are separated by such framework amino acids. Thus, the light and heavy chains of an antibody comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminal to the C-terminal direction. The FRs adopt a β-sheet conformation and the CDRs can form loops connecting the β-sheet structure. The CDRs in each chain are fixed by the FRs in their three-dimensional structure and together with the CDRs from the other chain form an "antigen binding site". In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding. CDR and FR regions are determined according to the standard definitions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0042] For the purposes of this article, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus sequence framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0043] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) wherein such alterations result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alterations.

[0044] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the 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 multispecific antibodies formed from antibody fragments.

[0045] As used herein, "Fab fragment" refers to an antibody fragment that comprises a light chain fragment containing the VL domain and the constant domain of the light chain (CL) and the VH domain and the first constant domain (CH1) of the heavy chain.

[0046] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0047] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0048] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is typically composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR sequences and FR sequences typically appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0049] The terms "full length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0050] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops"), and / or contain antigen contact residues ("antigen contacts"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0051] (a) hypervariable loops occurring 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));

[0052] (b) CDRs occurring 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));

[0053] (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0054] (d) a combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0055] The complementarity determining regions (CDRs) referred to herein for anti-minipig IgG antibodies are determined according to the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-212 (1999)).

[0056] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing spaces as needed to achieve maximum percent sequence identity and for comparison purposes, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be accomplished in a variety of ways within the capabilities of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA packages. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full-length sequence being compared. However, for the purposes of this article, % amino acid sequence identity values ​​are generated using the FASTA package version 36.3.8c or later, the ggsearch program in conjunction with the BLOSUM50 comparison matrix. The FASTA package was originally developed by W. R. Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W. R. Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36 and is publicly available from fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml. Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare sequences using the ggsearch (global protein:protein) program and default options (BLOSUM50; opening penalty: -10; extension penalty: -2; Ktup=2) to ensure that a global rather than a local alignment is performed. The percent amino acid identity is given in the output alignment title.

[0057] In one embodiment, the antibody of the invention is a monoclonal antibody. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single amino acid composition.

[0058] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to an antibody produced by a human or human cell or is derived from a non-human source utilizing a human antibody library or other sequence encoding a human antibody. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0059] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one and generally two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of non-human antibodies and all or substantially all of the FR regions correspond to those of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0060] As used herein, the term "antibody showing no relevant cross-reactivity with IgG from human" or "no relevant cross-reactivity with IgG from mouse" or their equivalents means that the antibody does not specifically bind to human IgG or mouse IgG, respectively, i.e., shows a (Kd) of 10 -7 mol / L or greater dissociation constant.

[0061] The term "minipig sample" includes any amount of material isolated from a minipig. Such material includes, but is not limited to, whole blood, serum, or plasma from such an individual, which are the most widely used sample sources in preclinical routine work. Such material also includes vitreous fluid or aqueous humor.

[0062] The term "support" or "solid phase" as used herein in connection with the method of the present invention means a solid substance suitable for immobilizing IgG molecules thereon. The two terms may be used interchangeably. Supports include particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass and ceramics; gel materials such as silica, alumina and polymer gels; capillaries that may be composed of polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; cuvettes, tubes or other spectrometer sample containers. The support may be a fixed component, such as a tube, strip, cuvette or microtiter plate, or may be a non-fixed component, such as beads and microparticles.

[0063] Technicians are familiar with immunoassays. Methods for implementing such assays as well as practical applications and procedures are summarized in relevant teaching materials. Examples of relevant teaching materials are Tijssen, P., Preparation of enzyme-antibody or other enzyme-macromolecule conjugates, cited in: Practice and theory of enzyme immunoassays, Burdon, RH and v. Knippenberg, PH (eds.), Elsevier, Amsterdam (1990) pp. 221-278; and Colowick, SP and Caplan, NO (eds.), "Methods in Enzymology", Academic Press, volumes relating to immunoassays, especially volumes 70, 73, 74, 84, 92, and 121.

[0064] As used herein, the term "detectable marker" refers to a substance that is detectable by means known in the art. Detectable markers can be directly detectable, such as chromogens (fluorescent or luminescent groups and dyes), or indirectly detectable, such as enzymes or haptens, such as digoxigenin. In one embodiment, the detectable marker is a hapten, and detection is performed using an enzyme coupled to a hapten-specific antibody, and the binding of the hapten-specific antibody to the hapten is visualized by assessing the enzyme activity in a color change assay.

[0065] 2. Detailed description of embodiments of the present invention

[0066] In one aspect, the present invention provides a method for determining the presence or absence of anti-drug antibodies (ADA) formed in response to a drug applied to a miniature pig from a sample isolated from a miniature pig. The method is an immunoassay that allows the detection of immune complexes ("drug / ADA complexes") containing a drug and ADA formed when the drug is contacted with a miniature pig sample. The drug / ADA complex is bound by an antibody that specifically binds to miniature pig IgG ("anti-miniature pig IgG antibody"), and in turn the binding of the anti-miniature pig IgG antibody to the drug / ADA complex is detected and the binding indicates the presence of ADA in the sample. This method allows the detection of drug / ADA complexes already present in the miniature pig sample and uncomplexed ADA from the initial sample.

[0067] Thus, in one aspect, the present invention relates to an in vitro method for determining the presence or absence of anti-drug antibodies (ADA) in a miniature pig sample, the method comprising:

[0068] a) contacting the sample with an excess of a drug, wherein the ADA binds to the drug to form a drug / ADA complex,

[0069] b) contacting the drug / ADA complex with an antibody that specifically binds to minipig IgG, wherein the antibody that specifically binds to minipig IgG does not bind to human IgG, and

[0070] c) Determine the binding of antibodies that specifically bind to minipig IgG to the drug / ADA complex to determine whether ADA is present in the sample.

[0071] In one embodiment, in step a), the drug is immobilized on a support.

[0072] In another aspect, the present invention provides a method for determining the presence or absence of a drug / ADA complex in a miniature pig sample. The method is an immunoassay that allows for direct detection of a drug / ADA complex from a miniature pig sample, excluding the step of pre-incubating the miniature pig sample with the drug. The drug / ADA complex from the sample is bound by an antibody that specifically binds to the drug, and in turn the binding of the drug / ADA complex to the antibody that specifically binds to the drug is detected and the binding indicates the presence of the drug / ADA complex in the sample.

[0073] Therefore, in this aspect, the present invention relates to an in vitro method for determining the presence or absence of an immune complex comprising a drug and an antibody that specifically binds to the drug (drug / ADA complex) in a miniature pig sample, the method comprising:

[0074] a) contacting the minipig sample with an immobilized antibody that specifically binds to the drug to immobilize the drug / ADA complex present in the minipig sample,

[0075] b) contacting the drug / ADA complex with an antibody that specifically binds to minipig IgG, wherein the antibody that specifically binds to minipig IgG does not bind to human IgG, and

[0076] c) Determine the binding of antibodies that specifically bind to minipig IgG to the drug / ADA complex to determine whether ADA is present in the sample.

[0077] The two methods of the present invention are carried out in the order shown, i.e. step a) is followed by step b), which in turn is followed by step c). The following embodiments apply to the two methods of the present invention, i.e. the method for detecting the presence or absence of ADA in a miniature pig sample and the method for detecting the presence or absence of a drug / ADA complex in a miniature pig sample:

[0078] In one embodiment of the method of the present invention, the antibody that specifically binds to minipig IgG does not specifically bind to human IgG. In one embodiment of the method of the present invention, the antibody that specifically binds to minipig IgG does not specifically bind to mouse IgG. In one embodiment of the method of the present invention, the antibody that specifically binds to minipig IgG does not specifically bind to human IgG and mouse IgG.

[0079] Thus, the sensitivity and reliability of the assay are improved, since background variations due to, for example, drug antibodies binding to human or mouse species can be minimized or even eliminated.

[0080] In one embodiment of the method of the present invention, the antibody that specifically binds to minipig IgG specifically binds to the Fc domain of minipig IgG.

[0081] In one embodiment of the method of the present invention, the antibody that specifically binds to minipig IgG specifically binds to the Fab domain of minipig IgG.

[0082] In one embodiment of the method of the present invention, at least one antibody that specifically binds to the Fab domain of minipig IgG and at least one antibody that specifically binds to the Fc domain of minipig IgG are used in step b).

[0083] In one embodiment of the method of the present invention, the drug used in step a) is immobilized on a support.

[0084] In one embodiment of the method of the invention, the drug is a drug antibody (DA). In one embodiment, the drug antibody is a human antibody or a humanized antibody. In one embodiment, the drug antibody is an IgG antibody. In one embodiment, the drug antibody is a human or humanized IgG antibody. In one embodiment, the drug antibody is a Fab fragment.

[0085] In one embodiment of the method of the present invention, wherein the drug antibody is a Fab fragment, step b) is performed using an antibody that specifically binds to the Fab domain of minipig IgG.

[0086] In one embodiment of the method of the present invention, the method is a sandwich assay, wherein the drug / ADA complex is contacted with an antibody that specifically binds to human IgG, wherein the antibody that specifically binds to human IgG is immobilized on a support, thereby immobilizing the drug / ADA complex on the support, and wherein according to step b) of the method of the present invention, the immobilized drug / ADA complex is subsequently contacted with an antibody that specifically binds to minipig IgG.

[0087] In one embodiment of the method of the invention, the antibody that specifically binds to minipig IgG is conjugated to a detectable marker. In one embodiment, the detectable marker is a hapten. In one embodiment, the detectable marker is digoxigenin.

[0088] The present invention also relates to antibodies that specifically bind to minipig IgG. The antibodies of the present invention specifically bind to the Fc or Fab portion of minipig IgG and show no relevant cross-reactivity with IgG from humans and mice. The antibodies of the present invention are particularly useful in the methods of the present invention for determining the presence or absence of anti-drug antibodies (ADA) in minipig samples.

[0089] In one aspect, the present invention relates to an antibody that specifically binds to minipig IgG, the antibody comprising the CDR1 of the heavy chain of SEQ ID NO: 3, the CDR2 of the heavy chain of SEQ ID NO: 4, and the CDR3 of the heavy chain of SEQ ID NO: 5, the CDR1 of the light chain of SEQ ID NO: 6, the CDR2 of the light chain of SEQ ID NO: 7, and the CDR3 of the light chain of SEQ ID NO: 8. In one embodiment, the antibody comprises a VH sequence and a VL sequence, the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 1 and the VL sequence of SEQ ID NO: 2. The CDRs and the VH and VL amino acid sequences correspond to the CDRs and variable domains of the antibody "2.8.14" as disclosed herein, and the antibody specifically binds to the Fc domain of minipig IgG.

[0090] In one aspect, the present invention relates to an antibody that specifically binds to minipig IgG, the antibody comprising the CDR1 of the heavy chain of SEQ ID NO: 11, the CDR2 of the heavy chain of SEQ ID NO: 12, and the CDR3 of the heavy chain of SEQ ID NO: 13, the CDR1 of the light chain of SEQ ID NO: 14, the CDR2 of the light chain of SEQ ID NO: 15, and the CDR3 of the light chain of SEQ ID NO: 16. In one embodiment, the antibody comprises a VH sequence and a VL sequence, the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 9, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 9 and the VL sequence of SEQ ID NO: 10. The CDRs and the VH and VL amino acid sequences correspond to the CDRs and variable domains of the antibody "1.2.6" as disclosed herein, and the antibody specifically binds to the Fab domain of minipig IgG.

[0091] In one aspect, the present invention relates to an antibody that specifically binds to minipig IgG, the antibody comprising the CDR1 of the heavy chain of SEQ ID NO: 19, the CDR2 of the heavy chain of SEQ ID NO: 20, and the CDR3 of the heavy chain of SEQ ID NO: 21, the CDR1 of the light chain of SEQ ID NO: 22, the CDR2 of the light chain of SEQ ID NO: 23, and the CDR3 of the light chain of SEQ ID NO: 24. In one embodiment, the antibody comprises a VH sequence and a VL sequence, the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 17, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 18. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 17 and the VL sequence of SEQ ID NO: 18. The CDRs and the VH and VL amino acid sequences correspond to the CDRs and variable domains of the antibody "2.3.8" as disclosed herein, and the antibody specifically binds to the Fab domain of minipig IgG.

[0092] In one aspect, the present invention relates to an antibody that specifically binds to minipig IgG, the antibody comprising the CDR1 of the heavy chain of SEQ ID NO: 27, the CDR2 of the heavy chain of SEQ ID NO: 28, and the CDR3 of the heavy chain of SEQ ID NO: 29, the CDR1 of the light chain of SEQ ID NO: 30, the CDR2 of the light chain of SEQ ID NO: 31, and the CDR3 of the light chain of SEQ ID NO: 32. In one embodiment, the antibody comprises a VH sequence and a VL sequence, the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 25, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 26. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 25 and the VL sequence of SEQ ID NO: 26. The CDRs and the VH and VL amino acid sequences correspond to the CDRs and variable domains of the antibody "2.11.16" as disclosed herein, and the antibody specifically binds to the Fab domain of minipig IgG.

[0093] Another aspect of the invention is the use of an antibody of the invention in a method for determining the presence or absence of anti-drug antibodies (ADA) in a minipig sample. In one embodiment, an antibody of the invention is used in a method of the invention.

[0094] 3. Specific embodiments of the present invention

[0095] The following specific embodiments of the present invention are listed below.

[0096] 1. An in vitro method for determining the presence or absence of anti-drug antibodies (ADA) in a miniature pig sample, the method comprising:

[0097] a) contacting the sample with an excess of a drug, wherein the ADA binds to the drug to form a drug / ADA complex,

[0098] b) contacting the drug / ADA complex with an antibody that specifically binds to minipig IgG, wherein the antibody that specifically binds to minipig IgG does not bind to human IgG, and

[0099] c) Determine the binding of antibodies that specifically bind to minipig IgG to the drug / ADA complex to determine whether ADA is present in the sample.

[0100] 2. The method of embodiment 1, wherein in step a), the drug is immobilized on a support.

[0101] 3. An in vitro method for determining the presence or absence of an immune complex comprising a drug and an antibody that specifically binds to the drug (drug / ADA complex) in a miniature pig sample, the method comprising:

[0102] a) contacting the minipig sample with an immobilized antibody that specifically binds to the drug to immobilize the drug / ADA complex present in the minipig sample,

[0103] b) contacting the drug / ADA complex with an antibody that specifically binds to minipig IgG, wherein the antibody that specifically binds to minipig IgG does not bind to human IgG, and

[0104] c) Determine the binding of antibodies that specifically bind to minipig IgG to the drug / ADA complex to determine whether ADA is present in the sample.

[0105] 4. The method of any one of embodiments 1 to 3, wherein the antibody that specifically binds to minipig IgG does not specifically bind to human IgG.

[0106] 5. The method of any one of embodiments 1 to 4, wherein the antibody that specifically binds to minipig IgG does not specifically bind to mouse IgG.

[0107] 6. The method of any one of embodiments 1 to 5, wherein the antibody that specifically binds to minipig IgG specifically binds to the Fc domain of minipig IgG.

[0108] 7. The method of embodiment 6, wherein the antibody that specifically binds to the Fc domain of miniature pig IgG comprises the CDR1 of the heavy chain of SEQ ID NO:3, the CDR2 of the heavy chain of SEQ ID NO:4, and the CDR3 of the heavy chain of SEQ ID NO:5, the CDR1 of the light chain of SEQ ID NO:6, the CDR2 of the light chain of SEQ ID NO:7, and the CDR3 of the light chain of SEQ ID NO:8 (which correspond to the six CDRs of antibody "2.8.14" as disclosed herein).

[0109] 8. The method of embodiment 6 or 7, wherein the antibody that specifically binds to the Fc domain of minipig IgG comprises a VH sequence of SEQ ID NO: 1 and a VL sequence of SEQ ID NO: 2 (which correspond to the variable domains of antibody "2.8.14" as disclosed herein).

[0110] 9. The method of any one of embodiments 1 to 5, wherein the antibody that specifically binds to minipig IgG specifically binds to the Fab domain of minipig IgG.

[0111] 10. The method of embodiment 9, wherein the antibody that specifically binds to the Fab domain of miniature pig IgG comprises the CDR1 of the heavy chain of SEQ ID NO: 11, the CDR2 of the heavy chain of SEQ ID NO: 12, and the CDR3 of the heavy chain of SEQ ID NO: 13, the CDR1 of the light chain of SEQ ID NO: 14, the CDR2 of the light chain of SEQ ID NO: 15, and the CDR3 of the light chain of SEQ ID NO: 16 (which correspond to the six CDRs of antibody "1.2.6" as disclosed herein).

[0112] 11. The method of embodiment 9 or 10, wherein the antibody that specifically binds to the Fab domain of minipig IgG comprises a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 10 (which correspond to the variable domains of antibody "1.2.6" as disclosed herein).

[0113] 12. The method of embodiment 9, wherein the antibody that specifically binds to the Fab domain of miniature pig IgG comprises the CDR1 of the heavy chain of SEQ ID NO: 19, the CDR2 of the heavy chain of SEQ ID NO: 20, and the CDR3 of the heavy chain of SEQ ID NO: 21, the CDR1 of the light chain of SEQ ID NO: 22, the CDR2 of the light chain of SEQ ID NO: 23, and the CDR3 of the light chain of SEQ ID NO: 24 (which correspond to the six CDRs of antibody "2.3.8" as disclosed herein).

[0114] 13. The method of embodiment 9 or 12, wherein the antibody that specifically binds to the Fab domain of minipig IgG comprises a VH sequence of SEQ ID NO: 17 and a VL sequence of SEQ ID NO: 18 (which correspond to the variable domains of antibody "2.3.8" as disclosed herein).

[0115] 14. The method of embodiment 9, wherein the antibody that specifically binds to the Fab domain of miniature pig IgG comprises the CDR1 of the heavy chain of SEQ ID NO:27, the CDR2 of the heavy chain of SEQ ID NO:28, and the CDR3 of the heavy chain of SEQ ID NO:29, the CDR1 of the light chain of SEQ ID NO:30, the CDR2 of the light chain of SEQ ID NO:31, and the CDR3 of the light chain of SEQ ID NO:32 (which correspond to the six CDRs of antibody “2.11.16” as disclosed herein).

[0116] 15. The method of embodiment 9 or 14, wherein the antibody that specifically binds to the Fab domain of minipig IgG comprises a VH sequence of SEQ ID NO: 25 and a VL sequence of SEQ ID NO: 26 (which correspond to the variable domains of antibody "2.11.16" as disclosed herein).

[0117] 16. The method according to any of the preceding embodiments, wherein at least one antibody that specifically binds to the Fab domain of minipig IgG and at least one antibody that specifically binds to the Fc domain of minipig IgG are used in step b).

[0118] 17. The method according to any one of embodiments 1 to 16, wherein the drug is a drug antibody (DA).

[0119] 18. The method of embodiment 17, wherein the drug antibody is a human antibody or a humanized antibody.

[0120] 19. The method of embodiment 17 or 18, wherein the drug antibody is an IgG antibody.

[0121] 20. The method of any one of embodiments 17 to 19, wherein the drug antibody is a Fab fragment.

[0122] 21. The method of embodiment 20, wherein an antibody that specifically binds to the Fab domain of minipig IgG is used in step b).

[0123] 22. The method of embodiment 21, wherein the antibody that specifically binds to the Fab domain of minipig IgG is selected from the antibodies defined in embodiments 7 to 13.

[0124] 23. The method of any one of embodiments 17 to 22, wherein the method is a sandwich assay, wherein in step a), the drug / ADA complex is contacted with an antibody that specifically binds to human IgG, wherein the antibody that specifically binds to human IgG is immobilized on a support, thereby immobilizing the drug / ADA complex on the support, and

[0125] Wherein according to step b) the immobilized drug / ADA complex is subsequently contacted with an antibody which specifically binds to minipig IgG.

[0126] 24. The method of any one of embodiments 1 to 23, wherein the antibody that specifically binds to minipig IgG is conjugated to a detectable label.

[0127] 25. The method of embodiment 24, wherein the detectable label is digoxigenin.

[0128] 26. An antibody that specifically binds to miniature pig IgG, the antibody comprising the heavy chain CDR1 of SEQ ID NO:3, the heavy chain CDR2 of SEQ ID NO:4, and the heavy chain CDR3 of SEQ ID NO:5, the light chain CDR1 of SEQ ID NO:6, the light chain CDR2 of SEQ ID NO:7, and the light chain CDR3 of SEQ ID NO:8 (which correspond to the six CDRs of antibody "2.8.14" as disclosed herein).

[0129] 27. An antibody according to embodiment 26, comprising a VH sequence and a VL sequence, wherein the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0130] 28. The antibody according to embodiment 26 or 27, comprising the VH sequence of SEQ ID NO: 1 and the VL sequence of SEQ ID NO: 2 (which correspond to the variable domains of antibody "2.8.14" as disclosed herein).

[0131] 29. An antibody that specifically binds to miniature pig IgG, the antibody comprising the CDR1 of the heavy chain of SEQ ID NO: 11, the CDR2 of the heavy chain of SEQ ID NO: 12, and the CDR3 of the heavy chain of SEQ ID NO: 13, the CDR1 of the light chain of SEQ ID NO: 14, the CDR2 of the light chain of SEQ ID NO: 15, and the CDR3 of the light chain of SEQ ID NO: 16 (which correspond to the six CDRs of antibody "1.2.6" as disclosed herein).

[0132] 30. The antibody of embodiment 29, comprising a VH sequence and a VL sequence, wherein the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:9, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0133] 31. The antibody of embodiment 29, comprising the VH sequence of SEQ ID NO: 9 and the VL sequence of SEQ ID NO: 10 (which correspond to the variable domains of antibody "1.2.6" as disclosed herein).

[0134] 32. An antibody that specifically binds to miniature pig IgG, the antibody comprising the heavy chain CDR1 of SEQ ID NO: 19, the heavy chain CDR2 of SEQ ID NO: 20, and the heavy chain CDR3 of SEQ ID NO: 21, the light chain CDR1 of SEQ ID NO: 22, the light chain CDR2 of SEQ ID NO: 23, and the light chain CDR3 of SEQ ID NO: 24 (which correspond to the six CDRs of antibody "2.3.8" as disclosed herein).

[0135] 33. The antibody of embodiment 32, comprising a VH sequence and a VL sequence, wherein the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:17, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:18.

[0136] 34. The antibody of embodiment 32, comprising the VH sequence of SEQ ID NO: 17 and the VL sequence of SEQ ID NO: 18 (which correspond to the variable domains of antibody "2.3.8" as disclosed herein).

[0137] 35. An antibody that specifically binds to miniature pig IgG, the antibody comprising the heavy chain CDR1 of SEQ ID NO:27, the heavy chain CDR2 of SEQ ID NO:28, and the heavy chain CDR3 of SEQ ID NO:29, the light chain CDR1 of SEQ ID NO:30, the light chain CDR2 of SEQ ID NO:31, and the light chain CDR3 of SEQ ID NO:32 (which correspond to the six CDRs of antibody "2.11.16" as disclosed herein).

[0138] 36. The antibody of embodiment 35, comprising a VH sequence and a VL sequence, wherein the VH sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:25, and the VL sequence has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:26.

[0139] 37. The antibody of embodiment 35, comprising the VH sequence of SEQ ID NO: 25 and the VL sequence of SEQ ID NO: 26 (which correspond to the variable domains of antibody "2.11.16" as disclosed herein).

[0140] 38. The antibody of any one of embodiments 26 to 37, for use in a method for determining the presence or absence of anti-drug antibodies (ADA) in a miniature pig sample.

[0141] 39. The antibody according to one of embodiments 26 to 37, for use in a method according to one of embodiments 1 to 25.

[0142] Description of amino acid sequence

[0143]

[0144] Example

[0145] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.It is understood that modifications can be made to the procedures described without departing from the spirit of the present invention.

[0146] Embodiment 1:

[0147] Production of IgG in miniature pigs

[0148] Serum from miniature pigs The mixture was degreased at 380 °C and precipitated with ammonium sulfate (to 2.0 M). The precipitate was homogenized in phosphate buffer and dialyzed against phosphate buffer, pH 7.0. The mixture was separated by DEAE ion exchange chromatography at pH 7.0 and the IgG in the flow-through was concentrated and purified by gel filtration.

[0149] Embodiment 2:

[0150] Generation of monoclonal anti-minipig IgG antibodies

[0151] Anti-minipig antibodies were generated by immunization of mice with minipig IgG derived from minipig serum and subsequent hybridoma selection. Appropriate clones were screened in the presence of human IgG to select the most potent antibodies for the intended application of detecting ADA against human or humanized IgG drug antibodies.

[0152] a) Immunization of mice

[0153] Female BALB / c or NMRI mice were immunized intraperitoneally for the first time with 90 μg of minipig IgG mixed with CFA (Freund's complete adjuvant). Six weeks and 10 weeks after the first immunization, 90 μg of minipig IgG prepared with IFA (Freund's incomplete adjuvant) were then further immunized intraperitoneally twice. Subsequently, three days before and two days before the animals were killed, 50 μg of minipig IgG was used for an intraperitoneal booster immunization and 25 μg of minipig IgG prepared in PBS buffer were used for an intravenous booster immunization, and spleen cells were used to produce hybridomas as described below.

[0154] b) Fusion and cloning

[0155] Splenocytes from mice immunized according to a) were fused with myeloma cells using standard procedures using polyethylene glycol. Briefly, approximately 1 x 10 8 splenocytes and approximately 2 x 10 7 Individual myeloma cells (P3x63-Ag8.653) are mixed in RPMI-1640 and centrifuged (10 minutes, 250x g). Wash cells once with RPMI-1640 and centrifuge again. After this, 1ml PEG (polyethylene glycol) is added dropwise to the cell pellet. After incubation at 37 ° C for 1 minute in a water bath, 5ml RPMI-1640 is added dropwise to the cells. Subsequently, cell suspension is filled with RPMI-1640 until 30ml. After centrifugation (10 minutes, 180x g), cells are resuspended in selection medium (RPMI-1640, supplemented with 10% FCS, 100U / ml IL-6, 2mM L-glutamine, 100 μM NEAA, 1mM sodium pyruvate, 24 μM 2-mercaptoethanol, 1x azaserine / hypoxanthine (Sigma-Aldrich)). After 24 hours of culture at 37°C, the cells were plated into 96-well cell culture plates. After 10 days of culture, the production of miniature pig IgG-specific antibodies in the primary culture was analyzed as described below. The selected primary culture was cloned by single cell sorting using a flow cytometer (FACSAria, BD Biosciences). Cell clones were cultivated in RPMI-1640 supplemented with 10% FCS, 50U / ml IL-6, 2mM L-glutamine, 100μM NEAA, 1mM sodium pyruvate, 24μM 2-mercaptoethanol. The specificity of the established monoclonal hybridoma cell line was repeated as described below.

[0156] c) Selecting hybridomas expressing anti-minipig antibodies with no relevant cross-reactivity with human IgG

[0157] Initial screening

[0158] To identify hybridomas secreting anti-minipig antibodies, streptavidin-precoated microtiter plates (MTPs) (MicroCoat, Bernried, Germany) were coated with 100 μl of biotinylated minipig IgG (250 ng / ml in PBS / 1.0% (w / v) BSA) per well at room temperature (RT) for 1 h. Subsequently, the plates were washed with a wash buffer (0.9% (w / v) NaCl / 0.05% 20) Wash each well 3 times. Thereafter, add 75 μL of hybridoma cell culture supernatant to each well and incubate at room temperature for 1 hour. After washing, add 100 μL of horseradish peroxidase-labeled F(ab')2 fragment of polyclonal sheep anti-mouse Fc antibody to detect bound antibody and incubate at room temperature for 1 hour. After washing, add 100 μL of (Roche, Germany) and the extinction (OD) was measured in a microtiter plate reader at 405 nm and 492 nm 405 / 492. This screening resulted in the selection of antibodies that bind to minipig IgG.

[0159] Second screening

[0160] To identify those anti-minipig antibodies that showed minimal cross-reactivity to human IgG, the following competition assay was performed. Recombinant streptavidin-precoated MTPs (MicroCoat) were coated with 100 μl per well of biotinylated minipig IgG (250 ng / ml in PBS / 1.0% BSA) at room temperature as described above. Subsequently, 0.9% NaCl / 0.05% The coated plates were washed. In the next step, a mixture of 37.5 μl of anti-minipig antibody (hybridoma cell culture supernatant) and 37.5 μl of polyclonal human IgG (final concentration 40 mg / ml) was added to each well. In a control experiment, a mixture of 37.5 μL of the corresponding anti-minipig antibody (hybridoma cell culture supernatant) and 37.5 μl of buffer (PBS / 1.0% BSA) was added to each well. The plates were incubated at room temperature for 1 hour. The plates were washed with 0.9% NaCl / 0.05% After washing, add 100 μl / well of horseradish peroxidase-labeled F(ab')2 fragment (100 ng / ml) of polyclonal sheep anti-mouse Fc antibody to detect bound sample antibodies. After incubation at room temperature for 1 hour, wash the plate as described above. Finally, add 100 μl / well (Roche Diagnostics GmbH) and the extinction (OD) was measured at 405 nm and 492 nm [405 / 492]. The anti-minipig IgG antibodies that showed the least signal decrease in the presence of polyclonal human IgG (compared to buffer control) showed the lowest cross-reactivity.

[0161] This screening approach resulted in the selection of antibodies that bound well to minipig IgG and showed no relevant cross-reactivity to human IgG.

[0162] d) Identification of the binding regions of generated anti-minipig antibodies

[0163] To identify the binding region of anti-minipig antibodies, recombinant streptavidin-precoated MTPs (MicroCoat) were coated with 100 μl of biotinylated minipig IgG (250 ng / ml in PBS / 1.0% BSA), 100 μl of biotinylated minipig Fab fragment (250 ng / ml in PBS / 1.0% BSA) or 100 μl of biotinylated minipig IgG Fc fragment (250 ng / ml in PBS / 1.0% BSA) per well. After washing the coated MTPs, 100 μL of anti-minipig antibody (hybridoma cell culture supernatant; 1:2 diluted in PBS / 1.0% BSA) was added to each well. The plate was incubated at room temperature for 1 hour. Subsequently, 0.9% NaCl / 0.05% Wash the coated plates with 0.9% NaCl / 0.05% After washing, 100 μl / well of horseradish peroxidase-labeled F(ab')2 fragment of polyclonal sheep anti-mouse Fcγ antibody (100 ng / ml) was added to detect bound antibody. After incubation at room temperature for 1 hour, the plate was washed as described above. Finally, 100 μl / well of (Roche Diagnostics GmbH) and the extinction (OD) was measured at 405 nm and 492 nm [405 / 492].

[0164] To confirm the results of the ELISA described above, a competition ELISA has been performed. Briefly, recombinant streptavidin pre-coated MTP (MicroCoat) was coated with 100 μl per well of biotinylated minipig IgG (250 ng / ml in PBS / 1.0% BSA), 100 μl per well of biotinylated minipig Fab fragment (250 ng / ml in PBS / 1.0% BSA), or 100 μl per well of biotinylated minipig IgG Fc fragment (250 ng / ml in PBS / 1.0% BSA). Subsequently, the cells were incubated with 0.9% NaCl / 0.05% The coated plates were washed. In the next step, a mixture of 50 μl of anti-minipig antibody (hybridoma cell culture supernatant) and 50 μl of polyclonal minipig Fab fragment (final concentration 5 μg / ml in PBS / 1.0% BSA, pre-incubated at room temperature for 30 minutes) was added to each well. In a control experiment, a mixture of 50 μL of the corresponding anti-minipig antibody (hybridoma cell culture supernatant) and 50 μl of buffer (PBS / 1.0% BSA) was added to each well. The plates were incubated at room temperature for 1 hour. The plates were washed with 0.9% NaCl / 0.05% After washing, add 100 μl / well of horseradish peroxidase-labeled F(ab')2 fragment (100 ng / ml) of polyclonal sheep anti-mouse Fcγ antibody to detect bound sample antibodies. After incubation at room temperature for 1 hour, the plate was washed as described above. Finally, add 100 μl / well (Roche Diagnostics GmbH) and the extinction (OD) was measured at 405 nm and 492 nm [405 / 492].

[0165] This ELISA screening method resulted in the identification of antibodies that bind to either the minipig Fab fragment or the Fc fragment.

[0166] The following clones (Table 1) were selected as suitable candidates.

[0167] Table 1: Anti-Miniature Pig IgG clones

[0168] clone IgG class and subclass Immunogen Responsiveness 2.8.14 IgG1, kappa Minipig IgG Minipig IgG, Fc fragment 1.2.6 IgG1, kappa Minipig IgG Minipig IgG, Fab fragment 2.3.8 IgG1, kappa Minipig IgG Minipig IgG, Fab fragment 2.11.16 IgG1, kappa Minipig IgG Minipig IgG, Fab fragment

[0169] e) Production of immunoglobulins

[0170] The generated monoclonal hybridoma cell lines were cultured at an initial cell density of 1.0 x 10 5 Up to 2.2x 10 5 Cells / mL were inoculated in hybridoma-SFM medium (Gibco) supplemented with 5% FCS, 50 U / ml IL-6, 50 μM β-mercaptoethanol and 20 μM ethanolamine. The cells were expanded in a spinner bottle for 7 to 14 days. Subsequently, the cell culture supernatant was harvested and antibodies were purified from the supernatant according to standard protein chemistry methods (see, e.g., Bruck, C. et al., Methods Enzymol. 121 (1986) 587-596).

[0171] Embodiment 3:

[0172] Cross-reactivity of generated monoclonal anti-minipig IgG antibodies with IgG from other species

[0173] The cross-reactivity of anti-minipig IgG antibody 2.8.14 generated as in Example 2 was evaluated and compared to two commercially available anti-minipig IgG antibodies: HRP-conjugated goat polyclonal anti-pig IgG antibody (AbD-Serotec, catalog number AAI41P) and in-house digoxigenylated mouse monoclonal anti-pig IgG antibody (AbD-Serotec, clone K139 3C8, catalog number MCA635GA). For the sake of clarity, it should be noted that since minipigs are pigs, the anti-pig antibodies are of course reactive with minipig antigens, i.e., the anti-pig IgG antibodies specifically bind to minipig-IgG. The following antibodies were compared:

[0174] - Digitized anti-minipig IgG antibody "2.8.14"

[0175] -HRP-conjugated polyclonal anti-porcine IgG from Abd-Serotec as indicated above under "pAb"

[0176] - Digitized monoclonal anti-porcine IgG "mAb"

[0177] Cross-reactivity was assessed in an immunoassay. Briefly, sera or plasma from 12 different species were bound to NUNC Maxisorp plates as dilutions of 5% matrix concentrate in alkaline NaHCO3 buffer. Two human samples and two miniature pig samples were used in addition to samples from baboons, rhesus monkeys, cynomolgus monkeys, marmosets, dogs, mice, rats, hamsters, guinea pigs and rabbits. Plates were incubated at 2 μg / ml with the three tested anti-miniature pig IgG antibodies, either digitoxinylated or conjugated to horseradish peroxidase (HRP) as indicated. Incubation with digitoxinylated anti-miniature pig IgG was followed by incubation with a polyclonal anti-digoxigenin Fab fragment conjugated to HRP (Roche Diagnostics, catalog number 11633716001). Use The substrate was added and the signal was detected at 405 nm (reference wavelength: 490 nm ([405 / 490] nm)) using a standard ELISA reader and detected by color change evaluation.

[0178] Figure 1 The results are shown in . The commercially available polyclonal antibodies tested significantly cross-reacted with IgG from a wide range of species. In this assay, the commercially available monoclonal antibodies tested did not show significant binding to any of the species tested, including miniature pigs.

[0179] In this assay, monoclonal antibody 2.8.14 of the invention showed no cross-reactivity against IgG from any of the species tested except minipigs.

[0180] Embodiment 4:

[0181] Cross-reactivity of generated monoclonal anti-minipig IgG antibodies with human IgG drug antibodies

[0182] The cross-reactivity of anti-minipig IgG antibodies 2.8.14 and 2.3.8 generated as in Example 2 with several human IgG drug antibodies was evaluated in an immunoassay and compared to a commercially available polyclonal anti-minipig IgG antibody ("pAb", as described in Example 3).

[0183] The assay was performed using a total of 13 different human IgG isotype drug antibodies, either full-length IgG with wild-type antibody domain arrangement (shown as "h-IgG"), full-length IgG CrossMabs (containing domain swaps as described in Klein et al. [MAbs. 2012 Nov-Dec; 4(6): 653-63]; shown as "XMab"), or Fab fragments (shown as "h-Fab"). Minipig IgG (an in-house preparation from minipig serum) was used as a positive control. For drugs 3, 5, 7, and 8, which were full-length IgG, the assay was performed using a Fab fragment (shown as "h-Fab"), which was ... Figure 2 Fragments of the indicated antibodies.

[0184] To detect cross-reactivity to anti-drug antibodies, positive controls and drug antibodies were bound to Maxisorp plates and detected using digoxigenin-ylated monoclonal antibodies 2.8.14 and 2.3.8, followed by incubation with HRP-conjugated anti-digoxigenin antibody and HRP-conjugated polyclonal anti-porcine IgG ("pAb", as described in Example 3) from Abd-Serotec. The substrate was added and the signal was detected at 405 nm (reference wavelength: 490 nm ([405 / 490] nm)) using a standard ELISA reader and detected by color change evaluation.

[0185] Figure 2 The results are shown in Figure 2. The monoclonal antibodies 2.8.14 and 2.3.8 of the present invention did not show cross-reactivity with the tested drug antibodies or their fragments. Under the conditions tested, the commercially available polyclonal anti-porcine antibodies tested significantly cross-reacted with all full-length IgG drug antibodies and their Fc fragments.

[0186] Embodiment 5:

[0187] Suitability of generated anti-minipig IgG monoclonal antibodies versus commercially available anti-pig IgG antibodies in the method of the present invention (without pre-incubation with drug) for detecting drug / ADA complexes in minipig samples

[0188] In order to evaluate the suitability of different anti-minipig IgG antibodies in the method of the present invention for detecting drug / ADA complexes formed in vivo from minipig samples, several commercially available antibodies were compared with the antibodies of the present invention. The following antibodies were used:

[0189] - Monoclonal anti-swine antibody MAb <sw-igg>M-F007-1241-IgG-Dig (BD Pharmingen, catalog number 552554) ("mAb1")

[0190] - Monoclonal anti-swine antibody MAb <sw-igg>M-K139-3E1-IgG (AbD Serotec, catalog number MCA633GA) ("mAb2")

[0191] - Monoclonal anti-swine antibody MAb <sw-igg>M-K139-3C8-IgG (AbD Serotec, catalog number MCA633GA) ("mAb3")

[0192] - Polyclonal anti-porcine antibody PAb <porcine-IgG-Fc> rabbit-IgG-HRP (Rockland, catalog number 614-4303) ("pAb1")

[0193] - Polyclonal anti-porcine antibody PAb <porcine-IgG-Fc> goat-IgG-HRP (Bethyl (Biomol), catalog number A100-104P) ("pAb2")

[0194] - Polyclonal anti-porcine antibody PAb <porcine-IgG-Fc> goat-IgG-HRP, (AbD-Serotec, catalog number AAI41P) ("pAb3")

[0195] - Monoclonal anti-Miniature Pig IgG antibody 2.8.14

[0196] - Monoclonal anti-minipig IgG antibody 2.3.8

[0197] - Monoclonal anti-minipig IgG antibody 2.11.16

[0198] To detect drug / ADA complexes formed in vivo from minipig samples, the following assay (of the present invention) was used:

[0199] In the first step, biotinylated anti-human IgG-Fab (κ) monoclonal antibody MAb<h-IgG-κ> M-1.7.10-Fab' (Roche, as disclosed in WO 2012 / 022682) was bound to each well of a streptavidin-coated microtiter plate (SA-MTP). Excess unbound Fab'-fragments were removed by washing.

[0200] In order to simulate the drug / ADA complex, a human IgG / pig IgG conjugate was prepared. Therefore, polyclonal human IgG (purified from human serum) was chemically conjugated to polyclonal pig IgG (purified from pig serum). The human IgG / pig IgG conjugate thus derived was spiked into 2% of minipig pooled plasma at low, medium and high concentrations and used as a positive control (wherein "LPC" refers to a low concentration positive control, "MPC" refers to a medium concentration positive control and "HPC" refers to a high concentration positive control). Minipig pooled plasma (not spiked with human IgG / pig IgG conjugate) was used as a negative control.

[0201] Therefore, controls were added to each well and incubated therein. After washing away unbound material, human IgG / porcine IgG conjugates (mimicking drig / ADA complexes) were detected with the indicated digoxigenin-ylated anti-minipig antibodies followed by incubation with horseradish peroxidase-labeled anti-digoxigenin antibodies. The color reaction of the substrate. The signal was measured at a wavelength of 405 nm (reference wavelength: 490 nm ([405 / 490] nm)) by a standard ELISA reader. The absorbance value of each sample was determined in duplicate.

[0202] Figure 3 A schematic diagram illustrating this assay system suitable for direct detection of drug / ADA complexes is shown in .

[0203] Figure 4 The results are shown in , "NC" refers to the negative control, "LPC" refers to the positive control with low level of human IgG / pig IgG conjugate, "MPC" refers to the positive control with medium level of human IgG / pig IgG conjugate; and "HPC" refers to the positive control with high level of human IgG / pig IgG conjugate.

[0204] The commercially available monoclonal antibodies tested showed either a rather high background (mAb2) or no significant signal at all (mAb1, mAb3). The commercially available polyclonal antibodies tested showed reasonable signal-to-noise ratios and seemed suitable for this detection method. All tested antibodies of the present invention showed slightly better signal-to-noise ratios than the tested polyclonal antibodies.

[0205] Embodiment 6:

[0206] Suitability of generated anti-minipig IgG monoclonal antibodies versus commercially available anti-pig IgG antibodies in the method of the invention (with pre-incubation with drug) for detecting ADA in minipig samples

[0207] To evaluate the suitability of different anti-minipig IgG antibodies for the methods of the invention for detecting ADA from minipig samples, several commercially available polyclonal antibodies as used in Example 5 were compared with the antibodies of the invention.

[0208] To detect ADA from minipig samples, the following assay (of the present invention) was used:

[0209] In the first step, biotinylated anti-human IgG-Fab (κ) monoclonal antibody MAb<h-IgG-κ> M-1.7.10-Fab' is bound to each well of a streptavidin-coated microtiter plate (SA-MTP). Excess unbound Fab'-fragments are removed by washing.

[0210] Control samples (LPC, MPC, HPC and NC) were used as described in Example 5.

[0211] For analysis, control samples were added to each well of a microtiter plate and incubated therein. After washing away unbound material, human IgG / porcine IgG conjugates (mimicking drig / ADA complexes) were detected with the indicated digoxigenin-ylated anti-minipig antibodies followed by incubation with horseradish peroxidase-labeled anti-digoxigenin antibodies. The color reaction of the substrate. The signal is measured by an ELISA reader at a wavelength of 405 nm (reference wavelength: 490 nm ([405 / 490] nm)). The absorbance value of each sample is determined in duplicate.

[0212] Figure 5 A schematic diagram illustrating this assay system suitable for detecting ADA and drug / ADA complexes from minipig samples is shown in FIG.

[0213] Figure 6 The results are shown in , "NC" refers to the negative control, "LPC" refers to the positive control with low level of human IgG / pig IgG conjugate, "MPC" refers to the positive control with medium level of human IgG / pig IgG conjugate; and "HPC" refers to the positive control with high level of human IgG / pig IgG conjugate.

[0214] The commercially available polyclonal antibodies tested showed low signal-to-noise ratios and considerable background noise, which probably occurred due to cross-reactivity with human IgG. All tested antibodies of the present invention showed better signal-to-noise ratios than antibodies in the prior art.

[0215] Embodiment 7:

[0216] Using monoclonal anti-minipig IgG 2.8.14, ADA was detected from minipig samples by the method of the present invention

[0217] The method of the invention using monoclonal anti-minipig IgG 2.8.14 was performed to detect ADA from minipig samples derived from different in vivo studies with human IgG1 drug antibodies.

[0218] The results were compared to a direct immunoassay ("bridging assay") for detection of ADA from minipig samples, using appropriately conjugated drug antibodies for detection.

[0219] The bridging assay (Comparative Example) was performed as follows:

[0220] Briefly, minipig samples (appropriately diluted depending on sample type) were pre-incubated simultaneously with biotinylated drug antibodies and digoxigenin-conjugated drug antibodies as capture antibodies. Binding of ADA from minipig samples to biotinylated drug antibodies and digoxigenin-conjugated drug antibodies was detected on streptavidin-coated microtiter plates by incubation with horseradish peroxidase-labeled anti-digoxigenin antibodies. Antibody-enzyme conjugates catalyzed Color reaction of the substrate. The signal is measured by an ELISA reader at a wavelength of 405 nm (reference wavelength: 490 nm ([405 / 490] nm)).

[0221] Detection of ADA from minipig samples using the method of the invention (also referred to herein as "IC assay with pre-incubation") was performed as described in Example 6, with the difference that instead of the minipig samples previously screened, the indicated samples from the in vivo study were used and diluted at a 1:20 ratio. The minipig samples were taken from in vivo studies of two different ophthalmic drug antibodies, drug A being a human full-length IgG1 CrossMab and drug B being a human IgG1 Fab fragment.

[0222] As a positive control, human IgG (purified from human serum, in-house preparation) chemically conjugated to porcine IgG (purified from porcine serum, in-house preparation) spiked in 2% minipig CTAD plasma was used. Minipig pooled plasma was used as a negative control.

[0223] The results are shown in Tables 2 to 10, where applicable, indicating the route and dose of application of the drug, sample type, subject identification code of the miniature pig receiving the treatment, sampling time after drug application, and ADA test results (classified as positive [pos] and negative [neg]). According to Shankar et al. (J Pharm Biomed Anal. 2008 Dec 15; 48 (5): 1267-81), the screening cutoff point (CP) was evaluated using a floating CP and non-parametric determination method. By calculating the plate-specific CP (a blank of the merged original plasma multiplied by the normalization factor), screening negative (< plate-specific CP, PSCP) samples and screening positive (≥PSCP) samples were identified. The method was validated in full accordance with current guidelines. The method validation was implemented separately for each assay of the present invention and the bridging assay used as a comparative example.

[0224] Figure 7 Also shown are the results from intravitreal injections of Drug B as shown below in Tables 7 and 9 for vitreous humor samples, indicating the absolute values ​​of OD assessed using both assays.

[0225] Table 2: Plasma samples for intravitreal injection of 1.5 mg / eye Drug A

[0226]

[0227]

[0228] Table 3: Vitreous humor samples for intravitreal injection of 1.5 mg / eye drug A

[0229]

[0230]

[0231] Table 4: Plasma samples after intravenous injection of 0.3 mg / kg drug A (human IgG1)

[0232]

[0233] Table 5: Plasma samples for intravitreal injection of 5 mg / eye Drug B

[0234]

[0235]

[0236] Table 6: Intravitreal injection of 10 mg / eye Drug B, plasma samples

[0237]

[0238]

[0239]

[0240] Table 7: Vitreous humor samples for intravitreal injection of 5 mg / eye drug B

[0241]

[0242] Table 8: Intravitreal injection of 5 mg / eye drug B, aqueous humor samples

[0243]

[0244] Table 9: Vitreous humor samples for intravitreal injection of 10 mg / eye of drug B

[0245]

[0246] Table 10: Intravitreal injection of 10 mg / eye drug B, aqueous humor samples

[0247]

[0248] In summary, the results show that the method of the present invention reliably detects ADA from minipig samples. The IC assay with pre-incubation appears to be more sensitive than the bridging assay, as some sample ADA was detected by the IC assay with pre-incubation (method of the present invention) but not by the bridging assay.

[0249] Example 8

[0250] Comparative study of the method of the present invention: detection of ADA in miniature pig samples using IC assay with pre-incubation and detection of drug / ADA complexes using IC assay without pre-incubation using monoclonal anti-miniature pig IgG 2.8.14

[0251] Detection of ADA from minipig samples derived from different in vivo studies with human IgG1 drug antibodies was performed with a method of the invention using monoclonal anti-minipig IgG 2.8.14 in an experimental setup as described in Example 7 (IC assay with pre-incubation).

[0252] Detection of drug / ADA complexes from minipig samples derived from different in vivo studies using human IgG1 drug antibodies (this method for detecting drug / ADA complexes formed in vivo is further referred to herein as "IC assay without pre-incubation") was performed with another method of the invention using monoclonal anti-minipig IgG 2.8.14, i.e., the setup described in Example 5. Deviating from the setup as described in Example 5, instead of pre-screening minipig samples with low, medium, and high levels of drug / ADA complexes, samples from the in vivo studies were used as indicated and diluted at a 1:20 ratio. The minipig samples were taken from in vivo studies of two different ophthalmic drug antibodies, drug A being a human full-length IgG1 CrossMab and drug B being a human IgG1 Fab fragment.

[0253] As a positive control, human IgG (purified from human serum, in-house preparation) chemically conjugated to porcine IgG (purified from porcine serum, in-house preparation) spiked in 2% minipig CTAD plasma was used. Minipig pooled plasma was used as a negative control.

[0254] The results are shown in Tables 11 to 16, where applicable, indicating the route and dose of application of the drug, sample type, subject identification code of the miniature pig receiving the treatment, sampling time after drug application, and ADA test results (classified as positive [pos] and negative [neg]). According to Shankar et al. (J Pharm Biomed Anal. 2008 Dec 15; 48 (5): 1267-81), the screening cutoff point (CP) was evaluated using a floating CP and a non-parametric determination method. By calculating the plate-specific CP (a blank of the merged original plasma multiplied by a normalization factor), screening negative (< plate-specific CP, PSCP) samples and screening positive (≥PSCP) samples were identified. The method was validated in full accordance with current guidelines. The method validation was implemented separately for each assay of the present invention and the bridging assay used as a comparative example.

[0255] Table 11: Plasma samples of drug B injected intravitreally at 5 mg / eye

[0256]

[0257]

[0258] Table 12: Plasma samples of drug B injected intravitreally at 10 mg / eye

[0259]

[0260]

[0261] Table 13: Vitreous humor samples of drug B injected intravitreally at 5 mg / eye

[0262]

[0263]

[0264] Table 14: Aqueous humor samples of drug B injected intravitreally at 5 mg / eye

[0265]

[0266] Table 15: Vitreous humor samples of drug B injected intravitreally at 10 mg / eye

[0267]

[0268]

[0269] Table 16: Aqueous humor samples of drug B injected intravitreally at 10 mg / eye

[0270]

Claims

1. An in vitro method for determining the presence or absence of anti-drug antibodies in a miniature pig sample, the method being a sandwich assay, wherein the drug is a human antibody or a humanized antibody, the abbreviation for the anti-drug antibodies being ADA, the method comprising: a) contacting a sample with an excess of a drug, wherein ADA binds to the drug to form a drug / ADA complex, then contacting the drug / ADA complex with an antibody that specifically binds to human IgG, wherein the antibody that specifically binds to human IgG is immobilized on a support, thereby immobilizing the drug / ADA complex on the support; contacting the immobilized drug / ADA complex with a monoclonal antibody that specifically binds to minipig IgG, the monoclonal antibody comprising: The heavy chain CDR1 of SEQ ID NO:3, the heavy chain CDR2 of SEQ ID NO:4, and the heavy chain CDR3 of SEQ ID NO:5, the light chain CDR1 of SEQ ID NO:6, the light chain CDR2 of SEQ ID NO:7, and the light chain CDR3 of SEQ ID NO:8; and wherein the antibody that specifically binds to miniature pig IgG does not bind to human IgG, and b) Determine the binding of antibodies that specifically bind to minipig IgG to the drug / ADA complex to determine if ADA is present in the sample.

2. An in vitro method for determining the presence or absence of an immune complex comprising a drug and an antibody that specifically binds to the drug in a miniature pig sample, wherein the drug is a human antibody or a humanized antibody and the immune complex is a drug / ADA complex, the method comprising: a) contacting the minipig sample with an immobilized antibody immobilized on a support, the immobilized antibody specifically binding to human IgG to immobilize the drug / ADA complex present in the minipig sample, b) contacting the drug / ADA complex with an antibody that specifically binds to minipig IgG, wherein the antibody is selected from a monoclonal antibody comprising The heavy chain CDR1 of SEQ ID NO:3, the heavy chain CDR2 of SEQ ID NO:4, and the heavy chain CDR3 of SEQ ID NO:5, the light chain CDR1 of SEQ ID NO:6, the light chain CDR2 of SEQ ID NO:7, and the light chain CDR3 of SEQ ID NO:8; and wherein the antibody that specifically binds to miniature pig IgG does not bind to human IgG, and c) Determine the binding of antibodies that specifically bind to minipig IgG to the drug / ADA complex to determine whether ADA is present in the sample.

3. The method according to claim 1 or 2, wherein the antibody that specifically binds to minipig IgG also does not specifically bind to mouse IgG.

4. The method according to claim 1 or 2, wherein the antibody that specifically binds to miniature pig IgG comprises a VH sequence of SEQ ID NO: 1 and a VL sequence of SEQ ID NO:

2.

5. The method according to claim 1 or 2, wherein the antibody that specifically binds to miniature pig IgG specifically binds to the Fab domain of miniature pig IgG.

6. The method according to claim 1 or 2, wherein the drug is a drug antibody, and the drug antibody is a Fab fragment.

7. The method according to claim 1 or 2, wherein the antibody that specifically binds to minipig IgG is conjugated to a detectable marker.

8. A monoclonal antibody that specifically binds to miniature pig IgG, for implementing the method according to claim 1 or 2, wherein the monoclonal antibody comprises The heavy chain CDR1 of SEQ ID NO:3, the heavy chain CDR2 of SEQ ID NO:4, and the heavy chain CDR3 of SEQ ID NO:5, the light chain CDR1 of SEQ ID NO:6, the light chain CDR2 of SEQ ID NO:7, and the light chain CDR3 of SEQ ID NO:

8.

9. The monoclonal antibody according to claim 8, for use in implementing the method according to claim 1 or 2, wherein the monoclonal antibody is selected from An antibody comprising a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1 and a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2.

Images