Multivalent anti-variant FC region antibodies and methods of use

Abstract

The present invention relates to an antibody comprising at least four binding sites which specifically bind to an immunoglobulin Fc region of the human IgG1 subclass which comprises one, two, three or four amino acid changes compared to a wild-type Fc region of said human IgG1 subclass, for use as a positive control and calibration standard in immunoassays for detecting and quantifying anti-drug antibodies against said one, two, three or four amino acid changes in the Fc region of a drug antibody. TIFF2024534067000017.tif69128

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to multivalent antibodies, in particular valency enhancement and multimers of antibodies that specifically bind variant Fc regions (multivalent anti-variant Fc region antibodies), which can be used as positive controls and calibration standards in bridging immunoassays and domain detection assays. The multivalent antibodies according to the invention do not bind the corresponding wild-type Fc regions, but specifically bind variant Fc regions, and can therefore specifically crosslink two of said variant Fc regions in immunoassays, for example as positive controls or calibration standards. Methods for their production and their use are also reported herein. [Background technology]

[0002] background Since the first monoclonal antibody was developed by Koehler and Milstein in 1974, much effort has been put into developing antibodies suitable for therapy in humans. The first monoclonal antibodies that became available were developed in mice and rats. These antibodies, when used in human therapy, caused undesirable side effects due to the induction of immune responses, i.e., the formation of anti-rodent antibodies. Much effort has been put into reducing or eliminating such undesirable side effects.

[0003] A large number of human or humanized monoclonal antibodies are currently under investigation and need to be studied in laboratory animals before administration to humans can be considered for initial testing.

[0004] In addition to standard antibodies with human wild-type Fc regions, antibodies with variant Fc regions are also being developed, and these variations may also elicit an immune response as being generally non-natural.

[0005] Important criteria such as bioavailability and immunogenicity, to name just two of them, must be studied with the help of laboratory animals. These studies require, among other things, the quantification of anti-drug antibodies in the background of the host's own antibodies. In most cases, mammals are used as laboratory animals. Toxicology is often first evaluated in rodents such as mice or rats. At more advanced stages of drug development, even monkeys must be included in such preclinical trials, especially before introducing the drug in humans.

[0006] Currently, the bridging format enzyme-linked immunosorbent sandwich assay (ELISA) represents the state-of-the-art assay format for immunogenicity testing due to its high throughput and sensitivity, as well as its easy adaptability to different projects (Mikulskis, A., et al., J. Immunol. Meth. 365 (2011) 38-49 (Non-Patent Document 1)).

[0007] A standard solid-phase anti-drug antibody immunoassay using monoclonal antibodies involves the formation of a complex between a drug antibody (capture antibody) adsorbed or bound to a solid phase, an anti-drug antibody, and a drug antibody (tracer antibody) conjugated to a detectable label, such as an enzyme. Thus, a sandwich of solid-phase capture antibody-anti-drug antibody-tracer antibody is formed. In the reaction catalyzed by the sandwich, the activity of the antibody-conjugated enzyme is proportional to the anti-drug antibody concentration. The standard sandwich method is also called a bridging immunoassay, since the anti-drug antibody bridges between the capture antibody and the tracer antibody, i.e., the drug antibody. Immunoassays such as bridging ELISA are a common assay type in the investigation of patients' immunogenic responses to antibody drugs.

[0008] Mire-Sluis, AR et al., J. Immunol. Methods 289 (2004) 1-16 (Non-Patent Document 2), summarized recommendations for the design and optimization of immunoassays used for the detection of host antibodies against biotechnology products.

[0009] Wadhwa, M. et al., J. Immunol. Methods 278 (2003) 1-17, reported a strategy for the detection, measurement and characterization of unwanted antibodies induced by therapeutic biologics.

[0010] The principles of the different immunoassays are described, for example, in Hage, DS, Anal. Chem. 71 (1999) 294R-304R.

[0011] Lu, B. et al., Analyst 121 (1996) 29R-32R (Non-Patent Document 5), reported oriented immobilization of antibodies for use in immunoassays.

[0012] Avidin-biotin mediated immunoassays are reported, for example, in Wilchek, M. and Bayer, EA, Methods Enzymol. 184 (1990) 467-469 (Non-Patent Document 6).

[0013] International Publication No. WO 2017 / 072210 (Patent Document 1) reports an anti-variant Fc region antibody and a method for using the same.

[0014] Wessels, U. et al., Bioanal. 9 (2017) 849-859 (Non-Patent Document 7) reported a novel drug and soluble target-resistant anti-drug antibody assay for therapeutic antibodies with the P329G mutation.

[0015] US Patent Application Publication No. 2008 / 0118939 reports the conjugate and its use as a standard in immunoassays. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2017 / 072210 [Patent Document 2] US Patent Application Publication No. 2008 / 0118939 [Non-patent literature]

[0017] [Non-Patent Document 1] Mikulskis, A., et al., J. Immunol. Meth. 365 (2011) 38-49 [Non-Patent Document 2] Mire-Sluis, AR et al., J. Immunol. Methods 289 (2004) 1-16 [Non-Patent Document 3] Wadhwa, M. et al., J. Immunol. Methods 278 (2003) 1-17 [Non-Patent Document 4] Hage, DS, Anal.Chem.71(1999)294R-304R [Non-Patent Document 5] Lu, B. et al., Analyst 121 (1996) 29R-32R [Non-Patent Document 6] Wilchek, M. and Bayer, E. A., Methods Enzymol. 184 (1990) 467-469 [Non-Patent Document 7] Wessels, U. et al., Bioanal. 9 (2017) 849-859 Summary of the Invention

[0018] The reliability of a bridging anti-drug antibody (ADA) assay is based on the availability of at least one functional positive control that provides a sufficient assay signal above background, as well as a calibration standard when using an ADA assay to quantify ADA. In particular, the detection of ADA for modifications in the Fc region is not trivial, especially when the modifications are present in both chains of the Fc region of a drug antibody (therapeutic antibody). In addition, domain detection assays also require a positive control with appropriate sensitivity.

[0019] An antibody that specifically binds to a variant Fc region and does not substantially bind to a wild-type Fc region is referred to as an anti-variant Fc region antibody.

[0020] Without being bound by this theory, a standard bivalent Y-type anti-variant Fc region antibody can bind to a single Fc region simultaneously with both of its binding specificities. This blocks both binding sites, and the formation of a cross-linked complex is no longer possible. Similarly, an anti-variant Fc region antibody can bind in a sterically unfavorable orientation, so that cross-linking, i.e., simultaneous binding to capture and tracer drug antibodies, is prevented. Therefore, a functional positive control as well as a calibration standard are needed in anti-drug antibody cross-linking ELISA.

[0021] The present invention is based at least in part on the finding that a monomer of a standard Y-shaped bivalent antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to the wild-type Fc region of the human IgG1 subclass (variant human IgG1 Fc region) is unable to bind two variant Fc regions simultaneously, i.e. to an extent suitable as a positive control or calibration standard in a cross-linking ADA assay to form a bridge between the two variant Fc regions. It has been found that in an antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to the wild-type Fc region of the human IgG1 subclass, by increasing the valency beyond two, for example by adding additional binding sites or by forming multimers, it is possible to simultaneously bind two variant Fc regions, i.e. to cross-link and link the two variant Fc regions, in contrast to a bivalent antibody.

[0022] The present invention provides drug antibodies that lack an Fc region effector function, e.g., ADCC, e.g., by introducing a Pro329Gly (PG) substitution within the Fc region, functional positive controls, and calibration standards for use in ADA assays. The functional positive controls and calibration standards according to the present invention are either valency-enhanced antibodies or multimers specific for substitutions within the Fc region of the drug antibody, e.g., tetravalent or multimeric anti-PG antibodies.

[0023] The multivalent antibodies according to the invention in combination with the bridging ADA assay allow a detailed ADA characterization of clinical samples, since on the one hand it is possible to determine the proper functioning of the assay and on the other hand it is possible to calibrate the assay. The bridging format ADA immunoassays using the multivalent antibodies according to the invention are complemented for a detailed characterization of individual ADA responses to Fc-region-modified drug antibodies.

[0024] One embodiment according to the invention is an antibody comprising at least three binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that comprises one, two, three or four amino acid changes compared to a wild-type Fc region of the human IgG1 subclass.

[0025] One embodiment according to the invention is a multimer of an antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to a wild-type Fc region of the human IgG1 subclass.

[0026] One embodiment according to the invention is an antibody comprising at least three binding sites which specifically bind to an immunoglobulin Fc region of the human IgG1 subclass which comprises the amino acid residue glycine at position 329 (numbering according to the Kabat EU index) or a multimer of (bivalent) (Fab')2 fragments of an antibody which specifically binds to an immunoglobulin Fc region of the human IgG1 subclass which comprises the amino acid residue glycine at position 329 (numbering according to the Kabat EU index), wherein the binding sites are (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or (4) Any combination of (1) to (3) Including, HVR is determined according to Kabat.

[0027] In certain embodiments of all aspects and embodiments of the invention, the at least trivalent antibody (an antibody comprising at least three binding sites) is a trivalent, tetravalent, hexavalent, octavalent or decavalent antibody. In a preferred embodiment, the at least trivalent antibody is a tetravalent antibody.

[0028] In certain embodiments of all aspects and embodiments of the invention, the at least trivalent antibody (an antibody comprising at least three binding sites) is an IgA or IgM antibody.

[0029] One embodiment of the present invention is a multimer of an antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (numbering according to the Kabat EU index), or a multimer of (bivalent) (Fab')2 fragments of an antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (numbering according to the Kabat EU index), wherein the antibody or (Fab')2 fragment is (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30 Including, HVR is determined according to Kabat.

[0030] In a preferred embodiment of all aspects and embodiments of the invention, the antibody or (Fab')2 fragment specifically binds to an immunoglobulin Fc region of the human IgG1 subclass comprising the amino acid residue glycine at position 329 and the amino acid residues alanine at positions 234 and 235 (numbering according to the Kabat EU index).

[0031] In certain embodiments of all aspects and embodiments of the invention, the multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer.

[0032] One aspect according to the invention is the use of an at least trivalent antibody according to the invention as a positive control in an in vitro (bridging) immunoassay.

[0033] One aspect according to the invention is the use of a multimer according to the invention as a positive control in an in vitro (bridging) immunoassay.

[0034] An aspect according to the present invention is the use of at least trivalent antibodies according to the present invention as a standard in an in vitro (bridge) immunoassay.In a particular embodiment, the use is for generating a calibration function.In a preferred embodiment, the calibration function is for quantitatively determining anti-drug antibodies against a drug antibody, the anti-drug antibody binding to one or more amino acid residues in the Fc region of the drug antibody that are altered compared to the wild-type Fc region.

[0035] An aspect according to the present invention is the use of the multimer according to the present invention as a standard in an in vitro (bridge) immunoassay. In a particular embodiment, the use is for generating a calibration function. In a preferred embodiment, the calibration function is for quantitatively determining an anti-drug antibody against a drug antibody, the anti-drug antibody binding to one or more amino acid residues in the Fc region of the drug antibody that are altered compared to the wild-type Fc region.

[0036] In certain embodiments of all aspects and embodiments of the invention, the in vitro (bridging) immunoassay is for determining anti-drug antibodies against a drug antibody, which anti-drug antibodies bind to the Fc region of the drug antibody. In certain embodiments, the anti-drug antibodies bind to one or more amino acid residues in the Fc region of the drug antibody that are altered compared to the wild-type Fc region.

[0037] In certain embodiments of all aspects and embodiments of the present invention, the in vitro immunoassay is an in vitro bridging ELISA.

[0038] In certain embodiments of all aspects and embodiments of the invention, the drug antibody comprises an immunoglobulin Fc region of the human IgG1 subclass comprising the amino acid residue glycine at position 329 (numbering according to the Kabat EU index).

[0039] In a preferred embodiment of all aspects and embodiments of the invention, the drug antibody comprises an immunoglobulin Fc region of the human IgG1 subclass comprising the amino acid residue glycine at position 329 and the amino acid residues alanine at positions 234 and 235 (numbering according to the Kabat EU index).

[0040] In a preferred embodiment of all aspects and embodiments of the present invention, the in vitro immunoassay is an in vitro bridging immunoassay for determining anti-drug antibodies comprising a drug antibody as a capture antibody and as a tracer antibody.

[0041] One embodiment according to the invention is an in vitro immunoassay for determining the presence and / or amount of anti-drug antibodies in a (serum-containing) sample, comprising: the anti-drug antibody binds to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region; The immunoassay comprises a drug antibody as a capture antibody and a tracer antibody, The at least trivalent antibody according to the invention or the multimer according to the invention is characterized in that it is used as a positive control or as a calibration standard in an immunoassay.

[0042] In certain embodiments of all aspects and embodiments of the invention, the use as calibration standard is for generating a calibration function. In a preferred embodiment, the calibration function is for quantitatively determining an anti-drug antibody to a drug antibody, which anti-drug antibody binds to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region.

[0043] In certain embodiments of all aspects and embodiments of the invention, the antibody that specifically binds to a variant immunoglobulin Fc region of the human IgG1 subclass is a monoclonal antibody.

[0044] In certain embodiments of all aspects and embodiments of the invention, the drug antibody is a human antibody, a humanized antibody or a chimeric antibody.

[0045] One aspect is a method for producing a multimer according to the present invention, comprising the steps of: Chemically cross-linking a full-length antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (numbering according to the Kabat EU index) using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS); The antibody is (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30 (HVR is determined according to Kabat) Including, This is the method. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1: Scheme of an immunoassay using multimers according to the invention as positive controls or calibration standards. [Diagram 2]Diagram of the signal obtained with monomeric anti-PG antibody clone 1.7.24 in a bridging immunoassay using the same but differently derivatized drug antibodies as capture and tracer antibodies. [Diagram 3] Various binding modes of anti-variant Fc region antibodies to Fc regions: (A) shows simultaneous binding to a single Fc region at both binding sites, and (B) shows steric hindrance. [Figure 4] Modes for multimerizing anti-variant Fc region antibodies: (A) is a diagram of recombinant expression and (B) is a diagram of chemical cross-linking. [Diagram 5] SEC-chromatograms of a pool of cross-linked anti-PG antibodies with different cross-linking degrees, i.e. molecular sizes. [Figure 6] Diagram of the signal obtained with multimeric anti-PG antibody clone 1.7.24 in a bridging immunoassay using the same but differently derivatized drug antibodies as capture and tracer antibodies. [Figure 7] FIG. 1 shows the signal of monomeric and multimeric anti-PG antibodies according to the invention shown as a direct 1:1 comparison in a bridging immunoassay using the same but differently derivatized drug antibodies as capture and tracer antibodies. [Figure 8] Signal-to-noise ratio of monomeric and multimeric anti-PG antibodies according to the invention shown as a direct 1:1 comparison in a bridging immunoassay using the same but differently derivatized drug antibodies as capture and tracer antibodies. [Figure 9] Diagram of multimeric anti-PG antibodies according to the invention in a bridging immunoassay with different formats of drug antibodies used as capture and tracer antibodies (both in the same format). [Figure 10] Diagram of the signal of a tetravalent anti-PG antibody used as a calibration standard in a bridging immunoassay with the same but differently derivatized Fc region of the drug antibody as capture and tracer antibody. [Figure 11]Diagram of the signal of an anti-PG antibody in IgM format used as a calibration standard in a bridging immunoassay with a variant Fc region as capture agent and drug antibody in TCB format as tracer antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description of the Invention Embodiments I. Definition As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as "Kabat numbering". Specifically, the Kabat numbering system (see pages 647-660) of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3).

[0048] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). In general, the affinity of a molecule X for its partner Y is determined by the dissociation constant (K dAffinity can be measured by common methods known in the art, including those described herein.

[0049] The term "(amino acid) change" refers to the replacement of at least one amino acid residue in a given parent amino acid sequence with a different "substitution" amino acid residue to generate a variant amino acid sequence. The substitution residue or residues may be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and may be selected from the group consisting of alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). In certain embodiments, the substitution residue is not cysteine. Substitution with a non-naturally occurring amino acid residue is also encompassed by the definition of an amino acid change herein. "Non-naturally occurring amino acid residue" refers to a residue other than the naturally occurring amino acid residues described above that can be covalently linked to adjacent amino acid residues in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202 (1991) 301-336. To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. (Science 244 (1989) 182) and / or Ellman et al., supra, can be used. Briefly, these procedures involve chemical activation of suppressor tRNAs bearing the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. Non-naturally occurring amino acids can also be incorporated into peptides by chemical peptide synthesis followed by fusion of these peptides with recombinantly produced polypeptides, such as antibodies or antibody fragments.

[0050] In this application, whenever an amino acid change is referred to, it is a deliberate amino acid change, not a random amino acid modification.

[0051] The terms "anti-variant (human) Fc region antibody" and "antibody that specifically binds to a variant (human) Fc region" refer to an antibody that can bind to a variant (human) Fc region with sufficient affinity such that the antibody is useful as a diagnostic agent in targeting the variant (human) Fc region. In certain embodiments, the extent of binding of an anti-variant (human) Fc region antibody to a corresponding wild-type (human) Fc region is less than about 10% of the binding of the antibody to a variant (human) Fc region. This can be determined, for example, using surface plasmon resonance. In certain embodiments, an antibody that specifically binds to a variant (human) Fc region has a binding affinity of less than about 10%. -8 M or less, e.g. 10 -8 M~10 -12 Dissociation constant of M (K D ).

[0052] The term "drug antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), so long as they exhibit the desired antigen-binding activity.

[0053] The term "bind" refers to the binding of a first entity to a second entity, such as the binding of an antibody to its antigen. This binding can be measured, for example, using a BIAcore® assay (GE Healthcare, Uppsala, Sweden).

[0054] For example, in one possible embodiment of a BIAcore® assay, antigen is bound to a surface and antibody binding is measured by surface plasmon resonance (SPR).

[0055] The binding affinity is expressed by the term k a(association constant: rate constant for binding to form a complex), k d (dissociation constant; rate constant for dissociation of the complex), and K D (k d / k a Alternatively, the binding signal of the SPR sensorgram can be directly compared to the response signal of a reference in terms of resonance signal height and dissociation behavior.

[0056] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0057] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0058] "Effector function" refers to the biological activities attributable to the Fc region of an antibody, which vary depending on the class of antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0059] Fc receptor binding-dependent effector functions can be brought about by the interaction of the Fc region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of corresponding antibody-coated red blood cells and various other cellular targets (e.g., tumor cells) through antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes, and the Fc receptors of IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ et al., Immunomethods 4 (1994) 25-34; de Haas, M. et al., J. Lab. Clin. Med. 126 (1995) 330-341; Gessner, JE et al., Ann. Hematol. 76 (1998) 231-248.

[0060] Cross-linking of receptors to the Fc region of IgG antibodies (FcγR) triggers a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as control of immune complex clearance and antibody production. In humans, three classes of FcγR have been characterized:

[0061] - FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modification of the Fc region IgG at at least one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbering according to the Kabat EU index) reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 into IgG1 and IgG4 reduces binding to FcγRI by 103-fold and abolishes the human monocyte response to antibody-sensitized erythrocytes (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624). - FcγRII (CD32) binds complexed IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, FcγRIIB appears to function to suppress the production of further immunoglobulins and isotype switching, for example to the IgE class. On macrophages, FcγRIIB acts to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B form may serve to suppress the activation of these cells through IgE binding to its other receptors. Reduced binding to FcγRIIA is seen, for example, in antibodies comprising an IgG Fc region having mutations in at least one of amino acid residues E233 to G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414 (numbering according to the Kabat EU index). - FcγRIII (CD16) binds IgG with moderate to low affinity and exists as two types. FcγRIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to FcγRIIIA is seen, for example, in antibodies that contain an IgG Fc region with mutations in at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbering according to Kabat EU index).

[0062] Mapping of the binding sites on human IgG1 for Fc receptors, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0063] The term "Fc receptor" as used herein refers to an activating receptor characterized by the presence of a cytoplasmic ITAM sequence associated with the receptor (see, e.g., Ravetch, JV and Bolland, S., Annu. Rev. Immunol. 19 (2001) 275-290). Such receptors are FcγRI, FcγRIIA and FcγRIIIA. The term "no FcγR binding" indicates that at an antibody concentration of 10 μg / ml, the binding of the antibodies reported herein to NK cells is 10% or less than that observed for the anti-OX40L antibody LC.001 reported in WO 2006 / 029879.

[0064] IgG4 shows reduced FcR binding, whereas antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, as well as 234, 235, 236 and 237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are residues that also reduce FcR binding when altered (Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J. et al., FASEB J. 9 (1995) 115-119; Morgan, A. et al., Immunology 86 (1995) 319-324; and European Patent No. 0307434).

[0065] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In certain embodiments, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0066] The Fc region of an antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. The effect of an antibody on the complement system depends on the specific conditions, but binding to C1q is caused by a defined binding site in the Fc region. Such binding sites are known in the state of the art and are described, for example, in Lukas, TJ et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR et al., Nature 288 (1980) 338-344; Thommesen, JE et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. et al., J. Virol. 75 (2001) 12161-12168; Morgan, A. et al., Immunology 86 (1995) 319-324; and European Patent No. 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the Kabat EU index; unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No. 0307434). The EU numbering system, also called the EU index, as described in US Pat. No. 91-3242. Antibodies of subclasses IgG1, IgG2, and IgG3 usually exhibit complement activation, C1q binding, and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q, and does not activate C3. "Antibody Fc region" is a term well known to those skilled in the art and is defined based on papain cleavage of an antibody. In certain embodiments, the Fc region is a human Fc region.In certain embodiments, the Fc region of the drug antibody is of the human IgG1 subclass containing the mutations L234A and L235A (numbering according to the Kabat EU index).

[0067] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences generally appear in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0068] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having a heavy chain that includes an Fc region as defined herein.

[0069] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to a non-human antibody and all or substantially all of the FRs correspond to a human antibody. 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, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0070] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that comprise stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Generally, an antibody comprises six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0071] HVR includes: (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, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); (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, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242); (c) antigen 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)); (d) combinations of (a), (b), and / or (c), including 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).

[0072] Unless otherwise specified, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al., supra.

[0073] An "isolated" multimer is one that is separated from the components of its natural environment. In certain embodiments, the multimer is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848 (2007) 79-87.

[0074] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, with the exception of a small percentage of antibodies that contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0075] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), with a hinge region located between the first and second constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0076] The term "variant (human) Fc region" refers to an amino acid sequence that differs from a "wild-type" (human) Fc region amino acid sequence by at least one "amino acid change." In certain embodiments, a variant Fc region has at least one amino acid change compared to a native Fc region, e.g., about 1 to about 10 amino acid changes, and in certain embodiments, about 1 to about 5 amino acid changes relative to a native Fc region. In certain embodiments, a (variant) Fc region has at least about 80% homology with a wild-type Fc region, and in certain embodiments, a variant Fc region has at least about 90% homology, and in a preferred embodiment, a variant Fc region has at least about 95% homology.

[0077] Variant Fc regions are defined by the amino acid changes they contain. Thus, for example, the term P329G refers to a variant Fc region that has a mutation from proline to glycine at amino acid position 329 compared to the parent (wild type) Fc region. The identity of the wild type amino acid may be unknown, in which case the variant is referred to as 329G. The term "alteration" refers to alterations to naturally occurring amino acids as well as alterations to non-naturally occurring amino acids (e.g., U.S. Pat. No. 6,586,207, WO 98 / 48032, WO 03 / 073238, U.S. Patent Application Publication No. 2004 / 0214988, WO 2005 / 35727, WO 2005 / 74524, Chin, JW et al., J. Am. Chem. Soc. 124 (2002) 9026-9027; Chin, JW and Schultz, PG, ChemBioChem 11 (2002) 1135-1137; Chin, JW et al., PICAS United States of America 99 (2002) 11020-11024; Wang, L. and Schultz, PG, Chem. (2002) 1-10).

[0078] The term "wild-type Fc region" refers to an amino acid sequence identical to that of an Fc region found in nature. Wild-type human Fc regions include naturally occurring human IgG1 Fc regions (non-A and A allotypes), naturally occurring human IgG2 Fc regions, naturally occurring human IgG3 Fc regions, and naturally occurring variants thereof.

[0079] The term "drug antibody" relates to any antibody preparation intended for use as a therapeutic agent in humans. Preferably, such a drug antibody is a monoclonal antibody. More preferably, such a monoclonal antibody is obtained from an ape or is a human monoclonal antibody. Preferably, such a monoclonal antibody is a humanized monoclonal antibody.

[0080] The term "valent" as used within this application denotes the presence of a certain number of binding sites in an (antibody) molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" denote the presence of two, four and six binding sites, respectively, in an (antibody) molecule. An at least trivalent antibody according to the invention is in a preferred embodiment "tetravalent". A "binding site" is formed by a cognate pair of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0081] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt, TJ et al., Kuby Immunology, 6th Edition, WH Freeman and Co., NY (2007), p.91.) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0082] II. Compositions and Methods The present invention is based, at least in part, on the finding that a monomeric bivalent antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to a wild-type Fc region of the human IgG1 subclass is not able to bind to the two variant Fc regions with sufficient sensitivity / quantity, i.e. with sufficient sensitivity / quantity to form a (detectable) bridge between the two variant Fc regions, and cannot be used as a positive control or calibration standard in an ADA assay.

[0083] In contrast to bivalent forms, it has been found that multivalent forms of antibodies that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to a wild-type Fc region of the human IgG1 subclass are capable of binding two variant Fc regions with sufficient sensitivity / amount, i.e., linking two variant Fc regions with sufficient amount / sensitivity, and therefore are suitable as positive controls and / or calibration standards in ADA assays.

[0084] For example, functional positive controls as well as calibration standards for use in ADA assays are reported for drug antibodies lacking binding to Fc receptors and / or Fc effector functions by introducing a Pro329Gly (PG) substitution in the Fc region. The functional positive controls and calibration standards according to the invention are multivalent forms of antibodies specific for the amino acid changes in the Fc region of the drug (therapeutic) antibody, such as at least trivalent or multimeric forms of anti-PG antibodies.

[0085] The multivalent antibodies according to the invention in combination with bridging assays allow for detailed ADA characterization of clinical samples, since proper functioning of the assay can be determined. Bridging anti-drug antibody immunoassays using the multivalent antibodies according to the invention are complemented for detailed characterization of individual ADA responses to Fc region-modified drug antibodies.

[0086] One embodiment according to the invention is a multivalent form of an antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to a wild-type Fc region of the human IgG1 subclass.

[0087] In the first example, the present invention is illustrated below using a chemically conjugated multimer according to FIG. 4C of an anti-PG antibody, i.e. an antibody that specifically binds to the human Fc region of the IgG subclass with the P329G change. This is presented merely to illustrate the present invention and should not be construed as limiting. The true scope is set forth in the appended claims.

[0088] More specifically, monomeric anti-PG antibody clone 1.7.24 was tested for use as a positive control in a bridging immunoassay using the same but differently derivatized drug antibodies as the capture and tracer antibodies. The results are shown in Figure 2.

[0089] It was found that the bivalent Y-shaped (=monomeric) anti-PG antibody clone 1.7.24 signal could not be detected up to a concentration of 1,000 ng / mL in 100% matrix. Moreover, at a concentration of 10,000 ng / mL, only a signal of 0.13 AU was obtained. At a concentration of 2,500 ng / mL of anti-PG antibody clone 1.7.24 (see Example 4A), the signal-to-noise ratio was more than 2 AU.

[0090] Regulatory agencies require a threshold of at least 100 ng / mL, meaning that the sensitivity of the ADA positive control should reach 100 ng / mL in 100% matrix (see, e.g., 2019 FDA guidance).

[0091] In general, a signal is considered sufficient for use if it is at least twice as high as a blank sample, ie, a sample containing no analyte.

[0092] Therefore, the monomeric anti-PG antibody clone 1.7.24 is not suitable as a positive control and calibration standard in anti-drug antibody assays due to its low sensitivity.

[0093] The same behavior was observed with a different clone, namely the anti-PG antibody clone 1.3.17. This monomeric antibody gave a signal at 100 ng / mL that was only 1.85 times the blank value (see Example 4B). Therefore, this clone is also in monomeric form and is not sensitive enough to be used as a positive control or calibration standard in the ADA assay.

[0094] Why are these monomeric anti-PG antibodies unable to form cross-links in the ADA assay? Without being bound by this theory, we hypothesize that either one anti-PG antibody binds to one drug-antibody molecule at both paratopes (see Figure 3A) or that only one binding site of the anti-PG antibody can bind to the Fc region, while the other is sterically blocked (see Figure 3B).

[0095] To address this problem, the use of multivalent anti-PG antibodies has been found to be advantageous.

[0096] Any kind of valency enhancement technique can be applied, such as recombinant production as a dimer or fusion molecule, changing to a different antibody format, such as IgA (see FIG. 4A) or IgM, adding binding sites (see FIG. 4B), or chemical conjugation, for example using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS) (see FIG. 4C).

[0097] An exemplary chemical conjugation of anti-PG antibody clone 1.7.24 using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS) was performed. The reaction proceeded smoothly. SEC was used to analyze the reaction products and different fractions were collected. As can be seen from Figure 5, multimers with different degrees of multimerization can be obtained, i.e. only dimers and trimers from highly cross-linked multimers (pool 2).

[0098] All pools were tested in a bridging ADA assay. Pools 2, 3 and 4 were mixed. Mixing pools together is a common procedure when using chemically conjugated proteins. Before mixing, an ADA ELISA assay was performed and the pools with the highest signal were combined. This results in a higher yield. The results are shown in Figure 6. It can be seen that an increased signal can be obtained in all pools compared to the monomeric antibody. This allows for the first time their use as positive controls and calibration standards in bridging ADA determination immunoassays.

[0099] Furthermore, it can be seen that the most significant improvements are obtained at higher cross-linking degrees, ie, pools 2 and 3.

[0100] In Figures 7 and 8, the monomeric bivalent anti-PG antibody clone 1.7.24 and the multivalent anti-PG antibody clone 1.7.24 according to the invention are shown as a direct 1:1 comparison in terms of signal and signal to noise ratio. In both cases, a substantial improvement can be seen. For example, the signal to noise ratio (S / N) of the multivalent anti-PG antibody clone 1.7.24 is 4.5 at a concentration of 80 ng / mL and 83.6 at a concentration of 1000 ng / mL. The S / N of the bivalent anti-PG antibody clone 1.7.24 is only 3.8 at a concentration of 1000 ng / mL.

[0101] Furthermore, multivalent anti-PG antibodies have been tested with different drug antibodies in different formats. Regardless of the format, an improvement in the signal can be seen (see Figure 9).

[0102] In the second example, the present invention is illustrated below according to Figure 4B using a recombinantly produced tetravalent form of anti-PG antibody, i.e. an antibody that specifically binds to the human Fc region of the IgG subclass with the P329G change. This is presented merely to illustrate the present invention and should not be construed as limiting in any way. The true scope is set forth in the appended claims.

[0103] More specifically, a tetravalent anti-PG antibody was used as a calibration standard in a bridging immunoassay using the same but differently derivatized Fc regions of the drug antibody as the capture and tracer antibodies. The results are shown in Figure 10.

[0104] In the third example, the invention is illustrated below using a recombinantly produced IgM variant anti-PG antibody, an antibody that specifically binds to the human Fc region of the IgG subclass with the P329G change, which is presented merely to illustrate the invention and should not be construed as limiting in any way. The true scope is set forth in the appended claims.

[0105] More specifically, an IgM variant anti-PG antibody was used as a calibration standard in a bridging immunoassay with the Fc region as the capture reagent and the whole drug in TCB format as the tracer antibody. The results are shown in Figure 11.

[0106] A. Exemplary Anti-Variant Fc Region Antibodies In the bridging assay, ADA is captured and detected by differentially labeled drug antibodies. However, the bridging assay can detect ADA of various Ig subtypes, including IgM, and is applicable to all kinds of therapeutic antibodies (Mire-Sluis, AR, et al., J. Immunol. Meth. 289 (2004) 1-16 (2004); Geng, D., et al., J. Pharm. Biomed. Anal. 39 (2005) 364-375). In the bridging assay, the complex between ADA and drug antibodies is detected independently of the binding region of the therapeutic antibody. Moreover, the assay according to the present invention is particularly suitable for drug antibodies with P329G modification in the Fc region. For this group of drug antibodies, the assay represents a general approach and can be easily applied.

[0107] In summary, the assay described herein offers the potential for robust and sensitive detection of ADA against Fc-modified drug antibodies, and can be used in combination with standard cross-linking assays to characterize the immune response in more detail.

[0108] The assay described herein is a general approach and is applicable to all drug antibodies, e.g., those with a Pro329Gly substitution, i.e., those in which FcγR binding is prevented / abolished. The assay according to the present invention detects ADA and is based on two different labeled drug antibodies, (i) a double labeled drug antibody and (ii) a dig labeled drug antibody.

[0109] Furthermore, several other Fc modifications beyond PG substitutions have been identified that affect the affinity of drug antibodies for both Fc receptors and complement, thereby altering their functional profile (Moore, GL et al., MAbs 2 (2010) 181-189; Richards, JO et al., Mol. Cancer. Ther. 7 (2008) 2517-2527; Lazar, GA et al., Proc. Natl. Acad. Sci. USA 103 (2010) 4005-4010; Schlothauer, T. et al., Prot. Eng. Des. Sel. 29 (2016) 457-466).

[0110] The principles of the assay reported here can be transferred to a wide range of Fc region alterations, provided that these allow the generation of specific antibodies.

[0111] In summary, the combination of traditional cross-linking assays with multivalent antibodies according to the invention helps to characterize the immunogenicity profile of drug antibodies with reduced or altered Fc effector functions.

[0112] One embodiment according to the invention is a multivalent antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to a wild-type Fc region of the human IgG1 subclass.

[0113] Specific binding means that the antibody binds to the Fc region of a wild-type immunoglobulin of the human IgG1 subclass by 10 -8 K in mol / l or more D This means to join by value.

[0114] One embodiment of the present invention is a multivalent antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (numbering according to the Kabat EU index), the multivalent antibody comprising: (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or (4) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 35; or (5) Any mixture or combination of (1) and / or (2) and / or (3) and / or (4) The nucleic acid comprises at least three binding sites, including

[0115] In a preferred embodiment of all aspects and embodiments of the invention, the multivalent antibody specifically binds to an immunoglobulin Fc region of the human IgG1 subclass comprising the amino acid residue glycine at position 329 and the amino acid residues alanine at positions 234 and 235 (numbering according to the Kabat EU index). In a preferred embodiment, the multivalent antibody is a tetravalent antibody or a multimeric form of a divalent antibody or a multimeric (Fab')2 fragment of a divalent antibody.

[0116] A bivalent (Fab')2 fragment has two antigen-binding sites linked together by disulfide bonds. Digestion of a full-length Y-shaped antibody with papain produces two individual Fab fragments. (Fab')2 fragments that retain part of the hinge region are produced by pepsin digestion of IgG or IgM antibodies.

[0117] In certain embodiments of all aspects and embodiments of the invention, the multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer.

[0118] One aspect according to the invention is the use of a multivalent antibody according to the invention as a positive control in a (bridging) immunoassay.

[0119] An aspect according to the invention is the use of a multivalent antibody according to the invention as a calibration standard in a (bridging) immunoassay. In a particular embodiment, the use is for generating a calibration function. In a preferred embodiment, the calibration function is for quantitatively determining an anti-drug antibody against a drug antibody, the anti-drug antibody binding to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region.

[0120] In certain embodiments of all aspects and embodiments of the invention, the (bridging) immunoassay is for determining anti-drug antibodies against a drug antibody, which anti-drug antibody binds to the Fc region of the drug antibody. In certain embodiments, the anti-drug antibody binds to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region.

[0121] In certain embodiments of all aspects and embodiments of the present invention, the immunoassay is a bridging ELISA.

[0122] In certain embodiments of all aspects and embodiments of the invention, the drug antibody comprises an immunoglobulin Fc region of the human IgG1 subclass comprising the amino acid residue glycine at position 329 (numbering according to the Kabat EU index).

[0123] In a preferred embodiment of all aspects and embodiments of the invention, the drug antibody comprises an immunoglobulin Fc region of the human IgG1 subclass comprising the amino acid residue glycine at position 329 and the amino acid residues alanine at positions 234 and 235 (numbering according to the Kabat EU index).

[0124] In certain embodiments of all aspects and embodiments of the invention, the drug antibody comprises an immunoglobulin Fc region of the human IgG1 subclass comprising the amino acid residues alanine at positions 253, 310 and 435 (numbering according to the Kabat EU index).

[0125] In a preferred embodiment of all aspects and embodiments of the present invention, the immunoassay is a bridging immunoassay for determining anti-drug antibodies comprising a drug antibody as a capture antibody and as a tracer antibody.

[0126] One embodiment according to the invention is an immunoassay for determining the presence and / or amount of anti-drug antibodies in a (serum-containing) sample, comprising: the anti-drug antibody binds to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region; The immunoassay comprises a drug antibody as a capture antibody and a tracer antibody, The multivalent antibodies according to the invention are characterized in that they are used as positive controls or as calibration standards in immunoassays.

[0127] In certain embodiments of all aspects and embodiments of the invention, the use as calibration standard is for generating a calibration function. In a preferred embodiment, the calibration function is for quantitatively determining an anti-drug antibody to a drug antibody, which anti-drug antibody binds to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region.

[0128] In certain embodiments of all aspects and embodiments of the invention, the antibody that specifically binds to a variant immunoglobulin Fc region of the human IgG1 subclass is a monoclonal antibody.

[0129] In certain embodiments of all aspects and embodiments of the invention, the drug antibody is a human antibody, a humanized antibody or a chimeric antibody.

[0130] One embodiment is a method for producing multimers according to the invention comprising chemically cross-linking full-length antibodies that specifically bind to a variant Fc region using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS).

[0131] One embodiment is a method for producing a multimer according to the invention comprising chemically crosslinking a full-length antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue Glycine at position 329 (numbering according to the Kabat EU index), The antibody is (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or (4) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 35; or (5) Any combination of (1) to (4) Including, A method for producing multimers according to the invention comprising chemical cross-linking using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS).

[0132] In a preferred embodiment of all aspects and embodiments of the present invention, the anti-variant Fc region antibody comprises: (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or (4) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 35; or (5) A mixture of any one of (1) to (4) Includes.

[0133] The antibodies used in the present invention have the following sequences (HVRs determined according to Kabat):

[0134] TIFF2024534067000002.tif31147 SEQ ID NO:36 (SEQ ID NO:05 without signal sequence): E VQLVESGGDL VKPGGSLKLS CAASGFTFSS YGMSWVRQTP DKRLEWVATI SSGGSYIYYP DSVKGRFTIS RDNAKNTLYL QMSSLKSEDT AMYYCARLGM ITTGYAMDYW GQGTSVTVSS SEQ ID NO: 06: DVLMTQTPLS LPVSLGDQAS ISCRSSQTIV HSTGHTYLEW FLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP YTFGGGTKLE IK SEQ ID NO:37 (SEQ ID NO:07 without signal sequence): EV KLLESGGGLV QPGGSLKLSC AASGFDFSRY WMNWVRQAPG KGLEWIGEIT PDSSTINYTP SLKDKFIISR DNAKNTLYLQ MIKVRSEDTA LYYCVRPYDY GAWFASWGQG TLVTVSA SEQ ID NO: 08: QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYANWVQE KPDHLFTGLI GGTNKRAPGV PARFSGSLIG DKAALTITGA QTEDEAIYFC ALWYSNHWVF GGGTKLTVL

[0135] In a particular embodiment of all aspects and embodiments of the invention, the anti-variant (human) Fc region antibody (anti-AAA antibody) used for the preparation of the multivalent antibody according to the invention is ● specifically binds to an epitope on the variant (human) Fc region of the IgG1 subclass that contains amino acid residues (A)253, (A)310 and (A)435 (numbering according to the Kabat EU index); ● Binds specifically to variant (human) Fc regions of the IgG1 subclass that have alanine amino acid residues at positions 253, 310 and 435 (numbering according to the Kabat EU index); does not (specifically) bind to wild-type (human) Fc regions of the IgG1 subclass having an isoleucine amino acid residue at position 253, a histidine amino acid residue at position 310, and a histidine amino acid residue at position 435 (numbering according to the Kabat EU index); does not (specifically) bind to a (human) Fc region of the IgG1 subclass having an isoleucine amino acid residue at position 253, a histidine amino acid residue at position 310, a histidine amino acid residue at position 435, a glycine amino acid residue at position 329, an alanine amino acid residue at position 234 and an alanine amino acid residue at position 235 (numbering according to the Kabat EU index); ● Does not (specifically) bind to variant (human) Fc regions of IgG1 subclass having an isoleucine amino acid residue at position 253, a histidine amino acid residue at position 310, a histidine amino acid residue at position 435, a proline amino acid residue at position 329, a leucine amino acid residue at position 234, and a leucine amino acid residue at position 235 (numbering according to the Kabat EU index).

[0136] The term "does not (specifically) bind" means that in an assay in which binding is determined, the results obtained do not differ significantly from the results obtained with samples that do not contain the antibody in question, i.e. blank or buffer samples.

[0137] In certain embodiments of all aspects and embodiments of the invention, the variant (human) Fc region is an Fc region of the human IgG1 or IgG4 subclass with the mutations I253A, H310A and H435A (numbering according to Kabat EU index).

[0138] In certain embodiments of all aspects and embodiments of the invention, the anti-Fc region antibodies that specifically bind to Fc regions of the IgG1 subclass comprising the amino acid residues alanine at positions 253, 310 and 435 (numbering according to Kabat EU index) used for the generation of multimers according to the invention comprise at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09 or 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12, 13 or 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28, 29 or 30.

[0139] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies of the invention comprise (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09 or 10, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12 or 13 or 14, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 16, 17 or 18; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23 or 24, (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28, 29 or 30.

[0140] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 28.

[0141] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 29.

[0142] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 30.

[0143] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise any one or more amino acids substituted at the following HVR positions: - in HVR-H1 (SEQ ID NO: 11): position 5; - in HVR-H2 (SEQ ID NO: 15): positions 3, 7, 8, 11, 12; - in HVR-H3 (SEQ ID NO: 19): positions 2, 10; - in HVR-L1 (SEQ ID NO: 25): positions 3, 14; - in HVR-L2 (SEQ ID NO: 27): at position 4; and - in HVR-L3 (SEQ ID NO: 31): positions 1, 6.

[0144] In certain embodiments, the substitutions are conservative substitutions as provided herein. In certain embodiments, any one or more of the following amino acid residues (alone or in any one of a combination independent of each other) may be present in any combination: - in HVR-H1 (SEQ ID NO: 11): at position 5, a neutral hydrophilic amino acid residue selected from the group of amino acid residues consisting of S, T, N and Q; - in HVR-H2 (SEQ ID NO: 15): at position 3, a neutral hydrophilic or acidic amino acid residue selected from the group of amino acid residues consisting of S, T, N, Q, D and E, at position 7, a neutral hydrophilic or basic amino acid residue selected from the group of amino acid residues consisting of S, T, N, Q, H, K and R, at position 8, a neutral hydrophilic amino acid residue or a residue influencing chain orientation selected from the group of amino acid residues consisting of S, T, N, Q, G and P, at position 11, a neutral hydrophilic or aromatic amino acid residue or a residue influencing chain orientation selected from the group of amino acid residues consisting of S, T, N, Q, G, P, W, Y and F, at position 12, a neutral hydrophilic amino acid residue or a residue influencing chain orientation selected from the group of amino acid residues consisting of S, T, N, Q, G and P; - in HVR-H3 (SEQ ID NO: 19): at position 2, a hydrophobic or aromatic amino acid residue selected from the group of amino acid residues consisting of M, A, V, L, I, W, Y and F, at position 10, a neutral hydrophilic or aromatic amino acid residue selected from the group of amino acid residues consisting of S, T, N, Q, W, Y and F; - in HVR-L1 (SEQ ID NO: 25): at position 3, a neutral hydrophilic amino acid residue selected from the group of amino acid residues consisting of S, T, N and Q, at position 14, a neutral hydrophilic or acidic amino acid residue selected from the group of amino acid residues consisting of S, T, N, Q, D and E; - in HVR-L2 (SEQ ID NO: 27): at position 4, an acidic or basic amino acid residue selected from the group of amino acid residues consisting of E, D, H, K and R; and - in HVR-L3 (SEQ ID NO: 31): at position 1, a hydrophobic amino acid residue selected from the group of amino acid residues consisting of M, A, V, L and I, at position 6, a neutral hydrophilic or acidic amino acid residue selected from the group of amino acid residues consisting of S, T, N, Q, D and E.

[0145] All possible combinations of the above substitutions are encompassed in the consensus sequences of SEQ ID NOs:11, 15, 19, 25, 27, and 31.

[0146] SEQ ID NO: 07 is the mouse sequence of the heavy chain variable domain containing an 18 amino acid signal peptide at the N-terminus. SEQ ID NO: 37 is SEQ ID NO: 07 without the signal sequence.

[0147] SEQ ID NO:02 and SEQ ID NO:04 are the murine sequences of the light chain variable domain, respectively, including a signal peptide of 19 amino acid residues at the N-terminus.

[0148] In a particular embodiment of all aspects and embodiments of the invention, the antibody used to generate the multivalent antibody according to the invention comprises a heavy chain variable domain amino acid sequence derived from SEQ ID NO: 01 and a light chain variable domain amino acid sequence derived from SEQ ID NO: 02, and the humanized antibody has the same binding specificity as a chimeric or murine antibody comprising the amino acid sequence of SEQ ID NO: 01 as the heavy chain variable domain and the amino acid sequence of SEQ ID NO: 02 as the light chain variable domain.

[0149] In a particular embodiment of all aspects and embodiments of the invention, the antibody used to generate the multivalent antibody according to the invention comprises a heavy chain variable domain amino acid sequence derived from SEQ ID NO: 03 and a light chain variable domain amino acid sequence derived from SEQ ID NO: 04, and the humanized antibody has the same binding specificity as a chimeric or murine antibody comprising the amino acid sequence of SEQ ID NO: 03 as the heavy chain variable domain and the amino acid sequence of SEQ ID NO: 04 as the light chain variable domain.

[0150] In a particular embodiment of all aspects and embodiments of the invention, the antibody used to generate the multivalent antibody according to the invention comprises a heavy chain variable domain amino acid sequence derived from SEQ ID NO: 07 and a light chain variable domain amino acid sequence derived from SEQ ID NO: 08, and the humanized antibody has the same binding specificity as a chimeric or murine antibody comprising the amino acid sequence of SEQ ID NO: 07 as the heavy chain variable domain and the amino acid sequence of SEQ ID NO: 08 as the light chain variable domain.

[0151] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 01, 03 and 37. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an anti-variant (human) Fc region antibody comprising the sequence retains the ability to bind to a variant (human) Fc region. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in any one of SEQ ID NOs: 01, 03 and 37. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-variant (human) Fc region antibody comprises a VH sequence of any one of SEQ ID NOs: 01, 03, and 37, including post-translational modifications of the sequence.

[0152] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 02, 04 or 08. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an anti-variant (human) Fc region antibody comprising the sequence retains the ability to bind to the variant (human) Fc region. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NOs: 02, 04 or 08. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-variant (human) Fc region antibody comprises a VL sequence of SEQ ID NO: 02, 04, or 08, including post-translational modifications of the sequence.

[0153] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In certain embodiments, the antibodies comprise (i) the VH and VL sequences of SEQ ID NO: 01 and SEQ ID NO: 02, or (ii) the VH and VL sequences of SEQ ID NO: 03 and SEQ ID NO: 04, respectively, or (iii) the VH and VL sequences of SEQ ID NO: 37 and SEQ ID NO: 08, including post-translational modifications of those sequences.

[0154] In a particular embodiment of all aspects and embodiments of the invention, the anti-variant (human) Fc region antibody (anti-PG antibody) used for the preparation of the multivalent antibody according to the invention described herein is ● specifically binds to an epitope on the variant (human) Fc region of the IgG1 subclass that contains amino acid residues (A)234, (A)235 and (G)329 (numbering according to the Kabat EU index); ● specifically binds to a variant (human) Fc region of the IgG1 subclass having alanine amino acid residues at positions 234 and 235 and a glycine amino acid residue at position 329 (numbering according to the Kabat EU index); ● specifically binds to a variant (human) Fc region of the IgG1 subclass having alanine amino acid residues at positions 234 and 235, a glycine amino acid residue at position 329, an isoleucine amino acid residue at position 253, a histidine amino acid residue at position 310, and a histidine amino acid residue at position 435 (numbering according to the Kabat EU index); ● specifically binds to a variant (human) Fc region of the IgG1 subclass having alanine amino acid residues at positions 234, 235, 253, 310 and 435, and a glycine amino acid residue at position 329 (numbering according to Kabat); does not (specifically) bind to wild-type (human) Fc regions of the IgG1 subclass that have leucine amino acid residues at positions 234 and 235 and a proline amino acid residue at position 329 (numbering according to Kabat); does not (specifically) bind to wild-type (human) Fc regions of the IgG1 subclass having leucine amino acid residues at positions 234 and 235, and a proline amino acid residue at position 329, and an isoleucine amino acid residue at position 253, and a histidine amino acid residue at position 310, and a histidine amino acid residue at position 435 (numbering according to Kabat); ● Does not (specifically) bind to variant (human) Fc regions of the IgG1 subclass having leucine amino acid residues at positions 234 and 235, and a proline amino acid residue at position 329, and an alanine amino acid residue at position 253, and an alanine amino acid residue at position 310, and an alanine amino acid residue at position 435 (numbering according to Kabat).

[0155] The immunization carried out for the preparation of anti-PG antibodies was carried out using human IgG1 with P329G, L234A and L235AFc region substitutions, so it was expected that an antibody that specifically binds to these amino acid residues would be obtained.Surprisingly, the obtained antibody specifically binds to human IgG1 and Fc region fragments with only the P329G mutation, regardless of the presence or absence of L234A and L235A mutations, while human wild-type IgG1 and human IgG1 with mutations L234A and L235A did not bind.Therefore, the anti-PG antibody used to prepare the multimer according to the present invention is specific to a single P329G substitution in the Fc region of human IgG1.

[0156] In a particular embodiment of all aspects and embodiments of the invention, the variant (human) Fc region is an Fc region of the human IgG1 or IgG4 subclass with the mutation P329G (numbering according to Kabat EU index).

[0157] In a particular embodiment of all aspects and embodiments of the invention, the antibody used to generate the multivalent antibody according to the invention is an anti-Fc region antibody that specifically binds to an Fc region of the IgG1 subclass comprising the amino acid residue glycine at position 329 (and optionally the amino acid residues alanine at positions 234 and 235) (numbering according to Kabat EU index) comprising at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0158] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise: (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 22; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0159] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 35.

[0160] In a particular embodiment of all aspects and embodiments of the invention, in the antibodies used to generate the multivalent antibodies according to the invention, the amino acids of the anti-variant (human) Fc region antibody are substituted at the following HVR positions: - in HVR-L1 (SEQ ID NO: 33): position 9.

[0161] In certain embodiments, the substitutions are conservative substitutions as provided herein. In certain embodiments, any one or more of the following amino acid residues (alone or in any one of a combination independent of each other) may be present in any combination: - in HVR-L1 (SEQ ID NO: 33): at position 9, a neutral hydrophilic amino acid residue or a residue that influences chain orientation selected from the group of amino acid residues consisting of S, T, N, Q, G and P.

[0162] All possible combinations of the above substitutions are encompassed in the consensus sequence of SEQ ID NO:33.

[0163] SEQ ID NO: 05 is a mouse sequence of a heavy chain variable domain including a 19 amino acid signal peptide at the N-terminus. SEQ ID NO: 36 is SEQ ID NO: 05 without the signal sequence.

[0164] In a particular embodiment of all aspects and embodiments of the invention, the antibody used to generate the multivalent antibody according to the invention comprises a heavy chain variable domain amino acid sequence derived from SEQ ID NO: 05 and a light chain variable domain amino acid sequence derived from SEQ ID NO: 06, and the humanized antibody has the same binding specificity as a chimeric or murine antibody comprising the amino acid sequence of SEQ ID NO: 05 as the heavy chain variable domain and the amino acid sequence of SEQ ID NO: 06 as the light chain variable domain.

[0165] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an anti-variant (human) Fc region antibody comprising the sequence retains the ability to bind to the variant (human) Fc region. In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 36. In certain embodiments, the substitutions, insertions or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-variant (human) Fc region antibody comprises a VH sequence such as SEQ ID NO:36, including post-translational modifications of the sequence.

[0166] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 06. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an anti-variant (human) Fc region antibody comprising the sequence retains the ability to bind to the variant (human) Fc region. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 06. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., in the FRs). Optionally, the anti-variant (human) Fc region antibody comprises the VL sequence of SEQ ID NO: 06, including post-translational modifications of the sequence.

[0167] In certain embodiments of all aspects and embodiments of the invention, the antibodies used to generate the multivalent antibodies according to the invention comprise a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In certain embodiments, the antibodies comprise the VH and VL sequences of SEQ ID NO: 36 and SEQ ID NO: 06, including post-translational modifications of the sequences.

[0168] In certain embodiments of all aspects and embodiments of the present invention, the anti-variant (human) Fc region antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In certain embodiments, the anti-variant (human) Fc region antibody is an antibody fragment, such as a diabody or a F(ab')2 fragment. In certain embodiments, the antibody is a full-length antibody, such as an intact antibody of the human IgG1 subclass as defined herein, or other antibody classes or isotypes.

[0169] One embodiment of the invention is an antibody comprising four or six binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three or four amino acid changes compared to a wild-type Fc region of the human IgG1 subclass.

[0170] One aspect of the invention is a method for producing a medicament comprising the steps of: i) a bivalent full-length antibody, each of which comprises two binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three or four amino acid changes compared to the wild-type Fc region of the human IgG1 subclass; or ii) a (Fab')2 fragment of a bivalent full-length antibody, each of which comprises two binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three or four amino acid changes compared to the wild-type Fc region of the human IgG1 subclass. It is an antibody multimer comprising:

[0171] One embodiment of the present invention is an antibody or antibody multimer according to the present invention, wherein the binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to a wild-type Fc region of the human IgG1 subclass is a binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (numbering according to the Kabat EU index).

[0172] One aspect of the invention is an antibody or antibody multimer according to the invention, wherein each of the binding sites, independently of the others, (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or (4) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 35; or (5) A mixture of any one of (1) to (4) Includes any of the following.

[0173] One embodiment of the present invention is an antibody or antibody multimer according to the present invention, wherein the binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass which comprises 1, 2, 3 or 4 amino acid changes compared to a wild-type Fc region of the human IgG1 subclass is a binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass which comprises the amino acid residue glycine at position 329 and the amino acid residues alanine at positions 234 and 235 (numbering according to the Kabat EU index).

[0174] One aspect of the invention is an antibody multimer according to the invention, wherein the multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer.

[0175] One aspect of the present invention is the use of an antibody or antibody multimer according to the present invention as a positive control in an in vitro (bridging) immunoassay.

[0176] One aspect of the present invention is the use of an antibody or antibody multimer according to the present invention as a standard in an in vitro (bridging) immunoassay.

[0177] One aspect of the invention is the use of an antibody or antibody multimer according to the invention for generating a calibration function for quantitatively determining anti-drug antibodies to a drug antibody, which anti-drug antibody binds to one or more amino acid residues in the Fc region of the drug antibody that are altered compared to the wild-type Fc region.

[0178] One aspect of the invention is an immunoassay for determining the presence and / or amount of anti-drug antibodies in a (serum-containing) sample, comprising: the anti-drug antibody binds to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region; The immunoassay comprises a drug antibody as a capture antibody and a tracer antibody, The antibodies or antibody multimers according to the invention are characterized in that they are used as positive controls or as calibration standards in immunoassays.

[0179] One aspect of the invention is an immunoassay using an antibody or antibody multimer according to the invention, wherein the immunoassay is a bridging ELISA.

[0180] One aspect of the invention is an immunoassay in which an antibody or antibody multimer according to the invention is used as a calibration standard and is used to generate a calibration function for quantitatively determining anti-drug antibodies to drug antibodies.

[0181] One aspect of the invention is a method for producing antibody multimers according to the invention by chemical conjugation of bivalent full-length antibodies, each comprising two binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains 1, 2, 3 or 4 amino acid changes compared to the wild-type Fc region of the human IgG1 subclass, using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS).

[0182] One aspect of the invention is a method for the production of an antibody multimer according to the invention, wherein the multimer is a multimer of full-length antibodies.

[0183] One aspect of the present invention is a method for producing an antibody multimer, comprising the steps of: Chemically cross-linking a full-length antibody that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (numbering according to the Kabat EU index) using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS); The antibody, (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30 Includes.

[0184] Antibodies comprising a binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three or four amino acid changes compared to a wild-type Fc region of the human IgG1 subclass can be generated using any method known in the art.

[0185] For example, antibodies can be prepared by administering to an experimental animal an immune gene that contains at least the respective variant portion of the Fc region. Suitable constructs for presenting variant Fc regions are reported, for example, in WO 2012 / 150320.

[0186] Antibodies can also be produced by hybridoma-based methods. For example, human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991)). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0187] Antibodies can also be generated by isolating variable domain sequences selected from phage display libraries of human origin, which can then be combined with the desired human constant domains.

[0188] Techniques for selecting antibodies from antibody libraries are known in the art.

[0189] In certain embodiments, an anti-variant (human) Fc region antibody according to any of the above embodiments may incorporate any of the features described in sections 1-3 below, either alone or in combination.

[0190] 1. Antibody fragment In certain embodiments, the antibodies used to generate the multivalent antibodies of the present invention are bivalent antibody fragments. Bivalent antibody fragments include, but are not limited to, F(ab')2 and other fragments described below, so long as they are bivalent. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134. For a discussion of Fab and F(ab')2 fragments that contain salvage receptors that bind epitope residues and have increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0191] Diabodies are antibody fragments that have two antigen binding sites, which can be bivalent or bispecific.See, for example, EP 0404097; WO 1993 / 01161; Hudson, PJ et al., Nat.Med.9 (2003) 129-134; and Holliger, P. et al., Proc.Natl.Acad.Sci.USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat.Med.9 (20039 129-134).

[0192] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0193] 2. Chimeric and humanized antibodies In certain embodiments, the antibody used to generate the multivalent antibody according to the present invention is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison, SL et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855. In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from those of the parent antibody. The chimeric antibody includes an antigen-binding fragment thereof.

[0194] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0195] Humanized antibodies and methods for their production are reviewed, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and are described, for example, in Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri, SV et al., Methods 36 (2005) 25-34 (describing grafting of specificity determining regions (SDRs)); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing"); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing a "guide selection" approach to FR shuffling).

[0196] Human framework regions that may be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims, MJ et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, LG et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening of FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (19969 22611-22618)).

[0197] 3. Antibody variants In certain embodiments, amino acid sequence variants of the antibodies used to generate the multivalent antibodies according to the invention provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, so long as the final construct has the desired properties, e.g., antigen binding.

[0198] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". Substantial additional changes are shown in the following table under the heading of "exemplary substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest, and the product screened for the desired activity, for example, retaining / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

[0199] TIFF2024534067000003.tif114139Amino acids can be classified according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0200] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0201] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further testing has an altered (e.g., improved) specific biological property compared to the parent antibody (e.g., improved affinity, reduced immunogenicity) and / or substantially retains a specific biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage are screened for a specific biological activity (e.g., binding affinity).

[0202] Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such alterations may be made in HVR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196), and / or residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from the secondary libraries is described, for example, in Hoogenboom, HR et al., Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0203] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind to antigen.For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.Such changes may, for example, be outside of the antigen contact residues within the HVRs.In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0204] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085. In this method, a residue or group of targeted residues (e.g., charged residues, e.g., Arg, Asp, His, Lys and Glu) are identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they have the desired properties.

[0205] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which extends the serum half-life of the antibody.

[0206] b) Glycosylation variants In certain embodiments, the antibodies used to generate the multivalent antibodies according to the invention are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0207] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32. The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the invention may be performed to generate antibody variants with specific improved properties.

[0208] In certain embodiments, the antibodies used to generate the multivalent antibodies according to the invention are antibody variants with carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region, although Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294-300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication Nos. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication Nos. 2003 / 0115614; 2002 / 0164328; 2004 / 0093621; 2004 / 0132140; 2004 / 0110704; 2004 / 0110282 ... 004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545; US Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 2006) 680-688; WO 2003 / 085107).

[0209] Further included are antibody variants with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Patent No. 6,602,684; and U.S. Patent Application Publication No. 2005 / 0123546. Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0210] c) Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody used to generate a multivalent antibody according to the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0211] In certain embodiments, it is contemplated that the antibodies used to generate the multivalent antibodies of the present invention will retain some but not all effector functions, making the antibody variants desirable candidates for applications where the half-life of the antibody in vivo is important but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 of Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492, page 464. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assay methods may be used (e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.) and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays may be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity (see, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vitro clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18 (2006: 1759-1769)).

[0212] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0213] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0214] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0215] In certain embodiments, modifications are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0216] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593, and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434) have been described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0217] For other examples of Fc region variants, see also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0218] The C-terminus of the heavy chain of the antibody used to generate the multimers according to the invention may be a complete C-terminus terminating in the amino acid residue PGK. The C-terminus of the heavy chain may also be a shortened C-terminus in which one or two of the C-terminal amino acid residues have been removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus terminating in PG.

[0219] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues are present at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which may be used to conjugate the antibody to other sites, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, any of one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be created, for example, as described in U.S. Pat. No. 7,521,541.

[0220] B. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In certain embodiments, isolated nucleic acids are provided that encode the anti-variant (human) Fc region antibodies described herein. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., transformed): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In certain embodiments, the host cell is a eukaryotic cell, for example, a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, PS20 cell). In certain embodiments, a method of making an anti-variant (human) Fc region antibody is provided, comprising culturing a host cell comprising nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0221] For recombinant production of anti-variant (human) Fc region antibodies, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0222] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237, U.S. Patent No. 5,789,199 and U.S. Patent No. 5,840,523. See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003) pp. 245-254, which describes the expression of antibody fragments in E. coli. After expression, the antibody may be isolated from the bacterial cell paste in an appropriate fraction and may be further purified.

[0223] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are suitable as cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains that have been "humanized" in their glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0224] Also suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that may be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0225] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0226] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham, FL, et al., J. GenVirol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); see, e.g., Mather, JP, et al., Annals Other useful mammalian host cell lines include DHFR cells, described in NYAcad.Sci.383(1982)44-68; MRC5 cells; and FS4 cells. - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0227] C. Assay The multivalent anti-variant (human) Fc region antibodies according to the present invention may be used in a variety of assays known in the art.

[0228] The multivalent antibodies according to the invention are particularly useful when therapeutic antibodies comprising the respective mutations in the Fc region or anti-drug antibodies directed against such therapeutic antibodies must be detected, for example, in a sample.

[0229] In certain embodiments of all aspects and embodiments according to the invention, the drug antibody comprises: i) the mutations P329G or P329G, L234A and L235A, and / or ii) Mutations I253A, H310A and H435A Includes.

[0230] In certain embodiments of all aspects and embodiments according to the invention, the antibody comprising the respective mutation has i) the mutations P329G or P329G, L234A and L235A, and / or ii) Mutations I253A, H310A and H435A The antibody comprises:

[0231] One embodiment according to the invention is the use of a multivalent antibody according to the invention in an (antigen bridging) immunoassay, either as a positive control or as a (calibration) standard for determining anti-drug antibodies binding to / specifically to a therapeutic antibody (in a sample) comprising the respective mutation in its Fc region (i.e. an antibody comprising the respective mutation in its Fc region). The respective other reagent required for detection and capture is the therapeutic antibody, derivatized, immobilized or labeled depending on the detection and capture.

[0232] This assay is applicable to any non-human serum, in a particular embodiment, the use is for measuring in serum samples of non-human laboratory animals.

[0233] Such an assay has a lower limit of quantitation (threshold) of less than 100 pg / mL, for example 40-80 pg / mL (assay concentration) in 10% cynomolgus monkey serum.

[0234] One embodiment according to the invention is the use of a multivalent antibody according to the invention in an immunoassay for determining anti-drug antibodies against a therapeutic antibody, the therapeutic antibody comprising the respective mutation in the Fc region (in the sample).

[0235] In certain embodiments of all aspects and embodiments of the present invention, the drug antibody is used as a capture antibody. The capture antibody is, in certain embodiments, immobilized on a solid surface, which in one preferred embodiment is the well (wall or bottom or both) of a multi-well plate.

[0236] In certain embodiments of all aspects and embodiments of the present invention, the drug antibody is used as a tracer antibody, which is conjugated to a suitable label in order to be detected.

[0237] In certain embodiments of all aspects and embodiments of the invention, the sample is obtained from an experimental animal selected from marmosets and tamarins, Old World monkeys, dwarf and mouse lemurs, gibbons and little apes, lemurs, and hybrids thereof, or from a human. In certain embodiments, the sample is obtained from a rhesus monkey, or a marmoset monkey, or a baboon monkey, or a cynomolgus monkey, or a human. In certain embodiments, the experimental animal is a macaque or a macaque monkey. In certain embodiments, the sample is obtained from a cynomolgus monkey, or a rhesus monkey, or a human.

[0238] In certain embodiments of all aspects and embodiments of the present invention, the immunoassay is a sandwich immunoassay.

[0239] In certain embodiments of all aspects and embodiments of the present invention, the drug (therapeutic) antibody is linked to its conjugation partner by chemical coupling via the N-terminus and / or ε-amino group (lysine), the ε-amino groups of different lysines, the carboxy-, sulfhydryl-, hydroxyl-, and / or phenolic functional groups of the amino acid backbone of the antibody, and / or the sugar alcohol groups of the carbohydrate structure of the antibody. In certain embodiments, the capture antibody is immobilized via a specific binding pair. In a preferred embodiment, the capture antibody is conjugated to biotin and immobilization is via immobilized avidin or streptavidin. In certain embodiments, the tracer antibody is conjugated to a detectable label via a specific binding pair. In a preferred embodiment, the tracer antibody is conjugated to digoxigenin and linkage to the detectable label is via an antibody against digoxigenin. In certain embodiments, the drug antibody is a human or humanized antibody. In certain embodiments, the human or humanized antibody is a monoclonal antibody.

[0240] One embodiment according to the invention is a method for determining the correct / proper functioning of anti-drug antibodies against a therapeutic antibody with an altered (effector function silent) Fc region or a sandwich / bridging immunoassay for determining said Fc region (in a sample), comprising: a) incubating a multivalent antibody according to the present invention with a drug (therapeutic) antibody or its Fc region (fragment) immobilized on a solid surface to form a dimeric complex; b) incubating the dimeric complex with a drug (therapeutic) antibody conjugated to a detectable label to form a ternary complex; and c) determining the correct / proper functioning of the sandwich / bridging immunoassay if a ternary complex is formed in step b) / if the ternary complex formed in step b) can be detected; The method includes:

[0241] One embodiment according to the invention is a method for calibrating a sandwich / bridging immunoassay for determining anti-drug antibodies against a therapeutic antibody with an altered (effector function silent) Fc region, or its Fc region (in a sample), comprising: a) separately incubating the multivalent antibody according to the present invention at at least two different concentrations with a drug (therapeutic) antibody or its Fc region (fragment) immobilized on a solid surface to form a dimeric complex; b) separately incubating each of said dimeric complexes with a drug (therapeutic) antibody conjugated to a detectable label to form a ternary complex; c) determining the amount of each of the ternary complexes formed in step c) by determining the amount of detectable label; and d) calculating a calibration curve based on the amounts determined in step c), thereby calibrating the sandwich / bridging immunoassay. The method includes:

[0242] One aspect of the invention is an anti-drug antibody immunoassay for determining the presence of anti-drug antibodies to Fc receptor binding inhibited human or humanized drug antibodies (i.e. antibodies comprising the respective mutations in the Fc region) (in a sample) / Fc region of Fc receptor binding inhibited human or humanized drug antibodies (i.e. antibodies comprising the respective mutations in the Fc region), the method comprising the following steps in the following order: a) incubating a solid phase having an Fc receptor binding-inhibiting human or humanized drug antibody or an Fc region fragment thereof immobilized thereon with a sample containing mammalian serum (so that a solid phase-bound drug antibody-anti-drug antibody complex is formed); b) incubating the solid phase (to which the drug antibody-anti-drug antibody complex formed in step a) is bound) with a drug antibody or its Fc region fragment conjugated to a detectable label; and c) determining the formation of a solid phase bound complex in step b) by determining the presence of a detectable label, thereby determining the presence of anti-drug antibodies against the Fc receptor binding-inhibiting human or humanized drug antibody in the sample. Including, Thereby, the correct functioning of an immunoassay using the multivalent antibody according to the invention has been determined.

[0243] One aspect of the invention is an anti-drug antibody immunoassay for determining the presence of anti-drug antibodies to Fc receptor binding inhibited human or humanized drug antibodies (i.e. antibodies comprising the respective mutations in the Fc region) (in a sample) / Fc region of Fc receptor binding inhibited human or humanized drug antibodies (i.e. antibodies comprising the respective mutations in the Fc region), the method comprising the following steps in the following order: a) incubating a solid phase having an immobilized Fc receptor binding-inhibiting human or humanized drug antibody with a sample containing mammalian serum (so that a solid phase-bound drug antibody-anti-drug antibody complex is formed); b) incubating the solid phase (to which the drug antibody-anti-drug antibody complex formed in step a) is bound) with a drug antibody conjugated to a detectable label; and c) determining the formation of a solid phase bound complex in step b) by determining the presence of a detectable label, thereby determining the presence of anti-drug antibodies against the Fc receptor binding-inhibiting human or humanized drug antibody in the sample. Including, Thereby, a standard / calibration curve for an immunoassay has been determined using the multivalent antibody according to the present invention.

[0244] In a particular embodiment of all aspects and embodiments according to the present invention, after each incubation step, one of the following steps is performed: - washing the solid phase to remove unbound compounds; continues.

[0245] In certain embodiments of all aspects and embodiments according to the invention, determining the presence or amount of a detectable label comprises: - determining the formation of a solid phase bound complex in the previous step by determining the presence of a detectable label, and determining the amount of complex by determining the amount of the determined label. This is carried out by.

[0246] In certain embodiments of all aspects and embodiments of the invention, the Fc receptor binding inhibiting human or humanized drug antibody is of the human IgG1 or IgG4 subclass.

[0247] In certain embodiments of all aspects and embodiments of the invention, the Fc receptor binding-inhibited human or humanized drug antibody is of the human IgG1 subclass and has the mutations L234A, L235A and P329G in both Fc region polypeptides, or the Fc receptor binding-inhibited human or humanized drug antibody is of the human IgG4 subclass and has the mutations S228P, L235E and P329G in both Fc region polypeptides (numbering according to the EU numbering system according to Kabat).

[0248] In certain embodiments of all aspects and embodiments of the invention, the Fc receptor binding-inhibited human or humanized drug antibody is of the human IgG1 subclass and has the mutations I253A, H310A and H435A in both Fc region polypeptides (numbering according to the EU numbering system according to Kabat).

[0249] In certain embodiments of all aspects and embodiments of the invention, the Fc receptor binding inhibited human or humanized drug antibody is a bispecific antibody, or a trispecific antibody, or a tetraspecific antibody, or a pentaspecific antibody, or a hexaspecific antibody. In a preferred embodiment, the Fc receptor binding inhibited human or humanized drug antibody is a bispecific antibody.

[0250] In certain embodiments of all aspects and embodiments of the invention, the mammalian serum is human serum or cynomolgus monkey serum or mouse serum.

[0251] In certain embodiments of all of the aspects and embodiments of the invention, the presence and / or amount of label is determined using an enzyme-linked color reaction, surface plasmon resonance, electrochemiluminescence, or radioimmunoassay.

[0252] In certain embodiments of all aspects and embodiments of the present invention, the solid phase is conjugated to a first member of a binding pair and the compound immobilized on the solid phase is conjugated to a second member of the binding pair. Such binding pairs (first member / second member) are in certain embodiments selected from streptavidin or avidin / biotin, antibody / antigen (see, for example, Hermanson, GT et al., Bioconjugate Techniques, Academic Press (1996)), lectin / polysaccharide, steroid / steroid binding protein, hormone / hormone receptor, enzyme / substrate, IgG / protein A and / or G, etc. In certain embodiments, the compound immobilized on the solid phase is conjugated to the second member of the binding pair by chemically binding via the N-terminus and / or ε-amino group (lysine), the ε-amino group of a different lysine, the carboxy-, sulfhydryl-, hydroxyl- and / or phenol functional groups of the amino acid backbone of the polypeptide, and / or the sugar alcohol group of the carbohydrate structure of the polypeptide.

[0253] Such conjugation through different amino groups can be carried out in a first step by acylation of some of the ε-amino groups with a chemical protectant, for example by citraconylation. In a second step, conjugation is carried out through the remaining amino groups. The citraconylation is then removed and the binding partner is immobilized on the solid phase through the remaining free amino groups, i.e. the resulting binding partner is immobilized on the solid phase through the amino groups that are not protected by citraconylation. Suitable chemical protectants form bonds at unprotected side chain amines, which are less stable than and different from N-terminal bonds. Many such chemical protectants are known (see, for example, EP 0651761). In certain embodiments, the chemical protectant comprises a cyclic dicarboxylic acid anhydride, such as maleic anhydride or citraconic anhydride.

[0254] In a preferred embodiment, the first member of the binding pair is streptavidin, and the second member of the binding pair is biotin.In certain embodiments, the solid phase is conjugated with streptavidin, and the compound immobilized on the solid phase is biotinylated.In certain embodiments, the solid phase is streptavidin-coated paramagnetic beads or streptavidin-coated sepharose beads or streptavidin-coated wells of multi-well plates.

[0255] In certain embodiments of all aspects and embodiments of the invention, the compound conjugated to the solid phase is a mixture comprising at least two compounds, the at least two compounds being conjugated to biotin and thereby immobilized on the solid phase at different sites.

[0256] In a particular embodiment of all aspects and embodiments according to the invention, multimerization of the multimers according to the invention is carried out by chemical conjugation via the N-terminus and / or ε-amino groups (lysine), the ε-amino groups of different lysines, the carboxy-, sulfhydryl-, hydroxyl- and / or phenolic functional groups of the amino acid backbone of the polypeptide and / or the sugar alcohol groups of the carbohydrate structure of the polypeptide.

[0257] Coupling through different amino groups can be carried out in a first step by acylation of some of the ε-amino groups with a chemical protecting agent, for example by citraconylation. In a second step, conjugation is carried out through the remaining amino groups. The citraconylation is then removed and the binding partner is conjugated to the solid phase through the remaining free amino groups, i.e. the resulting binding partner is conjugated to the solid phase through the amino groups that are not protected by citraconylation. Suitable chemical protecting agents form bonds at unprotected side chain amines, which are less stable than and different from N-terminal bonds. Many such chemical protecting agents are known (see, for example, EP 0651761). In certain embodiments, the chemical protecting agent comprises a cyclic dicarboxylic acid anhydride, such as maleic anhydride or citraconic anhydride.

[0258] In certain embodiments of all aspects and embodiments of the present invention, the drug antibody or its Fc region is conjugated to the solid phase by passive adsorption, as described, for example, in "Solid Phases in Immunoassay" by Butler, JE (1996) 205-225 and "Immunoassay" by Diamandis, EP and Christopoulos, TK (eds.) (1996) Academic Press (San Diego).

[0259] The term "drug antibody" refers to an antibody that is or has been tested in clinical trials for approval as a human therapeutic and can be administered to an individual for the treatment of a disease. In certain embodiments, the drug antibody is a monoclonal antibody. In further embodiments, the drug antibody is obtained from an ape, or an animal transformed with human antibody loci, or is a human monoclonal antibody, or is a humanized monoclonal antibody. In certain embodiments, the drug antibody is a human monoclonal antibody. In certain embodiments, the drug antibody is a humanized monoclonal antibody. Drug antibodies are widely used in the treatment of various diseases, such as oncological diseases (e.g., hematological and solid malignancies, including non-Hodgkin's lymphoma, breast cancer, and colorectal cancer), immunological diseases, central nervous system diseases, vascular diseases, or infectious diseases.

[0260] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to conformational epitopes is lost in the presence of denaturing solvents, but not to non-conformational epitopes.

[0261] The principles of different immunoassays are described, for example, in Hage, DS (Anal. Chem. 71 (1999) 294R-304R). Lu, B. et al. (Analyst 121 (1996) 29R-32R) report the oriented immobilization of antibodies for use in immunoassays. Avidin-biotin mediated immunoassays are reported, for example, in Wilchek, M. and Bayer, EA, Methods Enzymol. 184 (1990) 467-469.

[0262] Polypeptides and monoclonal antibodies and their constant domains contain several reactive amino acid side chains for binding to members of binding pairs such as polypeptides / proteins, polymers (e.g., PEG, cellulose or polystyrol), or enzymes. Chemically reactive groups of amino acids are, for example, amino groups (lysine, alpha-amino groups), thiol groups (cystine, cysteine ​​and methionine), carboxylic acid groups (aspartic acid, glutamic acid), and sugar alcohol groups. Such methods are described, for example, in "Bioconjugation", MacMillan Ref. Ltd., 1999, pp. 50-100.

[0263] One of the most common reactive groups on polypeptides and antibodies is the aliphatic ε-amine of the amino acid lysine. In general, nearly all polypeptides and antibodies contain abundant lysine. Lysine amines are reasonably good nucleophiles above pH 8.0 (pKa=9.18), and therefore react easily and cleanly with a variety of reagents to form stable bonds. Amine-reactive reagents react primarily with α-amino groups of lysine and proteins. Reactive esters, especially N-hydroxy-succinimide (NHS) esters, are among the most commonly used reagents for the modification of amine groups. The optimal pH for reactions in an aqueous environment is pH 8.0-9.0. Isothiocyanates are amine-modifying reagents that form thiourea bonds with proteins. Isothiocyanates react with protein amines in aqueous solution (optimally at pH 9.0-9.5). Aldehydes react with aliphatic and aromatic amines, hydrazines, and hydrazides under mild aqueous conditions to form imine intermediates (Schiff bases). Schiff bases can be selectively reduced with mild or strong reducing agents (such as sodium borohydride or sodium cyanoborohydride) to induce stable alkylamine linkages. Other reagents that have been used to modify amines are acid anhydrides. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a bifunctional chelating agent that contains two amine-reactive anhydride groups. It can react with the N-terminus and ε-amine groups of amino acids to form amide bonds. The anhydride ring opens to generate multivalent metal chelating arms that can strongly bind to metals in coordination complexes.

[0264] Another common reactive group in polypeptides and antibodies is the thiol residue from the sulfur-containing amino acid cystine and its reduction product cysteine ​​(or half-cystine). Cysteine ​​is more nucleophilic than amines and contains a free thiol group, which is generally the most reactive functional group in proteins. Thiols are generally reactive at neutral pH, so they can selectively bind to other molecules in the presence of amines. Because free sulfhydryl groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide groups or disulfide bonds. In such proteins, reduction of the disulfide bonds with reagents such as dithiothreitol (DTT) is necessary to generate reactive free thiols. Thiol-reactive reagents are reagents that bind to thiol groups on polypeptides to form thioether-linked products. These reagents react rapidly at slightly acidic to neutral pH, and therefore can react selectively in the presence of amine groups. The literature reports the use of several thiolated cross-linking reagents, such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA) and sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (Sulfo-LC-SPDP), to provide an efficient method to introduce multiple sulfhydryl groups via reactive amino groups. Haloacetyl derivatives, such as iodoacetamide, form thioether bonds and are also reagents for thiol modification. Further useful reagents are maleimides. The reaction of maleimides with thiol-reactive reagents is essentially the same as with iodoacetamide. Maleimides react rapidly at slightly acidic to neutral pH.

[0265] Another common reactive group in antibodies is carboxylic acid. Polypeptides and antibodies contain carboxylic acid groups at the C-terminal position and in the side chains of aspartic acid and glutamic acid. The relatively low reactivity of carboxylic acids in water usually makes it difficult to use these groups to selectively modify polypeptides and antibodies. In this case, the carboxylic acid group is usually converted to a reactive ester using a water-soluble carbodiimide and reacted with a nucleophilic reagent such as an amine, hydrazide, or hydrazine. The amine-containing reagent must be weakly basic to selectively react with the activated carboxylic acid in the presence of the more highly basic ε-amine of lysine to form a stable amide bond. If the pH is increased above 8.0, protein cross-linking can occur.

[0266] Sodium periodate can be used to oxidize the alcohol moiety of the sugar within the carbohydrate moiety attached to the antibody to an aldehyde. Each aldehyde group can be reacted with an amine, hydrazide, or hydrazine as described for carboxylic acids. Because the carbohydrate moieties are found primarily on the fragment crystallizable (Fc) region of the antibody, conjugation can be achieved by site-specific modification of the carbohydrate away from the antigen binding site. A Schiff base intermediate is formed that can be reduced to an alkylamine by reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents.

[0267] The term "sample" includes, but is not limited to, any quantity of material from an organism or former organism. Such organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. In certain embodiments, the sample is obtained from monkeys, particularly cynomolgus monkeys, or rabbits, or mice, or rats, or humans. In certain embodiments, such material includes, but is not limited to, whole blood or serum from an individual, which are the most widely used sample sources in clinical routine.

[0268] "Solid phase" refers to non-fluid substances, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass, and ceramics; gel substances such as silica, alumina, and polymer gels; capillaries, which may be made from polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. Solid phase components are distinguished from inert solid surfaces in that the "solid phase" includes at least one moiety on its surface that is intended to interact with substances in the sample. The solid phase may be a stationary component, such as a chip, tube, strip, cuvette, or microtiter plate, or a non-stationary component, such as beads and microparticles. A variety of microparticles may be used that allow either non-covalent or covalent attachment of proteins and other substances. Such particles include polymer particles, such as polystyrene and poly(methyl methacrylate); gold particles, such as gold nanoparticles, gold colloids; and ceramic particles, such as silica, glass, metal oxide particles, and the like. See, for example, Martin, CR et al., Analytical Chemistry-News & Features, 70 (1998) 322A-327A, or Butler, JE, Methods 22 (2000) 4-23.

[0269] In certain embodiments, the detectable label is selected from chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups, metal particles or haptens, such as digoxigenin. The detectable label may also be a photoactivatable crosslinking group, such as an azide or azirine group. Metal chelates that can be detected by electrochemiluminescence are also signal-emitting groups in certain embodiments, such as ruthenium chelates, such as ruthenium(bispyridyl)3. 2+ Chelates are particularly preferred.Suitable ruthenium labelling groups are described, for example, in EP 0 580 979, WO 90 / 05301, WO 90 / 11511 and WO 92 / 14138.

[0270] Some compounds used in the immunoassays and methods described herein are conjugated to members of binding pairs. Conjugation is, in certain embodiments, performed by chemical binding via the N-terminus and / or ε-amino group (lysine), the ε-amino group of different lysines, the carboxy-, sulfhydryl-, hydroxyl-, and / or phenolic functional groups of the amino acid backbone of the compound, and / or the sugar alcohol group of the carbohydrate structure of the compound. The conjugated compound is, in certain embodiments, a mixture of at least two compounds conjugated to members of a binding pair, where at least two compounds in the mixture differ in the sites at which they are conjugated to members of the binding pair. For example, the mixture may include conjugation via an amino acid of the amino acid backbone and conjugation via a sugar alcohol group of the carbohydrate. Also, for example, the mixture may include compounds conjugated to members of a binding pair via different amino acid residues of the amino acid backbone. The expression "different amino acid residues" refers to either two different types of amino acids, such as lysine and aspartic acid, or tyrosine and glutamic acid, or two amino acid residues of the amino acid backbone that differ in their positions in the amino acid sequence of the compound. In the latter case, the amino acids may be of the same type or different types. The term "different moieties" refers to differences in either the type of moiety, e.g., amino acid or sugar alcohol group, or the number of amino acids, e.g., of the amino acid backbone, at which the compound is conjugated to a member of a binding pair.

[0271] For direct detection, the labeling group can be selected from any known detectable marker group, such as dyes, luminescent labeling groups, such as chemiluminescent groups, such as acridinium esters or dioxetanes, or fluorescent dyes, such as fluoresceins, coumarins, rhodamines, oxazines, resorufins, cyanines and their derivatives.Other examples of labeling groups are luminescent metal complexes, such as ruthenium or europium complexes, enzymes, such as those used in ELISA or CEDIA (cloned enzyme donor immunoassays, e.g., EP-A-0061888), and radioisotopes.

[0272] Indirect detection systems include, for example, that the detection reagent, such as the detection antibody, is labeled with the first partner of a bioaffine binding pair. Examples of suitable binding pairs are hapten or antigen / antibody, biotin or biotin analogue, such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analogue / complementary nucleic acid, and receptor / ligand, such as steroid hormone receptor / steroid hormone. Preferred first binding pair members include haptens, antigens and hormones. Haptens such as digoxin and biotin and analogues thereof are particularly preferred. The second partner of such binding pair, such as antibody, streptavidin, etc., is usually labeled to allow direct detection, for example by labeling as described above.

[0273] Immunoassays are well known to those skilled in the art. Methods for carrying out such assays as well as practical applications and procedures are summarized in relevant textbooks. Examples of relevant textbooks are Tijssen, P., Preparation of enzyme-antibody or other enzyme-macromolecule conjugates ("Practice and theory of enzyme immunoassays" (1990), pages 221-278, edited by RH Burdon and vPH Knippenberg, Elsevier, Amsterdam) and various volumes of "Methods in Enzymology" (edited by S. P Colowick, NO Caplan, Academic Press), dealing with immunological detection methods, in particular volumes 70, 73, 74, 84, 92 and 121.

[0274] In all the above immunological detection methods, reagent conditions are selected that allow the binding of the reagents used, for example the binding of antibodies to their corresponding antigens. Those skilled in the art refer to the result of such binding events by using the term complex. The complexes formed in the assay method according to the present invention are correlated with the corresponding concentration of said therapeutic antibody by state-of-the-art procedures. Depending on the detection reagents used, this correlation step results in the concentration of total therapeutic antibody, active antibody or antigen-bound therapeutic antibody.

[0275] The methods and immunoassays according to the present invention are in vitro methods and immunoassays. EXAMPLES

[0276] III. Examples The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that other various embodiments may be practiced.

[0277] Example 1 material and method General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is available in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains are numbered and referenced according to the numbering according to Kabat (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0278] Recombinant DNA Technology Standard methods can be used to manipulate DNA, as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents are used according to the manufacturer's instructions.

[0279] Gene synthesis From the oligonucleotides made by chemical synthesis, desired gene segments can be prepared.By annealing and ligating oligonucleotides, including PCR amplification, long gene segments can be constructed that can be flanked by single restriction endonuclease cleavage sites, and then cloned via the indicated restriction sites.By DNA sequencing, the DNA sequence of the subcloned gene fragments can be confirmed.

[0280] DNA sequencing The DNA sequence can be determined by double stranded sequencing.

[0281] DNA and protein sequence analysis and sequence data management The GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 can be used for sequence creation, mapping, analysis, annotation and illustration.

[0282] Expression vector For the expression of the antibodies, expression plasmids for transient expression (e.g. in HEK293 cells) based either on a cDNA construct with or without the CMV-intron A promoter or on a genomic construct with the CMV promoter can be applied.

[0283] In addition to the antibody expression cassette, the vector contains: - an origin of replication that allows the plasmid to replicate in E. coli, - The β-lactamase gene that confers ampicillin resistance in E. coli may include.

[0284] The transcription unit for an antibody gene contains the following elements: - A unique restriction site at the 5' end - immediate early gene and promoter from human cytomegalovirus, - intron A sequence in the case of a cDNA construct, - a 5'-untranslated region derived from a human antibody gene, - immunoglobulin heavy chain signal sequence, - nucleic acids encoding each antibody chain, either as cDNA or in a genomic exon-intron configuration; - a 3' untranslated region carrying a polyadenylation signal sequence, - A unique restriction site at the 3' end It may be composed of:

[0285] The fusion genes encoding the antibody chains can be generated by PCR and / or gene synthesis and assembled by known recombinant methods and techniques by joining the nucleic acid segments according to the unique restriction sites in each vector. The subcloned nucleic acid sequences can be verified by DNA sequencing. For transient transfection, larger quantities of the plasmid can be prepared by plasmid preparation from transformed E. coli cultures.

[0286] Cell culture technology Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc. can be used.

[0287] Recombinant antibody production by transient transfection in the HEK293 system Antibodies can be produced by transient expression. Thus, transfection with the respective plasmids can be performed using the HEK293 system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293 cells (Invitrogen) grown in suspension in serum-free FreeStyle™ 293 Expression Medium (Invitrogen) in either shake flasks or stirred fermenters can be transfected with a mixture of the respective expression plasmids and 293fectin™ or fectin (Invitrogen). HEK293 cells were cultured at 1.0×10 in 600 mL in 2L shake flasks (Corning). 6 Cells can be seeded at a density of 1.5 x 10 cells / mL and incubated at 120 rpm and 8% CO2. The next day, approximately 1.5 x 10 6At a cell density of 10000000 cells / mL, cells can be transfected with a mixture of A) 20 mL of Opti-MEM medium (Invitrogen) containing 600 µg of total plasmid DNA (1 µg / mL) and B) 20 mL of Opti-MEM medium supplemented with 1.2 mL of 293 fectin or fectin (2 µL / mL). Glucose solution can be added during the course of fermentation according to glucose consumption. The supernatant containing the secreted antibody is generally harvested after 5-10 days and the antibody can be purified directly from the supernatant or the supernatant can be frozen and stored.

[0288] Protein determination The protein concentration of purified antibodies and derivatives can be determined by determining the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science 4 (1995) 2411-1423.

[0289] Determination of antibody concentration in the supernatant The concentration of antibodies and derivatives in cell culture supernatants can be estimated by immunoprecipitation with protein A agarose beads (Roche Diagnostics GmbH, Mannheim, Germany). Thus, 60 µL of protein A agarose beads can be washed three times with TBS-NP40 (150 mM Tris buffer, pH 7.5 supplemented with 50 mM NaCl and 1% Nonidet-P40). Then, 1 to 15 mL of cell culture supernatant can be applied to the protein A agarose beads pre-equilibrated in TBS-NP40. After 1 h of incubation at room temperature, the beads can be briefly washed once with 0.5 mL of TBS-NP40, twice with 0.5 mL of 2x phosphate-buffered saline (2x PBS, Roche Diagnostics GmbH, Mannheim, Germany) and four times with 0.5 mL of 100 mM Na-citrate buffer (pH 5.0) on an Ultrafree-MC-filter column (Amicon). Bound antibodies can be eluted by adding 35 μL of NuPAGE® LDS sample buffer (Invitrogen). Half of the sample can be combined with NuPAGE® sample reducing agent or left unreduced, respectively, and heated at 70 °C for 10 min. 5-30 μl of the result can be applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE gel (Invitrogen) (containing MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie blue.

[0290] The concentration of antibodies in cell culture supernatants can be quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies that bind to Protein A can be applied to an Applied Biosystems Poros A / 20 column in 200 mM KH2PO4, 100 mM sodium citrate (pH 7.4) and eluted with 200 mM NaCl, 100 mM citric acid (pH 2.5) on an Agilent HPLC 1100 system. The eluted antibodies can be quantified by UV absorbance and peak area integration. Purified standard IgG1 antibody was used as the standard.

[0291] Alternatively, the concentration of antibodies and derivatives in cell culture supernatants can be measured by sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96-well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) can be coated with 100 μL / well of biotinylated anti-human IgG capture molecule F(ab')2-anti-human Fcγ antibody-BI (Dianova) at 0.1 μg / mL for 1 h at room temperature or overnight at 4°C, followed by washing three times with 200 μL / well of PBS, 0.05% Tween (PBST, Sigma). Then, 100 μL / well of a dilution series of the respective antibody-containing cell culture supernatant in PBS (Sigma) can be added to the wells and incubated for 1-2 h on a shaker at room temperature. Wells can be washed three times with 200 μL / well PBST and bound antibodies were detected using 100 μL F(ab')2-anti-human Fcγ antibody-POD (Dianova) at 0.1 μg / mL as detection antibody by incubating for 1-2 hours on a shaker at room temperature. Unbound detection antibodies can be removed by washing three times with 200 μL / well PBST. Bound detection antibodies can be detected by adding 100 μL ABTS / well followed by incubation. Absorbance determination was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).

[0292] Preparative antibody purification Antibodies can be purified from filtered cell culture supernatants with reference to standard protocols. Briefly, antibodies can be applied to a Protein A Sepharose column (GE Healthcare) and washed with PBS. Elution of antibodies can be achieved at pH 2.8 and then immediately neutralized. Aggregated proteins can be separated from monomeric antibodies by size-exclusion chromatography (Superdex 200, GE Healthcare) in PBS or 20 mM histidine buffer (pH 6.0) containing 150 mM NaCl. Monomeric antibody fractions can be pooled and concentrated (if necessary), for example using a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen, and stored at -20°C or -80°C. A portion of the sample can be provided for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, size-exclusion chromatography (SEC) or mass spectrometry.

[0293] SDS-PAGE The NuPAGE® Pre-Cast Gel System (Invitrogen) can be used according to the manufacturer's instructions. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and NuPAGE® MES (reducing gels, with NuPAGE® antioxidant running buffer additive) or MOPS (non-reducing gels) running buffer can be used.

[0294] CE-SDS Microfluidic Labchip technology (PerkinElmer, USA) can be used to analyze purity and antibody integrity by CE-SDS. Thus, 5 μl of antibody solution can be prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according to the manufacturer's instructions and analyzed on a LabChip GXII system using the HT Protein Express Chip. Data can be analyzed using LabChip GX Software.

[0295] Analytical Size Exclusion Chromatography Size exclusion chromatography (SEC) for determining the aggregation and oligomeric state of antibodies can be performed by HPLC chromatography. Briefly, Protein A purified antibodies can be applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4 / K2HPO4 buffer (pH 7.5) on a Dionex Ultimate® system (Thermo Fischer Scientific) or to a Superdex 200 column in 2×PBS on a Dionex HPLC-System (GE Healthcare). Eluted antibodies can be quantified by UV absorbance and peak area integration. BioRad Gel Filtration Standard 151-1901 served as the standard.

[0296] mass spectrometry Antibodies can be deglycosylated with N-glycosidase F in phosphate or Tris buffer at a protein concentration of 1 mg / ml for up to 17 h at 37 °C. Restrictive LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestion can be performed with 100 μg of deglycosylated antibody in Tris buffer (pH 8) for 120 h at room temperature or 40 min at 37 °C, respectively. Prior to mass spectrometry, samples can be desalted by HPLC on a Sephadex G25 column (GE Healthcare). Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0297] Using hybridomas to produce antibodies Hybridoma cell lines were cultured at 1.0 × 10 5 ~2.2×10 5 The cells are seeded at an initial cell density (viable cells) of 10 ...

[0298] Example 2 Characterization of the Monomers of the Multimers According to the Invention A capture plate was prepared by binding biotinylated anti-PG Fc region antibody or biotinylated anti-AAA Fc region antibody to the wells of a streptavidin-coated multiwell plate (SA-MTP). Excess unbound antibody was removed by washing. Sample / standard antibodies (10% final concentration) spiked with human and cynomolgus serum were added to the wells of the capture plate-coated SA-MTP multiwell plate and incubated at room temperature for 1 hour. After washing, the wells were incubated with digoxigenated anti-human kappa antibody M1.7.10 (see, for example, WO 2011 / 048043, which is incorporated herein by reference). After washing, the bound digoxigenated anti-human kappa antibody complex was incubated with horseradish peroxidase (HRP)-labeled anti-digoxigenin antibody. After another washing step, ABTS solution was added to the wells and incubated. The color reaction product was measured by an Elisa reader at a wavelength of 405 nm (reference wavelength: 490 nm). The absorbance value of each sample or standard was determined in triplicate.

[0299] The following table shows absorbance values ​​determined for anti-VEGF / ANG2 antibodies with mutations P329G, L234A, L235A, I253A, H310A and H435A in serum containing the anti-variant (human) Fc region antibody M1.3.17 (SEQ ID NOs: 03 and 04) reported herein as the capture antibody. TIFF2024534067000004.tif54128

[0300] The table below shows the absorbance values ​​determined for antibodies of different specificities with different mutations in the Fc region using different antibodies reported herein as capture and tracer antibodies.

[0301] Assay B: Capture antibodies: M1.6.22-Bi / M1.7.24-Bi / M1.3.17-Bi Tracer antibody: 1.7.10-Dig Assay C: Capture antibodies: M1.6.22-Bi / M1.7.24-Bi / M1.3.17-Bi Tracer compound: FcγRI-Dig M1.6.22=Anti-AAA variant Fc region antibody, M1.7.10=anti-IgG1κ antibody, M1.7.24=Anti-PG variant Fc region antibody, M1.3.17=Anti-PG variant Fc region antibody, sample: 1) Anti-VEGF / ANG2 antibody (IgG1 subclass with mutations P329G / L234A / L235A / I253A / H310A / H435A), 2) anti-VEGF / ANG2 antibody (IgG1 subclass with mutations P329G / L234A / L235A), 3) anti-IGF-1R antibody (IgG1 subclass with mutations I253A / H310A / H435A), 4) anti-P-selectin antibody (IgG4 subclass with mutation S228P / L235E), 5) Anti-VEGF / ANG2 antibody (wild-type IgG1 subclass).

[0302] TIFF2024534067000005.tif122151TIFF2024534067000006.tif233151TIFF2024534067000007.tif233151 TIFF2024534067000008.tif233151TIFF2024534067000009.tif233151TIFF2024534067000010.tif223151

[0303] Assay D: Capture antibody: M1.7.24-Bi / M1.3.17-Bi Tracer antibody: 1.7.10-Dig / M1.19.31-Dig M1.7.10=Anti-IgG1κ antibody M1.19.31=Anti-IgG1κ antibody M1.7.24=Anti-PGLALA variant Fc region antibody M1.3.17=Anti-PGLALA variant Fc region antibody sample: 6) Anti-Dig antibody (IgG1 subclass with mutations P329G / L234A / L235A)

[0304] TIFF2024534067000011.tif187151

[0305] Example 3 Formation of multivalent antibodies according to the present invention Preparation of anti-PG antibody clone 1.7.24SATP Monomeric bivalent anti-PG antibody clone 1.7.24 was dialyzed against 100 mM potassium phosphate buffer containing 150 mM NaCl, pH 7.8 and adjusted to a protein concentration of approximately 15 mg / mL. N-Succinimidyl-3-acetylthiopropionate (SATP) was dissolved in DMSO and added to the antibody solution at a molar ratio of 1:5 (monomeric antibody:SATP). The pH was adjusted to pH 7.1 and the mixture was incubated at 25 °C for 60 min. The reaction was stopped by adding L-lysine to a final concentration of 10 mM, and excess SATP was removed by dialysis against 10 mM potassium phosphate buffer containing 200 mM NaCl, 1 mM EDTA, pH 6.1.

[0306] Preparation of anti-PG antibody clone 1.7.24MH Monomeric bivalent anti-PG antibody clone 1.7.24 was dialyzed against 30 mM potassium phosphate buffer, pH 7.4, and then adjusted to a protein concentration of approximately 25 mg / mL. Maleimidohexanoyl-N-hydroxysuccinimide ester (MHS) was dissolved in DMSO and added to the antibody solution at a molar ratio of 1:6 (monomeric IgG:MHS). The pH was adjusted to pH 7.1, and the mixture was incubated at 25°C for 60 min. The reaction was stopped by adding L-lysine to a final concentration of 10 mM, the pH was adjusted to pH 6.2, and excess MHS was removed by dialysis against 10 mM potassium phosphate buffer containing 200 mM NaCl, 1 mM EDTA, pH 6.1.

[0307] Chemical conjugation of anti-PG antibody clone 1.7.24SATP and anti-PG antibody clone 1.7.24MH Anti-PG antibody clone 1.7.24SATP was deacetylated by incubation with 2% (v / v) 1 M hydroxylamine, pH 7.5, and incubated at 25° C. for 45 min. The deacetylated antibody was mixed with anti-PG antibody clone 1.7.24MH (molar ratio of deacetylated IgG:IgG-MH=1:3) and diluted with 10 mM potassium phosphate buffer containing 200 mM NaCl, 1 mM EDTA, pH 6.1 to a final concentration of 1.5 mg / mL deacetylated anti-PG antibody clone 1.7.24 and 4.5 mg / mL anti-PG antibody clone 1.7.24MH. The pH was adjusted to pH 7.1, and the mixture was incubated at 25° C. The conjugation process was analyzed with an analytical gel filtration column (e.g., TSK3000). The conjugation was stopped after 45 min by adding cysteine ​​to a final concentration of 1 mM. After a further 30 min incubation, N-methylmaleimide (NMM) was added to a final concentration of 5 mM and the pH was adjusted to pH 7.5. After 60 min incubation at 25° C., the conjugate was purified and size fractionated by S300 gel filtration chromatography to remove unconjugated antibodies. Six pools were obtained. Pool 6 contained only the monomeric anti-PG antibody clone 1.7.24.

[0308] Example 4 - Comparative Example Bivalent monomeric antibodies as calibration standards in anti-drug antibody assays A dilution series of monomeric full-length anti-PGFc domain antibody clone 1.3.17 was prepared as a standard to confirm the feasibility of generating a standard curve.

[0309] Biotinylated anti-VEGF / ANG2 antibodies with the mutations P329G, L234A, L235A, I253A, H310A, and H435A, and digoxigenated anti-VEGF / ANG2 antibodies with the mutations P329G, L234A, L235A, I253A, H310A, and H435A were preincubated with the standards overnight at room temperature. After preincubation, the samples were transferred to a streptavidin-coated multiwell plate and incubated for 1 hour at room temperature. Excess unbound antibody was removed by washing. After washing steps, the bound digoxigenated complexes containing biotinylated and digoxigenated anti-VEGF / ANG2 antibodies with the mutations P329G, L234A, L235A, I253A, H310A, and H435A, as well as the monomeric full-length anti-PG Fc region antibody M1.3.17 (SEQ ID NOs: 03 and 04), were detected with a horseradish peroxidase (HRP)-labeled anti-digoxigenin antibody. After washing steps and upon incubation with the respective substrate, the HRP present in the formed complexes catalyzes the conversion of ABTS into a colored product. The signal was measured by an Elisa reader at a wavelength of 405 nm (reference wavelength: 490 nm). The absorbance values ​​of each serum sample were determined in triplicate.

[0310] The table below shows the absorbance values ​​determined for anti-VEGF / ANG2 antibody with mutations P329G, L234A, L235A, I253A, H310A and H435A in serum as capture antibody (biotinylated) and tracer antibody (digoxigenated) with monomeric full-length anti-variant (human) Fc region antibody M1.3.17 (SEQ ID NOs: 03 and 04) as standard. TIFF2024534067000012.tif66128

[0311] At a concentration of 125 ng / mL, the signal-to-noise ratio is 1.85. The regulatory agency requirement for sensitivity in immunogenicity assays is 100 ng / mL. Therefore, the low-sensitivity monomeric bivalent anti-PG antibody M1.3.17 is not suitable either as a positive control or as a calibration standard for ADA assays. It can be seen that at a concentration of 125 ng / mL, the signal is only 1.85 times that of the blank and therefore does not reach the threshold required by the authorities for a valid assay.

[0312] Example 5 Use of the multivalent antibodies according to the invention obtained by chemical conjugation as positive controls and calibration standards Common assays: All steps were performed at room temperature (RT) and samples and quality controls were analyzed in the presence of 5% HPS (pooled human serum). Samples were adjusted to the respective concentrations by dilution with Low Cross Buffer® containing Drug-BI (biotinylated drug antibody) and Drug-DIG (digoxigenated drug antibody). Incubation with the respective biotinylated capture and digoxigenated detection antibodies (= drug (therapeutic) antibodies) at the respective concentrations was performed for 2 h at RT (room temperature) and shaking at 450 rpm, either directly on SA-MTPs (streptavidin-coated multititer (well) plates) or in underivatized MTPs (pre-incubation plates). Afterwards, if necessary, samples (100 μL) were transferred to SA-coated MTPs and incubated for 1 h at RT (450 rpm). Wells were then washed three times (300 μL each with washing buffer). 100 μL of polyclonal anti-DIG-S-Fab-HRP conjugate (50 mU / mL) was added and incubated for 1 h, after which the plates were washed again (3 times with 300 μL of washing buffer each). Finally, 100 μL of ABTS substrate was added per well and the color reaction was evaluated photometrically at 405 nm (reference wavelength 490 nm). Samples were measured in duplicate and averaged. Measurements were accepted as valid if the precision of the replicates was below 20% of the coefficient of variation (CV).

[0313] Pool Selection For pool selection, a bridging ELISA was used with 0.115 μg / mL biotinylated non-targeting antibody IL2 fusion (non-targeting IgG-IL2) and 0.230 μg / mL digoxigenated non-targeting IgG-IL2 without any matrix. The assay was performed directly in streptavidin-coated microtiter plates (SA-MTP) with a 2 hour incubation.

[0314] In a first step, pools 1 to 5 were tested separately in assay buffer at concentrations ranging from 10,000 ng / mL to 100 ng / mL in 10-fold dilution steps. TIFF2024534067000013.tif62143

[0315] The pool was diluted 1:20 with biotinylated and digoxigenated non-targeting IgG-IL2 conjugate.

[0316] Pools 2, 3 and 4 were pooled together and all three showed high binding signals in the ADA assay described above. The purified multivalent anti-PG antibody pool derived from clone 1.7.24 was concentrated to approximately 0.5 mg / mL and stored at -80°C.

[0317] Multimer vs. Monomer To compare multimers with multimers, the following assay was used: A mixed pool 2, 3, 4 of multivalent anti-PG antibodies from clone 1.7.24 and monomeric bivalent anti-PG antibodies clone 1.7.24 was diluted 1:2 starting from 10,000ng / mL and down to 0.6ng / mL. These samples were then diluted with a dilution factor of 20 into biotinylated and digoxigenated non-targeting IgG-IL2 conjugate and incubated for 2 hours on a preincubation plate before being transferred to SA-MTP. This was followed by peroxidase-conjugated polyclonal anti-DIG antibody and substrate ABTS. Optical density (OD) was measured at 405nm (reference wavelength 490nm). TIFF2024534067000014.tif160131

[0318] Drug-antibody in different formats To confirm the general applicability of the multivalent anti-PG antibody, i.e., if it can be used as a positive control and calibration standard independent of the drug antibody format, three different formats of drug antibodies were tested: non-targeting IgG-IL2 (interleukin-2 fused to the C-terminus of the heavy chain of a germline antibody that does not bind to a target), targeted IgG-IL2 (interleukin-2 fused to the C-terminus of the heavy chain of an antibody that specifically binds to a therapeutic target) and TCB (T cell bispecific format). The concentrations of targeted IgG-IL2-BI and -DIG and TCB-BI and -DIG were 0.5 μg / mL, respectively. For non-targeting IgG-IL2, the same concentrations were used as those used in the pool selection example above (0.115 μg / mL drug-BI and 0.230 μg / mL drug-DIG).

[0319] Polyvalent anti-PG antibodies derived from clone 1.7.24 were diluted in a 1:2 dilution series from 10 μg / mL to 0.6 ng / mL in pooled human serum.

[0320] Therefore, diluted multivalent anti-PG antibodies from clone 1.7.24 were diluted with Drug-BI and Drug-DIG by adding 12.5 μL of sample and 237.5 μL of Drug-BI / Drug-DIG solution to the preincubation plate. After 2 h of incubation, the complexes formed were added to the SA-MTP and further processed as described above.

[0321] TIFF2024534067000015.tif175133

[0322] Example 6 Use of the multivalent antibodies of the invention produced by recombinant expression as tetravalent fusion proteins as calibration standards In this example, a tetravalent form of the anti-PG antibody clone 1.7.24 was used, which was obtained by fusing additional Fabs of the anti-PG antibody clone 1.7.24 to the C-terminus of each heavy chain.

[0323] Variant Fc region-BI (biotinylated variant Fc region fragment) and variant Fc region-DIG (digoxigenated variant Fc region fragment) were used as capture and tracer molecules.

[0324] The results are shown in Figure 10.

[0325] Example 7 Use of the multivalent antibody of the invention produced by recombinant expression as IgM as a calibration standard In this example, an anti-PG antibody of the IgM type was used. This IgM type was recombinantly produced. The IgM was used in the ELISA as unpurified supernatant of cell culture. The supernatant was tested undiluted and serially diluted 1:5 in buffer.

[0326] Variant Fc region-BI (biotinylated variant Fc region fragment) and drug antibody in TCB format (digoxigenated drug antibody) were used as capture and tracer molecules.

[0327] The results are shown in FIG. 11 and in the table below. TIFF2024534067000016.tif60128

Claims

1. An antibody comprising four or six binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three or four amino acid changes compared to the wild-type Fc region of said human IgG1 subclass.

2. At least two covalently bonded i) a bivalent full-length antibody, each comprising two binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three or four amino acid changes compared to the wild-type Fc region of said human IgG1 subclass; or ii) a (Fab')2 fragment of a bivalent full-length antibody, each of which contains two binding sites that specifically bind to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three, or four amino acid changes compared to the wild-type Fc region of the human IgG1 subclass. An antibody multimer comprising:

3. The antibody or antibody multimer according to any one of claims 1 to 2, wherein the binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains one, two, three, or four amino acid changes compared to a wild-type Fc region of the human IgG1 subclass is a binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (numbering according to the Kabat EU index).

4. each of said binding sites independently of the other: （１） (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or （２） (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or （３） (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or （４） (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 35; or （５） A mixture of any one of (1) to (4) The antibody or antibody multimer according to any one of claims 1 to 2, comprising any one of:

5. The antibody or antibody multimer according to any one of claims 1 to 2, wherein the binding site that specifically binds to an immunoglobulin Fc region of the human IgG1 subclass that comprises one, two, three, or four amino acid changes compared to a wild-type Fc region of the human IgG1 subclass comprises the amino acid residue glycine at position 329 and the amino acid residues alanine at positions 234 and 235 (numbering according to the Kabat EU index).

6. The antibody multimer of claim 2, wherein the multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer or decamer.

7. Use of an antibody or antibody multimer according to any one of claims 1 to 2 as a positive control in an in vitro (bridging) immunoassay.

8. Use of an antibody or antibody multimer according to any one of claims 1 to 2 as a standard in an in vitro (bridging) immunoassay.

9. 9. The use of claim 8, wherein the use is for generating a calibration function for quantitatively determining anti-drug antibodies to a drug antibody, wherein the anti-drug antibody binds to one or more amino acid residues in the Fc region of the drug antibody that are altered compared to the wild-type Fc region.

10. 1. An immunoassay for determining the presence and / or amount of anti-drug antibodies in a (serum-containing) sample, comprising: the anti-drug antibody binds to at least one amino acid residue in the Fc region of the drug antibody that is altered compared to the wild-type Fc region; the immunoassay comprises the drug antibody as a capture antibody and as a tracer antibody; The antibody or antibody multimer according to any one of claims 1 to 2 is used as a positive control or a calibration standard in the immunoassay. The immunoassay.

11. The immunoassay of claim 10, wherein the immunoassay is a bridging ELISA.

12. 11. The immunoassay of claim 10, wherein the antibody of claim 1 is used as a calibration standard and to generate a calibration function for quantitatively determining anti-drug antibodies to drug antibodies.

13. An immunoassay as described in claim 10, wherein the antibody multimer as described in claim 2 is used as a calibration standard and is used to generate a calibration function for quantitatively determining anti-drug antibodies relative to drug antibodies.

14. 3. A method for producing the antibody multimer of claim 2 by chemical conjugation using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS).

15. The method of claim 14, wherein the multimer is a multimer of a full-length antibody.

16. A method for producing an antibody multimer, comprising: chemically cross-linking a full-length antibody that specifically binds to an Fc region of an immunoglobulin of the human IgG1 subclass that contains the amino acid residue glycine at position 329 (Kabat EU index numbering) using N-succinimidyl-3-acetylthiopropionate (SATP) and maleimidohexanoyl-N-hydroxysuccinimide (MHS); The antibody （１） (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 09; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28; or （２） (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29; or （３） (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30 Including, The method.

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