Method for determining free antigen of antibodies in a sample

Abstract

Reported herein is a method for determining the free antigen of an antibody in an undiluted serum sample, comprising the steps of: a) applying the undiluted sample to a solid phase on which a capture antibody is immobilized to form a capture antibody-antigen complex, wherein the capture antibody competes with the antibody for binding to a first epitope on the antigen; b) applying a tracer antibody to the solid phase to form a capture antibody-antigen-tracer antibody complex, wherein the tracer antibody specifically binds to a second epitope on the antigen, and wherein the epitope of the tracer antibody does not overlap with the epitope of the capture antibody on the antigen; and c) determining the free antigen of the antibody by determining the tracer antibody in the capture antibody-antigen-tracer antibody complex.

Description

[Technical Field]

[0001] The present invention is in the field of pharmacokinetics. More specifically, an assay for determining free antigen of a therapeutic antibody in a sample, particularly in the presence of the therapeutic antibody at high serum concentrations, is reported herein. [Background technology]

[0002] Lee, JW, et al. (AAPS J. 13 (2011) 99-110) reported that the primary driver of bioanalysis in supporting drug development is the intended use of the data. A reliable methodology for measuring circulating mAbs and their antigen ligands (L) is important for assessing exposure-response relationships and dose selection in support of efficacy and safety assessment. Ligand binding assays (LBAs) are widely used for analyzing protein biotherapeutics and antigen ligands (L) to support pharmacokinetic / pharmacodynamic (PK / PD) and safety assessment. In particular, for monoclonal antibody drugs (mAbs) that noncovalently bind to L, multiple forms of mAb and L may exist in vivo, including free mAb, free L, and monovalent and / or bivalent complexes of mAb and L. Considering the complexity of the dynamic binding equilibrium that occurs in the body after administration and the multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from their in vivo counterparts. LBA reagents and assay formats can, in principle, be designed to measure either the total or free forms of mAb and L. However, confirmation of the form being measured under specified conditions can be technically challenging.

[0003] Generally, commercially available assays for the detection of analytes are performed at a minimum required dilution (MRD) of 1:2 or higher (Gyrolab's affinity flow-through format). The drawback of applying a diluent is, among other things, that complexes in the sample, such as antibody-antigen complexes, are forced to dissociate by the applied solution. As a result, the assay results no longer reflect the true situation in the sample.

[0004] WO 2018 / 075758 (Patent Document 1) reported a method for quantifying free (unbound) human C5 complement protein (C5) from a sample, comprising binding a biotinylated anti-C5 capture antibody to streptavidin-coated particles; capturing free (unbound) C5 in the sample; detecting the captured free C5; and quantifying the captured free C5 using laser-induced fluorescence detection.

[0005] Takashi, I. et al. reported increased cerebrospinal fluid complement C5 levels in patients with major depressive disorder and schizophrenia (Biochem. Biophys. Res. Commun. 497 (2018) 683-688 (Non-Patent Document 2)).

[0006] Roth, A. et al. reported the complement C5 inhibitor clovalimab in paroxysmal nocturnal hemoglobinuria (Blood 135 (2020) 912-920 (Non-Patent Document 3)).

[0007] Haringman, J. et al. reported a randomized controlled trial using anti-CCL2 (anti-monocyte chemoattractant protein 1) monoclonal antibody in patients with rheumatoid arthritis (Arth) (Arth. Rheum. 54 (2006) 2387-2392 (Non-Patent Document 4)).

[0008] Therefore, there is a need for an assay to specifically determine free, i.e., uncomplexed, antigen in the presence of complexed antigen and antibody that specifically binds to the antigen. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2018 / 075758 [Non-patent literature]

[0010] [Non-Patent Document 1] Lee, JW et al., AAPS J.13(2011)99-110 [Non-patent document 2] Takashi, I. et al., Biochem. Biophys. Res. Commun. 497 (2018) 683-688 [Non-patent document 3] Roth, A. et al., Blood 135(2020)912-920 [Non-patent document 4] Haringman, J. et al., Arth. Rheum. 54 (2006) 2387-2392 Summary of the Invention

[0011] Herein we report a method for detecting the presence and determining the amount of free antigen of a (therapeutic) antibody, i.e., antigen in uncomplexed form, in a serum sample, wherein the serum sample contains the antigen, the (therapeutic) antibody and the complexed antigen, i.e., the antigen in a (therapeutic) antibody-antigen complex. The antigen may be specifically bound by the therapeutic antibody, such as by the first binding specificity of the (multispecific, therapeutic) antibody.

[0012] The present invention is based, at least in part, on the discovery that free antigen determination in qualitative and quantitative form can be performed without sample dilution, i.e., in 100% serum, thereby avoiding complex dissociations and falsification of the determination.

[0013] The present invention is based, at least in part, on the discovery that falsification of results in the determination of free antigen can be reduced or even prevented by omitting the pre-analysis sample dilution step. This is particularly true when the antibody-antigen complex has a short half-life, i.e., is very unstable. This is particularly true when the complex half-life is less than 600 seconds, less than 300 seconds, and particularly less than 100 seconds. Without being bound by this theory, it is believed that due to the kinetic properties of the complex, i.e., dilution due to the short half-life of the complex or the low affinity of the (monovalent) antibody (binding site) for the antigen, the complex present in the sample tends to dissociate or is forced to dissociate during / between the assay. This increases the amount of free antigen, leading to falsification of the assay results.

[0014] The present invention is based, at least in part, on the discovery that the use of capture and / or tracer antibodies that bind to the same or overlapping epitope as that of a therapeutic antibody, in combination with a short incubation time in a bridging assay format, can prevent displacement of the (therapeutic) antibody in the antigen-antibody complex, thereby reducing or even preventing falsification of the results in the determination of free antigen. Without being bound by this theory, it is assumed that the use of capture and / or tracer antibodies that bind to the same or overlapping epitope as the therapeutic antibody achieves elimination of the antigen-(therapeutic) antibody complex in the determination of free antigen. This prevents an increase in the amount of free antigen, resulting in better assay results.

[0015] Thus, the present invention includes at least the following aspects and embodiments.

[0016] 1. A method for determining free antigens of antibodies in a serum sample, comprising the steps of: a) applying a sample to a solid phase on which a capture antibody is immobilized to form a capture antibody-antigen complex; the capture antibody competes with the antibody for binding to the first epitope on the antigen; b) applying a tracer antibody to the solid phase to form a capture antibody-antigen-tracer antibody complex; the tracer antibody specifically binds to a second epitope on the antigen; the epitope of the tracer antibody does not overlap with the epitope of the capture antibody on the antigen; and c) determining the free antigen of the antibody by determining the tracer antibody in the capture antibody-antigen-tracer antibody complex.

[0017] 2. The method according to item 1, wherein the sample contains free antigen, free antibody, and antigen-antibody complexes.

[0018] 3. The method of any one of items 1 or 2, wherein the method is an antigen bridging assay.

[0019] 4. The method according to any one of items 1 to 3, wherein the applying in step a) is under conditions in which at most 10% of the antibodies bound to the antigen are displaced by the capture antibody, or in step a) in which at most 10% of the antibodies bound to the antigen are displaced by the capture antibody.

[0020] 5. The method according to any one of items 1 to 4, wherein the applying in step a) is under conditions in which at most 5% of the antibodies bound to the antigen are displaced by the capture antibodies, or in step a) in which at most 5% of the antibodies bound to the antigen are displaced by the capture antibodies.

[0021] 6. The method according to any one of items 1 to 5, wherein the applying in step a) is under conditions in which at most 1% of the antibodies bound to the antigen are displaced by the capture antibody, or in step a) in which at most 1% of the antibodies bound to the antigen are displaced by the capture antibody.

[0022] 7. The method according to any one of items 1 to 6, wherein the applying in step a) is under conditions such that the antibody bound to the antigen is not substantially displaced by the capture antibody.

[0023] 8. The method according to any one of items 1 to 7, wherein the serum sample is an undiluted serum sample.

[0024] 9. The method of any one of items 1 to 7, wherein the sample comprises about 100% serum.

[0025] 10. Step a) applying the sample to a solid phase on which a capture antibody is immobilized to form a capture antibody-antigen complex; the capture antibody competes with the antibody for binding to the first epitope on the antigen; The sample is incubated with the solid phase for 300 seconds or less and removed from the solid phase within 300 seconds. 10. The method according to any one of items 1 to 9, wherein

[0026] 11. The method according to any one of items 1 to 10, wherein the sample is incubated with the solid phase for no more than 240 seconds / the sample is removed from the solid phase within 240 seconds.

[0027] 12. The method according to any one of items 1 to 11, wherein the sample is incubated with the solid phase for 100 seconds or less / the sample is removed from the solid phase within 100 seconds.

[0028] 13. The method according to any one of items 1 to 12, wherein the sample is incubated with / removed from the solid phase, more preferably incubated for 10 seconds or less / removed after 10 seconds or less.

[0029] 14. The method according to any one of items 1 to 13, wherein the sample is incubated with the solid phase for no more than 2 seconds / the sample is removed from the solid phase within 2 seconds.

[0030] 15. The method according to any one of items 1 to 14, wherein the sample is incubated with the solid phase for 1 second or less / removed from the solid phase within 1 second.

[0031] 16. The method according to any one of items 1 to 15, wherein the stability / half-life of a complex (monovalent non-avid interaction) between the antigen and an antigen-binding site of an antibody that specifically binds to a first epitope on the antigen is 200 seconds or less.

[0032] 17. The method according to any one of items 1 to 16, wherein the stability / half-life of the complex (monovalent non-avido interaction) between the antigen and the antigen-binding site of an antibody that specifically binds to a first epitope on the antigen is 100 seconds or less.

[0033] 18. The method of any one of items 1 to 17, wherein the antibody is a bispecific antibody, the bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on the antigen and a second, different antigen-binding site that (specifically) binds to a second epitope on the antigen, and the tracer antibody competes with the bispecific antibody for binding to the second epitope on the antigen.

[0034] 19. The method according to item 18, wherein the stability / half-life of the complex between the antigen and the antigen-binding site of the bispecific antibody that specifically binds to a second epitope on the antigen (monovalent, non-avido interaction) is less than the stability / half-life of the complex between the antigen and the first binding site of the bispecific antibody that specifically binds to a first epitope on the antigen (monovalent, non-avido interaction).

[0035] 20. The method according to any one of items 18 to 19, wherein the stability / half-life of the complex (monovalent, non-avido interaction) between the antigen and the antigen-binding site of the bispecific antibody that specifically binds to a second epitope on the antigen is 100 seconds or less.

[0036] 21. The method according to any one of items 18 to 20, wherein the stability / half-life of the complex (monovalent, non-avido interaction) between the antigen and the antigen-binding site of the bispecific antibody that specifically binds to a second epitope on the antigen is 20 seconds or less.

[0037] 22. The method according to any one of items 1 to 21, wherein the capture antibody and the tracer antibody are non-human, non-humanized antibodies.

[0038] 23. The method according to any one of items 1 to 22, which is an enzyme-linked immunosorbent assay, in which the sample is incubated with the solid phase for 180 to 240 seconds / the sample is removed from the solid phase after 180 to 240 seconds.

[0039] 24. The method of any one of items 1 to 23, wherein the tracer antibody is incubated with the capture antibody-antigen complex for less than 1200 seconds.

[0040] 25. The method according to any one of items 1 to 22, wherein the method is a nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection, in which the sample is incubated with the solid phase for 2 seconds or 1 second or less and the sample is removed from the solid phase within 2 seconds or 1 second.

[0041] 26. The method according to any one of items 1 to 22 and 25, wherein the tracer antibody is incubated with the capture antibody-antigen complex for less than 2 seconds or less than 1 second.

[0042] 27. The method of any one of items 1 to 26, wherein the antibody is a therapeutic antibody.

[0043] 28. The method of any one of items 1 to 27, wherein the antibody is a multispecific antibody.

[0044] 29. The method of any one of items 1 to 28, wherein the antibody is a bispecific antibody.

[0045] 30. The method of any one of items 1 to 29, wherein the antibody is a bispecific antibody having a first binding site that specifically binds to a first epitope on the antigen and a second binding site that specifically binds to a second epitope on the antigen.

[0046] 31. The method is for determining the amount of free antigen, wherein step c) comprises: determining the amount of free antigen in the sample by determining the amount of tracer antibody in the capture antibody-antigen-tracer antibody complex; 31. The method according to any one of items 1 to 30,

[0047] 32. The method according to any one of items 1 to 31, wherein determining the free antigen is by incubating the capture antibody-antigen-tracer antibody complex with a detection antibody conjugated to a detectable label and determining the signal generated by the detectable label.

[0048] 33. The method according to any one of items 31 to 32, wherein determining the amount of free antigen is by using a calibration curve to correlate the signal generated by the detectable label of the detection antibody bound to the capture antibody-antigen-tracer antibody complex with the amount of free antigen.

[0049] 34. The method of any one of items 1 to 33, wherein the antigen is human CCL2.

[0050] 35. The method according to any one of items 1 to 34, wherein the (therapeutic) antibody is an antibody that specifically binds to human CCL2 as described herein.

[0051] 36. The method of any one of items 1 to 33, wherein the antigen is human C5.

[0052] 37. The method according to any one of items 1 to 33 and 36, wherein the (therapeutic) antibody is an antibody that specifically binds to human C5 as described herein.

[0053] 38. The method of any one of items 1 to 37, wherein the serum is human serum.

[0054] 39. An in vitro method for determining the presence and / or amount of an antigen of a bispecific antibody that can be specifically bound by the bispecific antibody in a sample, wherein the antigen to be detected is capable of being specifically bound by at least a first binding specificity of the bispecific antibody and the antigen is free antigen, the method comprising the step(s) disclosed herein.

[0055] 40. The method according to item 39, wherein the sample is an undiluted serum sample. DETAILED DESCRIPTION OF THE INVENTION

[0056] Detailed Description of the Invention Herein, we report an in vitro method for detecting free antigen of a multispecific binder, such as a bispecific antibody / drug, in preclinical and clinical samples in the presence of the multispecific binder.

[0057] Definition: The terms "therapeutic antibody" and "drug" are used interchangeably herein. These terms are used in the broadest sense and encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0058] In certain embodiments of the invention, the antibody is a multispecific antibody, e.g., at least a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different antigens. In certain embodiments of the invention, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain embodiments of the invention, a bispecific antibody can bind to two different epitopes on the same antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. In certain embodiments of the invention, the antibody is a bispecific antibody that specifically binds to a first and a second antigen. In certain embodiments of the invention, a bispecific antibody has i) a first binding specificity that specifically binds to a first antigen or a first epitope on an antigen, and ii) a second binding specificity that specifically binds to a second antigen or a second epitope on the (same) antigen. In certain embodiments of the invention, the second epitopes on the same antigen are non-overlapping epitopes. In certain embodiments of the invention, the antibody is a bispecific, bivalent antibody. In a preferred embodiment, the antibody is a monoclonal, bispecific, bivalent antibody.

[0059] The antibody or fragment may also be a multispecific antibody as described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 or WO 2010 / 145793.

[0060] The terms "anti-C5 antibody" and "antibody that (specifically) binds to C5" refer to an antibody that can bind to C5 with sufficient affinity so as to be useful as a diagnostic and / or therapeutic agent targeted to C5. In certain embodiments of the invention, the extent of binding of the anti-C5 antibody to unrelated non-C5 proteins is less than about 10% of the binding of the antibody to C5. In certain embodiments of the invention, the anti-C5 antibody binds to an epitope of C5 that is conserved among C5 from different species. In a preferred embodiment, the C5 is human C5.

[0061] As used herein, the term "C5" encompasses any native C5 from any vertebrate source, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). Unless otherwise indicated, the term "C5" refers to a human C5 protein having the amino acid sequence set forth in SEQ ID NO: 30 and containing the beta chain sequence set forth in SEQ ID NO: 31. The term encompasses "full-length," unprocessed C5 and any form of C5 resulting from intracellular processing. The term also encompasses naturally occurring variants of C5, such as splice variants or allelic variants. An exemplary amino acid sequence of human C5 is set forth in SEQ ID NO: 30 ("wild-type" or "wt" C5). An exemplary amino acid sequence of the beta chain of human C5 is set forth in SEQ ID NO: 31. The amino acid sequences of exemplary MG1, MG2, and MG1-MG2 domains of the beta chain of human C5 are set forth in SEQ ID NOs: 32, 33, and 34, respectively. Exemplary cynomolgus monkey and mouse C5 amino acid sequences are set forth in SEQ ID NOs: 35 and 96, respectively. Amino acid residues 1-19 of SEQ ID NOs: 30, 31, 34, 35, and 96 correspond to signal sequences that are removed during intracellular processing and are therefore missing from the corresponding exemplary amino acid sequences.

[0062] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention 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.

[0063] The principles of different immunoassays are described, for example, by 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, by Wilchek, M., and Bayer, EA, in Methods Enzymol. 184 (1990) 467-469.

[0064] Monoclonal antibodies and their constant domains contain several reactive amino acid side chains for attachment to members of binding pairs such as polypeptides / proteins, polymers (e.g., PEG, cellulose, or polystyrene), or enzymes. Chemically reactive groups of amino acids are, for example, amino groups (lysine, alpha-amino group), 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, pages 50-100.

[0065] One of the most common reactive groups on antibodies is the aliphatic ε-amine of the amino acid lysine. Generally, nearly all antibodies contain abundant lysines. Lysine amines are reasonably good nucleophiles above pH 8.0 (pKa = 9.18), so they react easily and cleanly with various reagents to form stable bonds. Amine-reactive reagents primarily react with the α-amino groups of lysine and proteins. Reactive esters, especially N-hydroxysuccinimide (NHS) esters, are one of the most commonly used reagents for modifying 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. They 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 yield 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 containing 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.

[0066] Another common reactive group in antibodies is the thiol residue from the sulfur-containing amino acid cystine and its reduction product, cysteine ​​(or half-cystine). Cysteine ​​is more nucleophilic than amines and generally contains a free thiol group, which is the most reactive functional group in proteins. Thiols are generally reactive at neutral pH, allowing them to 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 bond with a reagent such as dithiothreitol (DTT) is required 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 can therefore 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 for introducing multiple sulfhydryl groups via reactive amino groups. Haloacetyl derivatives, such as iodoacetamide, form thioether bonds and are also useful reagents for thiol modification. Another useful reagent is maleimide. The reaction of maleimide with thiol-reactive reagents is essentially the same as that of iodoacetamide. Maleimide reacts rapidly at slightly acidic to neutral pH.

[0067] Another common reactive group in antibodies is carboxylic acid. Antibodies contain carboxylic acid groups at the C-terminus and in the side chains of aspartic acid and glutamic acid. The relatively low reactivity of carboxylic acids in water typically makes it difficult to use these groups to selectively modify polypeptides and antibodies. To do this, the carboxylic acid group is typically converted to a reactive ester using a water-soluble carbodiimide and then reacted with nucleophiles such as amines, hydrazides, and hydrazines. Amine-containing reagents must be weakly basic to selectively react with activated carboxylic acids to form stable amide bonds in the presence of the more highly basic ε-amine of lysine. Protein cross-linking can occur when the pH is increased above 8.0.

[0068] 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 carbohydrate moieties are primarily found on the crystallizable fragment region (Fc region) of antibodies, conjugation can be achieved by site-specific modification of the carbohydrate away from the antigen-binding site. Reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents forms a Schiff base intermediate that can be reduced to an alkylamine.

[0069] The conjugation of the tracer and / or capture and / or detection antibody with its conjugation partner can be achieved by different methods, such as chemical bonding or bonding via a binding pair. The term "conjugation partner" as used herein refers to, for example, a solid support, a polypeptide, a detectable label, or a member of a specific binding pair. In certain embodiments of the present invention, the capture and / or tracer and / or detection antibody is bonded to its binding partner by chemical bonding via the N-terminus and / or ε-amino group (lysine), the ε-amino group of a different lysine, the carboxy-, sulfhydryl-, hydroxyl-, and / or phenolic functional groups of the antibody's amino acid backbone, and / or the sugar alcohol group of the antibody's carbohydrate structure. In certain embodiments of the present invention, the capture antibody is conjugated to its conjugation partner via a binding pair. In one preferred embodiment, the capture antibody is conjugated to biotin and immobilized to the solid support via avidin- or streptavidin-immobilized solid support. In certain embodiments of the present invention, the capture antibody is conjugated to its conjugation partner via a binding pair. In a preferred embodiment, the tracer antibody is covalently conjugated to digoxigenin as a detectable label.

[0070] The term "sample" includes, but is not limited to, any quantity of material from a living organism or former living organism. Such organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. In certain embodiments of the present invention, the sample is obtained from a monkey, particularly a cynomolgus monkey, or a rabbit, or a mouse, or a rat, or a human. In one embodiment, the sample is a clinical sample. Such materials include, but are not limited to, whole blood, plasma, or serum from an individual, which in certain embodiments are the most widely used sample sources in clinical routines.

[0071] "Solid phase" refers to a non-fluid substance, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic and ferromagnetic particles), glass, and ceramics; gel materials 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 a "solid phase" contains at least one moiety on its surface intended to interact with substances in a sample. A 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 for either non-covalent or covalent attachment of proteins and other substances. Such particles include polymeric particles such as polystyrene and poly(methyl methacrylate); gold particles, such as gold nanoparticles and gold colloids; and ceramic particles, such as silica, glass, and metal oxide particles. See, for example, Martin, CR et al., Analytical Chemistry - News & Features, 70 (1998) 322A-327A, or Butler, JE, Methods 22 (2000) 4-23.

[0072] Chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles, haptens, such as digoxigenin, are examples of "detectable labels." Detectable labels can also be photoactivatable crosslinking groups, such as azide or azirine groups. Metal chelates that can be detected by electrochemiluminescence are also preferred signal-emitting groups, including ruthenium chelates, such as ruthenium(bispyridyl)3. 2+Chelates are particularly preferred.Suitable ruthenium labeling groups are described, for example, in EP0580979, WO90 / 05301, WO90 / 11511, and WO92 / 14138.For direct detection, the labeling group can be selected from any known detectable marker group, such as dyes, luminescent labeling groups, for example, chemiluminescent groups, such as acridinium esters or dioxetanes, or fluorescent dyes, such as fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine, 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 immunoassay, e.g., EP-A-0061888), and radioisotopes.

[0073] Indirect detection systems involve, for example, labeling a detection reagent, such as a detection antibody, with a first partner of a binding pair. Examples of suitable binding pairs include antigen / antibody, biotin or biotin analogs, such as aminobiotin, iminobiotin, or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analog / complementary nucleic acid, and receptor / ligand, such as steroid hormone receptor / steroid hormone. In a preferred embodiment, the first binding pair member comprises a hapten, an antigen, and a hormone. In a preferred embodiment, the hapten is selected from the group consisting of digoxin, digoxigenin, and biotin and analogs thereof. The second partner of such a binding pair, such as an antibody or streptavidin, is usually labeled to allow direct detection, e.g., by a label as described above.

[0074] The term "immunoassay" refers to any technique that utilizes a specific binding molecule, such as an antibody, to capture and / or detect a specific target for qualitative or quantitative analysis. Generally, an immunoassay is characterized by the following steps: 1) immobilization or capture of the analyte, and 2) detection and measurement of the analyte. The analyte can be captured, i.e., bound, to any solid surface, such as a membrane, a plastic plate, or any other solid surface.

[0075] Immunoassays can generally be performed in three different formats: direct detection, indirect detection, or sandwich assays. Direct detection immunoassays use a detector (or tracer) antibody that can be measured directly. An enzyme or other molecule generates a signal that produces color, fluorescence, or luminescence, allowing the signal to be visualized or measured (although radioisotopes can also be used, although not commonly used today). Indirect assays use a primary antibody that binds to the analyte to provide a defined target for a secondary antibody (called a detector or tracer antibody) that specifically binds to the target provided by the primary antibody. The secondary antibody generates a measurable signal. Sandwich assays utilize two antibodies: a capture antibody and a tracer (detector) antibody. The capture antibody is used to bind (immobilize) the analyte from or in solution, allowing the analyte to be specifically removed from the sample. The tracer (detector) antibody is used in a second step to generate a signal (either directly or indirectly, as described above). The sandwich format requires two antibodies, each with a different epitope on the target molecule. Furthermore, both antibodies must bind to the target simultaneously, so they must not interfere with each other.

[0076] The term "free antigen" refers to an antigen that can be specifically bound by the binding specificity of an antibody, but is not currently bound to this binding specificity. In certain embodiments of the invention, the free antigen is a non-antibody-bound or non-antibody-complexed antigen, i.e., an antigen that is not covalently or non-covalently complexed with (any) therapeutic antibody.

[0077] The principles of different immunoassays are described, for example, by 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, by Wilchek, M., and Bayer, EA, in Methods Enzymol. 184 (1990) 467-469.

[0078] The term "biparatopic antibody" refers to an antibody that has at least two binding sites and specifically binds to two non-overlapping epitopes on the same antigen.

[0079] Specific embodiments of the method according to the invention A single interaction between the first binding site of a therapeutic antibody and an antigen results in the formation of an antigen-antibody complex. The half-life of this single interaction depends on a simple affinity-driven interaction, i.e., no avid participation. Only the interaction of the second binding site of the antibody with the antigen results in the formation of a long-term stable complex with affine and avid binding interactions.

[0080] This feature makes the determination of free antigen in the presence of therapeutic antibodies challenging.

[0081] Generally, for the determination of free antigens in samples obtained from, for example, human laboratory animals, the bridging principle is used, whereby the antigen is bound (captured) to a solid phase (via a first epitope) by the use of a so-called capture antibody and detected via a second, non-overlapping epitope by the use of a so-called tracer antibody.

[0082] Therefore, a positive assay result can only be obtained if the bridge complex containing the two exclusive affinity-driven interactions is sufficiently stable.

[0083] Furthermore, if the assay is required to detect free antigen in the presence of therapeutic antibodies (or even more complex in the presence of different therapeutic antibodies that bind to the same antigen), the capture and tracer antibodies should bind to the same or at least partially overlapping epitopes as bound by the therapeutic antibody(ies).

[0084] Furthermore, the formation of the detection complex should not change the proportion of free antigen, i.e., not by replacing, for example, the therapeutic antibody. Without being bound by this theory, it is assumed that the capture antibody or the detection antibody should not affect the amount of detection complex. It is assumed that the incubation time should be in line with the dissociation rate of the complex, and preferably shorter.

[0085] Thus, this assay allows for the sensitive determination of free antigen of a therapeutic antibody, even in the presence of the therapeutic antibody, given the short half-life of the individual interactions.

[0086] The present invention is based at least in part on the finding that for the determination of free antigen of therapeutic antibodies, assays with no sample dilution and short interaction times achieve the best results.

[0087] The following exemplification of the methods according to the invention is presented using exemplary bispecific anti-CCL2 antibodies. It is presented as an example only and should not be construed as a limitation of the methods according to the invention. The true scope is set forth in the appended claims.

[0088] Corresponding examples are Examples 4 to 10, which demonstrate the properties of an immunoassay for determining free CCL2 (not in complex with anti-CCL2 antibodies) with a sensitivity of 10 pg / mL, supporting proof-of-concept (POC) testing in cynomolgus monkeys.

[0089] The following was found: - In ELISA format, the combination of dilution and long incubation times leads to erroneous results (a value of ratio 1 is expected if there is no effect of dilution, see Example 4): In the TIFF0007824236000001.tif68170-ELISA format, a short incubation time of approximately 3-4 minutes without dilution (100% horse serum) results in an assay working range of 20 pg / mL antigen to 1000 pg / mL antigen (see Figure 1 and Example 5): TIFF0007824236000002.tif89148 - In a nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection, the assay functions without dilution, despite the manufacturer's minimum required dilution of 1:2, and has a working range of 313 pg / mL to 40,000 pg / mL. In a nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection, the assay functions without dilution, despite the manufacturer's minimum required dilution of 1:2, and has an LLOQ of 10 pg / mL when the detection antibody is directly labeled with a dye (see Figure 2 and Example 7). TIFF0007824236000003.tif86170 - Using capture and detection antibodies with non-human backbones avoids a) false positive and b) false negative results -Anti-drug antibodies (ADAs) against the constant region of human IgG can form a bridge between the human capture and detection antibodies, potentially leading to false-positive free antigen assay results. ADAs against the CDRs of the drug antibody can bind to the capture antibody in a neutralizing manner, potentially causing false-negative free antigen assay results because the capture antibody can no longer capture free antigen; The assay also gives the same results when using a non-human scaffold antibody that is different from the humanized antibody but binds to the same epitope as shown in the table below: TIFF0007824236000004.tif110170

[0090] The assay according to the invention has been used to analyze samples from a cynomolgus monkey pharmacokinetic study. The results obtained for the control samples are shown in the table below (see also Example 8). TIFF0007824236000005.tif106170

[0091] The same assay setup was used for the determination of human antigens in B16 mice (see Figure 4 and Example 9).

[0092] In a particular embodiment of the invention, the antigen is human CCL2 and the antibody is a bispecific anti-CCL2 antibody that binds to two different epitopes on human CCL2.

[0093] In certain embodiments of the invention, a bispecific antibody comprises a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second, different antigen-binding site that (specifically) binds to a second epitope on human CCL2.

[0094] In a particular embodiment of the invention, a bispecific antibody comprises a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a different second antigen-binding site that (specifically) binds to a second epitope on human CCL2; where: A) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 142; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 143; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 144; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 153, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 154, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 155, or or B) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 142; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 143; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 144; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 133, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 134, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 135, or or C) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 142; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 143; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 144; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 127, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 128, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129, or or D) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 133, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 134, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 135, ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 153, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 154, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 155, or or E) i) the first antigen-binding site is A VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 136; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 137; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 138; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 141, ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 153, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 154, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 155; or or F) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 158; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 159; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 160; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 161, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 162, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 163, ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 153, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 154, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 155; or or G) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 127, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 128, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129, ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 133, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 134, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 135, or or H) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 125; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 127, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 128, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129, ii) the second antigen-binding site comprises: A VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 136; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 137; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 138; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 141, or or I) i) the first antigen-binding site is a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 118; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 119; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120; and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 122, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 123, ii) the second antigen-binding site comprises: A VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 136; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 137; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 138; and (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 141.

[0095] In certain embodiments of the invention, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

[0096] In a particular embodiment of the invention, the bispecific antibody comprises a constant heavy chain domain of the human IgG1 isotype.

[0097] In a particular embodiment of the invention, the bispecific antibody is an (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, where: i) the first antigen-binding site binds to the same epitope on CCL2 as an antibody comprising: A VH domain comprising the amino acid sequence of SEQ ID NO: 148, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 142, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 143, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 144; and a VL domain comprising the amino acid sequence of SEQ ID NO: 149, comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; and ii) the second antigen-binding site binds to the same epitope on CCL2 as an antibody comprising: A VH domain comprising the amino acid sequence of SEQ ID NO: 156, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and a VL domain comprising the amino acid sequence of SEQ ID NO: 157, comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 153, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 154, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 155.

[0098] In a particular embodiment of the invention, the bispecific antibody is an (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, where: i) the first antigen-binding site comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3a VH domain comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167, (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168, and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and, ii) the second antigen-binding site comprises: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and, (d)X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187; and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R.

[0099] In a particular embodiment of the invention, the bispecific antibody is an (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, where: i) the first antigen-binding site comprises: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3is H or G, 1 IX 2 IFX 3 a VH domain comprising (a) a CDR-H2 comprising TANYAQKFQG, (b) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166, (c) a CDR-H3 comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO: 170, (d) an FR-H1 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO: 171, (e) an FR-H2 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO: 171, (f) an FR-H3 comprising the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVY YCAR of SEQ ID NO: 172, and (g) an FR-H4 comprising the amino acid sequence WGQGTLVTVSS of SEQ ID NO: 173; and, (h) CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (i) CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168, and (j) CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169, (k) FR-L1 comprising the amino acid sequence EIVLTQSPATLSLSPGERATLSC of SEQ ID NO: 174, (l) FR-L2 comprising the amino acid sequence WYQQKPGQAPRLLIY of SEQ ID NO: 175, (m) FR-L3 comprising the amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC of SEQ ID NO: 176, and (n) FR-L4 comprising the amino acid sequence GQGTKVEIK of SEQ ID NO: 177; and, ii) the second antigen-binding site comprises: (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; (d) an FR-H1 comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO: 189; (e) an FR-H2 comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID NO: 190; (f) an FR-H3 comprising the amino acid sequence RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO: 191; and (g) an FR-H4 comprising the amino acid sequence WGQGTTVTVSS of SEQ ID NO: 192; and, (h)X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A VL domain comprising: (i) a CDR-L1 comprising the amino acid sequence GATSLEH of SEQ ID NO: 187; (j) a CDR-L3 comprising the amino acid sequence QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; (k) an FR-L1 comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO: 193; (l) an FR-L2 comprising the amino acid sequence WYQQKPGKAPKLLIH of SEQ ID NO: 194; (m) an FR-L3 comprising the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO: 195; and (n) an FR-L4 comprising the amino acid sequence FGGGTKVEIK of SEQ ID NO: 196.

[0100] In a particular embodiment of the invention, the bispecific antibody is an (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, where: A) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 178; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or B) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 178; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 198; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or C) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 178; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 201; or or D) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 179; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 201; or or E) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 180; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or F) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 180; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 201; or or G) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 180; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 199; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or H) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 180; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 198; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or I) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 179; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or J) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 179; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 199; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or K) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 179; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 198; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or L) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 181; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or M) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 181; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 197; and a VL domain comprising the amino acid sequence of SEQ ID NO: 201; or or N) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 181; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 199; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or O) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 181; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 198; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200; or or P) i) the first antigen-binding site is a VH domain comprising the amino acid sequence of SEQ ID NO: 178; and a VL domain comprising the amino acid sequence of SEQ ID NO: 182; and ii) the second antigen-binding site comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 199; and a VL domain comprising the amino acid sequence of SEQ ID NO: 200.

[0101] In a particular embodiment of the invention, the bispecific antibody is an (isolated) bispecific antibody comprising a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen-binding site that (specifically) binds to a second epitope on human CCL2, where: A) i) the first antigen-binding site is a VH domain 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: 178, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I; (b) X 1 is V and X 2 is P and X 3 is H, the amino acid sequence GX of SEQ ID NO: 165 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D; and a VL domain 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: 200, wherein (d) X 1 is F and X 2 is R, the amino acid sequence KAX of SEQ ID NO: 186 1EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W; or or B) i) the first antigen-binding site is a VH domain 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: 178, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I; (b) X 1 is V and X 2 is P and X 3 is H, the amino acid sequence GX of SEQ ID NO: 165 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 198, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is E; and a VL domain 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: 200, wherein (d) X 1 is F and X 2 is R, the amino acid sequence KAX of SEQ ID NO: 186 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W; or or C) i) the first antigen-binding site is a VH domain 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: 178, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 201, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or D) i) the first antigen-binding site is a VH domain 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: 179, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 201, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or E) i) the first antigen-binding site is a VH domain 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: 180, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or F) i) the first antigen-binding site is a VH domain 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: 180, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 201, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or G) i) the first antigen-binding site is a VH domain 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: 180, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 199, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or H) i) the first antigen-binding site is a VH domain 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: 180, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 198, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or I) i) the first antigen-binding site is a VH domain 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: 179, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or J) i) the first antigen-binding site is a VH domain 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: 179, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 199, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or K) i) the first antigen-binding site is a VH domain 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: 179, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 198, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or L) i) the first antigen-binding site is a VH domain 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: 181, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or M) i) the first antigen-binding site is a VH domain 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: 181, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 197, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 201, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or N) i) the first antigen-binding site is a VH domain 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: 181, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 199, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or O) i) the first antigen-binding site is a VH domain 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: 181, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 198, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187, (e) a CDR-L2 comprising the amino acid sequence of GATSLEH of SEQ ID NO: 187, and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R; or or P) i) the first antigen-binding site is a VH domain 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: 178, wherein: (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain sequence comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and a VL domain 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: 182, comprising: (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168; and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; and ii) the second antigen-binding site comprises: a VH domain 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: 199, comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183; (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E; and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and a VL domain 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: 200, wherein (d) X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2A) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187; and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R.

[0102] In certain embodiments of the invention, the bispecific antibodies described herein bind to a first and a second epitope on human CCL2 in an ion-dependent manner.

[0103] In certain embodiments of the invention, the bispecific antibodies described herein bind to human CCL2 in a pH-dependent manner, with both the first antigen-binding site and the second antigen-binding site binding to CCL2 with higher affinity at neutral pH than at acidic pH.

[0104] In certain embodiments of the invention, the bispecific antibodies described herein bind to human CCL2 with 10-fold higher affinity at pH 7.4 than at pH 5.8.

[0105] In a particular embodiment of the invention, the bispecific antibody is an (isolated) (monospecific) antibody that (specifically) binds to human CCL2, wherein the antibody A) (a) CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 164, wherein X is I or T; (b) X 1 is V, I or H, and X 2 is P or H, and X 3 is H or G, 1 IX 2 IFX 3 a VH domain comprising (c) a CDR-H2 comprising TANYAQKFQG, and (c) a CDR-H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 166; and, (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 167, (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 168, and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of SEQ ID NO: 169; or B) a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence HTYMH of SEQ ID NO: 183, (b) a CDR-H2 comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 184, wherein X is D or E, and (c) a CDR-H3 comprising the amino acid sequence GVFGFFXH of SEQ ID NO: 185, wherein X is D or E; and, (d)X 1 is F or T, and X 2 is R or L, 1 EDIYNRX 2 A) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 187; and (f) a CDR-L3 comprising the amino acid sequence of QQFXSAPYT of SEQ ID NO: 188, wherein X is W or R.

[0106] The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments of the invention, epitopic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and may, in certain embodiments of the invention, have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody.

[0107] Whether an antibody binds to or competes for binding to the same epitope as a reference anti-CCL2 antigen-binding site can be easily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-CCL2 antigen-binding site of the present invention, the reference antibody is bound to its CCL2 domain under saturating conditions. The ability of the test antibody to bind to human CCL2 is then evaluated. If the test antibody can bind to human CCL2 after saturation binding with the reference anti-CCL2 antigen-binding site, it can be concluded that the test antibody binds to a different epitope from the reference anti-CCL2 antigen-binding site. On the other hand, if the test antibody cannot bind to human CCL2 after saturation binding with the reference anti-CCL2 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-CCL2 antibody of the present invention. Further routine experiments (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance (e.g., BIAcore), flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).

[0108] Alternatively, two antibodies are considered to bind to the same epitope if substantially all of the amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0109] To determine whether an antibody competes with a reference anti-CCL2 antibody for binding, the above binding methodology is performed in two ways. In the first way, the reference antibody is allowed to bind to CCL2 under saturating conditions, followed by assessing the binding of the test antibody to human CCL2. In the second way, the test antibody is allowed to bind to CCL2 molecules under saturating conditions, followed by assessing the binding of the reference antibody to human CCL2. If, in both ways, only the first (saturating) antibody can bind to CCL2 molecules, it is concluded that the test and reference antibodies compete for binding to CCL2. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0110] As used herein, the term "CCL2," also known as "MCP-1," "human CCL2," refers to the 76 amino acid sequence referenced in NCBI record accession number NP_002973 (SEQ ID NO: 117) and variously known as CCL2, MCP-1 (monocyte chemotactic protein 1), SMC-CF (smooth muscle cell chemotactic factor), LDCF (lymphocyte-derived chemotactic factor), GDCF (glioma-derived monocyte chemotactic factor), TDCF (tumor-derived chemotactic factor), HCl1 (human cytokine 11), and MCAF (monocyte chemotactic and activating factor). The gene symbol is SCYA2, the JE gene on human chromosome 17, and its new name is CCL2 (Zlotnik, Yoshie 2000. Immunity 12:121-127). JE is the mouse homolog of human MCP-1 / CCL2.

[0111] Handel et al. (Biochemistry. 1996;35:17269-6584) determined the solution structure of the CCL2 dimer. These studies showed that the secondary structure of CCL2 consists of four β-sheets. Furthermore, residues responsible for the dimerization interface of CCL2 were described by Zhang and Rollins (Mol Cell Biol. 1995;15:15751-4855). The protein complex appears elongated, with the two monomers oriented to form a large pocket. The structures of two crystal forms, the so-called I and P forms, of monomeric and dimeric CCL2 have also been determined (Lubkowski et al., Nat Struct Biol. 1997;4:171-69). Paolini et al. (J. Immunol. 1994 Sep 15;153(6):2704-17) described the existence of MCP1 / CCL2 as monomers at physiologically relevant concentrations: by analyzing rec.CCL2 protein (purchased from Peprotech) by size-exclusion HPLC, sedimentation equilibrium ultracentrifugation, and chemical cross-linking, they were able to show that the weight fraction of MCP-1 monomers and dimers depends on their in vitro cooperation. Finally, Seo and coworkers (J. Am. Chem. Soc. 2013 Mar 20;135(11):4325-32) were able to demonstrate the presence of both monomeric and dimeric forms of injected CCL2 under physiological conditions by ion mobility mass spectrometry.

[0112] Thus, "wild-type CCL-2" (wt CCL2) can exist as a monomer but can also form dimers at physiological concentrations. This monomer-dimer equilibrium is certainly different and must be carefully considered for all described in vitro experiments in which different concentrations may be used. To avoid uncertainty, we created a point-mutation CCL2 variant: the "P8A" variant of CCL2 harbors a mutation at the dimerization interface and, as a result, is unable to form dimers, resulting in a defined, pure CCL2 monomer. In contrast, the "T10C" variant of CCL2 results in a fixed dimer of CCL2 (J Am Chem Soc. 2013 Mar 20;135(11):4325-32).

[0113] The CCL2 / CCR2 axis is a major mediator of immature myeloid cell recruitment to tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM), establishing a chemoattractant gradient. Once in the tumor, myeloid-derived suppressor cells (MDSCs) contribute to a pro-tumorigenic environment by secreting and upregulating anti-inflammatory cytokines and receptors that inhibit the initiation of anti-tumor T cell responses. In this way, MDSCs can reduce or even impair the efficacy of any T cell-activating therapy (Meyer et al., 2014). Therefore, specific inhibition of the recruitment of these immature myeloid cells enhances the efficacy of checkpoint inhibitors, T cell bispecifics, and cancer immunotherapy. Furthermore, CCL2 is also involved in promoting angiogenesis, metastasis, and tumor growth, suggesting that neutralization of CCL2 may contribute to several anti-tumor interventions.

[0114] Targeting CCL2, as opposed to its receptor, specifically inhibits undesirable CCL2-mediated effects, sparing those that may signal through the same receptor (CCR2) but different ligands (e.g., CCL7, CCL8, CCL13) involved in the recruitment of other immune cell populations such as Th1 and NK cells.

[0115] Clinically, CCL2 has been the preferred antibody target in several studies aimed at neutralizing its elevated levels caused by different inflammatory diseases, such as rheumatoid arthritis (Haringman et al., 2006), idiopathic pulmonary fibrosis (Raghu et al., 2015), diabetic nephropathy (Menne et al., 2016), and cancer (Sandhu et al., 2013). However, its high synthesis rate, together with the observed high in vivo antibody-antigen dissociation constant (KD), has proven to be a major obstacle preventing the inhibition of free CCL2 by conventional antibodies at clinically viable doses (Fetterly et al., 2013).

[0116] CCL2 neutralization appears to be more clearly relevant in patients with elevated serum levels of CCL2, which has been observed in several cancer types, such as breast cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer, and prostate cancer. However, even patients within these indications who do not exhibit this serology but whose tumors are highly infiltrated with immune cells of the myeloid lineage may benefit greatly from this novel treatment due to the many roles that CCL2 plays in the tumor context, as discussed above.

[0117] As used herein, an antibody that "binds to human CCL2," "specifically binds to human CCL2," "binds to human CCL2," or "anti-CCL2" is an antibody that is 5.0 x 10 -8 K in mol / l or less D value, in a particular embodiment 1.0 x 10 -9 K in mol / l or less D value, in a particular embodiment 5.0 x 10 -8 mol / l ~ 1.0 × 10 -13 K in mol / l D This refers to an antibody that specifically binds to the human CCL2 antigen with a binding affinity of 0.01%.

[0118] Binding affinity is determined in standard binding assays, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare, Uppsala, Sweden), using constructs comprising the CCL2 extracellular domain (e.g., its naturally occurring three-dimensional structure). In certain embodiments of the invention, binding affinity is determined in standard binding assays using exemplary soluble CCL2.

[0119] Antibody specificity refers to the antibody's selective recognition of a particular epitope of an antigen. For example, natural antibodies are monospecific.

[0120] As used herein, the terms "bispecific antibody that binds to (human) CCL2," "biparatopic antibody that binds to (human) CCL2," "bispecific anti-CCL2 antibody," and "biparatopic anti-CCL2 antibody" mean that the antibody can specifically bind to at least two different epitopes on (human) CCL2. Typically, such a bispecific antibody contains two different antigen-binding sites (two different paratopes), each specific for a different epitope on (human) CCL2. In certain embodiments of the invention, the bispecific antibody can bind to two different, non-overlapping epitopes on CCL2, meaning that the two different antigen-binding sites do not compete for binding to CCL2.

[0121] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule to which an antigen-binding moiety / site binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virally infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0122] The following exemplification of the method according to the invention is presented using an exemplary anti-C5 antibody. It is presented as an example only and should not be construed as a limitation of the method according to the invention. The true scope is set forth in the appended claims.

[0123] The illustration shows the in vitro determination of free human C5 in a 100% human serum sample.

[0124] To detect free C5 in human serum samples, we used a nanoliter-scale microfluidic affinity flow-through format with a laser-induced fluorescence detection assay (Gyrolab® Workstation assay), which allows for quantitative detection of free C5 in 100% human serum.

[0125] Test samples, quality control samples, and positive control standards are analyzed in 100% serum. Quality control samples and standards are prepared in 100% horse serum (non-cross-reactive C5). Relative quantification of analytes is performed by back-calculation of fluorescence values ​​using the corresponding standard curve, e.g., using a nonlinear 4-parameter Wiemer-Rodbard fitting function.

[0126] Typical calibration curves are shown in Figure 5 (Examples 1 and 10).

[0127] In a particular embodiment of the invention, the therapeutic antibody is an anti-C5 antibody and the antigen is human C5.

[0128] The terms "anti-C5 antibody" and "antibody that (specifically) binds to C5" refer to an antibody that can bind to C5 with sufficient affinity so as to be useful as a diagnostic and / or therapeutic agent targeted to C5. In certain embodiments of the invention, the extent of binding of the anti-C5 antibody to unrelated non-C5 proteins is less than about 10% of the binding of the antibody to C5. In certain embodiments of the invention, the anti-C5 antibody binds to an epitope of C5 that is conserved among C5 from different species. In a preferred embodiment, the C5 is human C5.

[0129] In certain embodiments of the invention, the anti-C5 antibody is eculizumab or crovalimab.

[0130] In certain embodiments of the invention, the determination of free C5 is carried out in the presence of eculizumab and / or crovalimab. In this embodiment, the term "free C5" refers to C5 of any length that is not bound by eculizumab or crovalimab.

[0131] As used herein, the term "C5" encompasses any native C5 from any vertebrate source, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). Unless otherwise indicated, the term "C5" refers to a human C5 protein having the amino acid sequence set forth in SEQ ID NO: 30 and containing the beta chain sequence set forth in SEQ ID NO: 31. The term encompasses "full-length," unprocessed C5 and any form of C5 resulting from intracellular processing. The term also encompasses naturally occurring variants of C5, such as splice variants or allelic variants. An exemplary amino acid sequence of human C5 is set forth in SEQ ID NO: 30 ("wild-type" or "wt" C5). An exemplary amino acid sequence of the beta chain of human C5 is set forth in SEQ ID NO: 31. The amino acid sequences of exemplary MG1, MG2, and MG1-MG2 domains of the beta chain of human C5 are set forth in SEQ ID NOs: 32, 33, and 34, respectively. Exemplary cynomolgus monkey and mouse C5 amino acid sequences are set forth in SEQ ID NOs: 35 and 96, respectively. Amino acid residues 1-19 of SEQ ID NOs: 30, 31, 34, 35, and 96 correspond to signal sequences that are removed during intracellular processing and are therefore missing from the corresponding exemplary amino acid sequences.

[0132] U.S. Patent Application Publication No. 2016 / 0167054 discloses anti-C5 antibodies and methods of using same. In some embodiments, the disclosed isolated anti-C5 antibodies bind to an epitope within the beta chain of C5 with higher affinity at neutral pH than at acidic pH.

[0133] C5 is a 181 kDa protein found in normal serum at approximately 71 μg / ml (0.4 μM). Approximately 1.5–3% of C5's mass is glycosylated, resulting from carbohydrates. Mature C5 is a heterodimer of a 106 kDa alpha chain disulfide-linked to a 66 kDa beta chain. C5 is synthesized as a 1577 amino acid single-chain precursor protein (pro-C5 precursor) (see, e.g., U.S. Pat. Nos. 6,355,245 and 7,432,356). The pro-C5 precursor is cleaved to yield the beta chain as an amino-terminal fragment and the α chain as an alpha-carboxyl-terminal fragment. The alpha and beta chain polypeptide fragments are linked to each other via disulfide bonds to form the mature C5 protein.

[0134] Mature C5 is cleaved into C5a and C5b fragments during activation of the complement pathway. C5a is cleaved from the alpha chain of C5 by C5 convertase as an amino-terminal fragment containing the first 65 amino acids of the alpha chain. The remaining portion of mature C5 is the fragment C5b, which contains the remainder of the alpha chain disulfide attached to the beta chain. Approximately 20% of the 11 kDa mass of C5a is attributable to carbohydrate.

[0135] C5a is an anaphylatoxin. C5b binds with C6, C7, C8, and C9 to form the membrane attack complex (MAC, C5b-9, terminal complement complex (TCC)) on the surface of target cells. When sufficient numbers of MACs are inserted into the target cell membrane, MAC pores are formed, mediating rapid osmotic lysis of the target cell.

[0136] Anaphylatoxins can induce mast cell degranulation, which releases histamine and other inflammatory mediators, resulting in smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory phenomena, including cell proliferation, leading to hypercellularity. C5a also functions as a chemotactic peptide that helps attract granulocytes, such as neutrophils, eosinophils, basophils, and monocytes, to the site of complement activation.

[0137] The activity of C5a is regulated by the plasma enzyme carboxypeptidase N, which removes the carboxy-terminal arginine from C5a to form the C5a-des-Arg derivative, which exhibits only 1% of the anaphylactic and polymorphonuclear chemotactic activities of unmodified C5a.

[0138] Although a properly functioning complement system provides a robust defense against infectious microorganisms, inappropriate regulation or activation of complement has been implicated in the pathogenesis of a variety of disorders, including, for example, rheumatoid arthritis (RA); lupus nephritis; ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; multiple sclerosis (MS); traumatic brain injury; and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis (see, e.g., Holers et al., Immunol. Rev. 223 (2008) 300-316). Therefore, inhibition of excessive or uncontrolled activation of the complement cascade may provide clinical benefit to patients with such disorders, particularly those with paroxysmal nocturnal hemoglobinuria (PNH).

[0139] Eculizumab is a humanized monoclonal antibody against the complement protein C5 and is the first therapy approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (see, e.g., Dmytrijuk et al., The Oncologist 13 (2008) 894-910). Eculizumab inhibits the cleavage of C5 by C5 convertase into C5a and C5b, which prevents the generation of the terminal complement complex C5b-9. Both C5a and C5b-9 trigger the terminal complement-mediated events characteristic of PNH and aHUS (see also WO 2005 / 065607, WO 2007 / 96586, WO 2008 / 060790, and WO 2010 / 054403). Several reports have described other anti-C5 antibodies. For example, WO 86 / 28707 describes an anti-C5 antibody that binds to the alpha chain of C5 but not to C5a, preventing C5 activation, while WO 2002 / 30886 describes an anti-C5 monoclonal antibody that inhibits C5a formation. WO 2004 / 006653 describes an anti-C5 antibody that recognizes the proteolytic site of C5 convertase on the alpha chain of C5 and inhibits the conversion of C5 to C5a and C5b. WO 2010 / 015608 describes an anti-C5 antibody with an affinity constant of at least 1 x 10E7 M-1. In a specific embodiment of the present invention, the drug is eculizumab.

[0140] In some embodiments, the anti-C5 antibody binds to an epitope within the beta chain of C5. In some embodiments, the anti-C5 antibody binds to an epitope within the MG1-MG2 domain of the beta chain of C5. In some embodiments, the anti-C5 antibody binds to an epitope within a fragment consisting of amino acids 27-115 of the beta chain of C5 (SEQ ID NO: 31). In some embodiments, the anti-C5 antibody binds to an epitope within the beta chain of C5 (SEQ ID NO: 31) comprising at least one fragment selected from the group consisting of amino acids 38-48, 61-67, and 98-101. In some embodiments, the anti-C5 antibody binds to an epitope within a fragment of the beta chain of C5 (SEQ ID NO: 31) comprising at least one amino acid residue selected from the group consisting of Glu48, Asp51, His61, His63, Lys100, and His101 of SEQ ID NO: 31. In further embodiments, the antibody binds to C5 with greater affinity at neutral pH than at acidic pH. In a further embodiment, the antibody binds to C5 with greater affinity at pH 7.4 than at pH 5.8. In another embodiment, the anti-C5 antibody binds to the same epitope as an antibody listed in Table 1. In a further embodiment, the antibody binds to the same epitope as an antibody listed in Table 1 with greater affinity at pH 7.4 than at pH 5.8. In a further embodiment, the anti-C5 antibody binds to the same epitope as an antibody listed in Table 2 or 3. In a further embodiment, the antibody binds to the same epitope as an antibody listed in Table 2 or 3 with greater affinity at pH 7.4 than at pH 5.8.

[0141] [Table 1]

[0142] [Table 2]

[0143] [Table 3]

[0144] In certain embodiments of the invention, the anti-C5 antibody competes for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (a) a VH of SEQ ID NO: 01 and a VL of SEQ ID NO: 11; (b) a VH of SEQ ID NO: 05 and a VL of SEQ ID NO: 15; (c) a VH of SEQ ID NO: 04 and a VL of SEQ ID NO: 14; (d) a VH of SEQ ID NO: 06 and a VL of SEQ ID NO: 16; (e) a VH of SEQ ID NO: 02 and a VL of SEQ ID NO: 12; (f) a VH of SEQ ID NO: 03 and a VL of SEQ ID NO: 13; (g) a VH of SEQ ID NO: 09 and a VL of SEQ ID NO: 19; (h) a VH of SEQ ID NO: 07 and a VL of SEQ ID NO: 17; (i) a VH of SEQ ID NO: 08 and a VL of SEQ ID NO: 18; and (j) a VH of SEQ ID NO: 10 and a VL of SEQ ID NO: 20.

[0145] In certain embodiments of the invention, anti-C5 antibodies are used to treat complement-mediated diseases or conditions involving excessive or uncontrolled activation of C5. In further embodiments, anti-C5 antibodies are used to treat diseases or disorders including, but not limited to, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration, myocardial infarction, rheumatoid arthritis, osteoporosis, osteoarthritis, and inflammation. Anti-C5 antibodies are used to enhance the clearance of C5 from plasma.

[0146] In certain embodiments of the invention, the method is for detecting free C5 in the presence of an anti-C5 antibody comprising a VH as in any of the embodiments provided above and a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 27, 28, 29, 105, 106, and 107. In certain embodiments of the invention, the method is for detecting free C5 in the presence of an anti-C5 antibody comprising a VL as in any of the embodiments provided above and a light chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 36, 37, and 38.

[0147] In certain embodiments of the invention, the method is for detecting free C5 in the presence of an anti-C5 antibody that competes for binding to C5 with an antibody comprising a VH and VL pair selected from the following: (a) a VH of SEQ ID NO: 01 and a VL of SEQ ID NO: 11; (b) a VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 25; (c) a VH of SEQ ID NO: 21 and a VL of SEQ ID NO: 24; (d) a VH of SEQ ID NO: 05 and a VL of SEQ ID NO: 15; (e) a VH of SEQ ID NO: 04 and a VL of SEQ ID NO: 16; (f) VH of SEQ ID NO: 06 and VL of SEQ ID NO: 16; (g) VH of SEQ ID NO: 02 and VL of SEQ ID NO: 12; (h) VH of SEQ ID NO: 03 and VL of SEQ ID NO: 13; (i) VH of SEQ ID NO: 09 and VL of SEQ ID NO: 19; (j) VH of SEQ ID NO: 7 and VL of SEQ ID NO: 17; (k) VH of SEQ ID NO: 8 and VL of SEQ ID NO: 18; (l) VH of SEQ ID NO: 23 and SEQ ID NO: 26; and (m) VH of SEQ ID NO: 10 and SEQ ID NO: 20.

[0148] In certain embodiments of the invention, the method is for detecting free C5 in the presence of an anti-C5 antibody that competes for C5 binding with an antibody comprising a VH and VL pair selected from the following: (a) a VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 25; (b) a VH of SEQ ID NO: 21 and a VL of SEQ ID NO: 24; (c) a VH of SEQ ID NO: 05 and a VL of SEQ ID NO: 15; (d) a VH of SEQ ID NO: 04 and a VL of SEQ ID NO: 14; (e) a VH of SEQ ID NO: 06 and a VL of SEQ ID NO: 16; (f) a VH of SEQ ID NO: 02 and a VL of SEQ ID NO: 12; (g) a VH of SEQ ID NO: 03 and a VL of SEQ ID NO: 13; (h) a VH of SEQ ID NO: 09 and a VL of SEQ ID NO: 19; (i) a VH of SEQ ID NO: 07 and a VL of SEQ ID NO: 17; (j) a VH of SEQ ID NO: 8 and a VL of SEQ ID NO: 18; (k) a VH of SEQ ID NO: 23 and a VL of SEQ ID NO: 26.

[0149] In one preferred embodiment of the invention, the method is for detecting free C5 in the presence of an anti-C5 antibody comprising a VH of SEQ ID NO:97 and a VL of SEQ ID NO:102.

[0150] The following examples, sequences and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. [Brief explanation of the drawings]

[0151] [Figure 1] Cynomolgus monkey CCL2 calibration curves were prepared with an assay according to the invention in 100% horse serum ranging from 4 to 1000 pg / mL of CCL2 serum concentrations and analyzed with the Elisa assay described in Example 4, involving variations in incubation times of samples on the assay plate between 75 seconds and 12 minutes. [Figure 2] Nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection assay using indirect and direct Alexa labeling of the detection antibody according to the present invention. [Figure 3] A nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection assay according to the present invention was used, using a therapeutic antibody and a competing rabbit monoclonal antibody. CCL2 values ​​were back-calculated with the standard curve, and CCL2 recovery (% free) was calculated relative to the unspiked 5 ng / mL CCL2 value. The top curve used the therapeutic antibody as the capture antibody, and the bottom curve used the competing rabbit antibody as the capture antibody. [Figure 4] The assay according to the invention was performed using recombinant human wild-type CCL2 as a calibrator. The calibration range of two runs is shown. [Figure 5] Calibration curve of an assay according to the invention for detecting free C5 in human serum samples in a nanoliter scale microfluidic affinity flow-through format using a laser-induced fluorescence detection assay (Gyrolab® Workstation assay). [Figure 6] 1. Scheme of the method according to the invention using ELISA. [Figure 7] Scheme of comparative ELISA in 25% serum (Example 4). [Figure 8] Scheme of the method according to the invention using a nanoliter scale microfluidic affinity flow-through format with laser-induced fluorescence detection, indirect format (Gyros assay) (Examples 6, 7). [Figure 9] Calibration curve for nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection (Gyros assay) (Example 6). [Figure 10] Scheme of the method according to the invention using a nanoliter scale microfluidic affinity flow-through format with laser-induced fluorescence detection, direct format (Gyros assay) (Example 7). [Figure 11] Scheme of the method according to the invention using a nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection (Gyros assay) (Example 9). [Example]

[0152] Example 1 General description of the method according to the invention 1) A nanoliter-scale microfluidic affinity flow-through format with an assay format based on laser-induced fluorescence detection. A Gyrolab® workstation assay was set up to detect free antigen in human serum samples. This test was used for quantitative detection of free antigen. Test samples, quality control samples, and positive control standards were analyzed in 100% serum. Quality control samples and standards were prepared in 100% horse serum (containing non-cross-reactive endogenous targets).

[0153] The following sequential steps were carried out: The capture reagent (mAb<target>rH-IgG-Bi, 5,000 ng / mL) was added, followed by the addition of the sample, and finally the detection reagent (mAb<target>M-Alexa 647, 10,000 ng / mL). After each step, a wash was performed to remove unbound reagent. Each wash step consisted of the addition of a wash solution (1x PBS containing 0.05% (v / v) Tween 20).

[0154] 2) ELISA-based assay format An ELISA assay is set up to detect free antigen in human serum samples. This test is used for the quantitative detection of free antigen. Test samples, quality control samples, and positive control standards are analyzed in 100% serum. Quality control samples and standards are prepared in 100% horse serum (containing non-cross-reactive endogenous targets).

[0155] The following successive steps are carried out: Different combinations of recombinant antigens and antibodies are prepared in 100% horse pool serum and incubated for 2 hours at room temperature. Standard curve samples are prepared in 100% horse serum. Briefly, biotinylated (bispecific) therapeutic antibodies as capture antibodies, test samples, and detection reagents (digoxylated (bispecific) therapeutic antibodies) are added stepwise to a 384-well streptavidin-coated microtiter plate and incubated for 3-4 minutes (target 3.5 minutes) on a gentle shaker. For detection of immobilized immune complexes, a polyclonal anti-digoxigenin-POD conjugate is added and the plate is incubated for 15-20 minutes. Optionally, the plate is washed three times after each step to remove unbound material. ABTS is added to the plate and incubated at room temperature with shaking. Absorbance is measured at wavelengths of 405 / 490 nm. The antigen concentration is calculated based on the standard curve response using the analysis software XLfit (IDBS).

[0156] Example 2 Determination of KD values ​​in 100% serum samples Based on the general method described in Example 1, the following experiment was designed to determine the KD value of a monoclonal antibody against its endogenous antigen. The calibration curve ranged from 0.55 ng / mL to 3000 ng / mL. Qualified control (QC) samples were prepared in horse serum at five concentrations: 0.55 ng / mL, 1.5 ng / mL, 100 ng / mL, 1200 ng / mL, and 3000 ng / mL. The recoveries of the QC samples met the criterion of + / - 20% from the nominal value and ranged from 97% to 112%. To determine KD values ​​for endogenous targets (following the method outlined in WO 2014 / 023655), three human serum samples were diluted 40-fold with horse serum to maintain a 100% serum concentration, spiked with 1000 ng / mL, 2000 ng / mL, 3000 ng / mL, 4000 ng / mL, and 5000 ng / mL of therapeutic antibody, and equilibrated. Similarly, unspiked human serum samples were analyzed at a dilution factor of 40 (using horse serum) to calculate the total target concentration in each individual serum sample. Based on the determined total target value, the free target fraction was determined for each spiked serum sample. Based on each free fraction, the KD value was calculated. TIFF0007824236000009.tif57170The average (av) KD was 0.05 nM with a standard deviation (cv) of 0.012 nM.

[0157] Example 3 Assay according to the invention in 100% human serum Based on Example 2, the following experiment was designed, focusing on the preparation and analysis of free QC samples in 100% human serum. Free QC samples were prepared by spiking 100 μg / mL of crovalimab into each of the individual human serum samples used in Example 2. Each serum sample was analyzed undiluted, and the free target concentrations were analyzed as follows: ID1 = 6.5 ng / mL; ID2 = 8.2 ng / mL; and ID3 = 7.6 ng / mL. Based on the KD values ​​and total target concentrations determined in Example 2, the estimated free target concentrations should be in the range of 2.41 ng / mL to 6.83 ng / mL for ID1, 3.38 ng / mL to 9.56 ng / mL for ID2, and 3.51 ng / mL to 9.93 ng / mL for ID3. The range calculations were based on two standard deviations of the KD values. All three individual serum samples fell within the calculated ranges, thus validating the validity of the method according to the present invention.

[0158] Example 4 Comparative example: ELISA in 25% serum Different combinations of recombinant cynomolgus monkey CCL2 and the biparatopic anti-CCL2 antibody CKLO2-SG1 were prepared in 100% pooled horse serum and incubated for 2 h at room temperature. Samples were diluted 1 to 4 in assay buffer (PBS, 0.1% Tween, 1% BSA) (to obtain the MRD, 25% matrix) and then further diluted 1 to 10 in assay buffer containing 25% horse serum. A calibration curve was prepared in assay buffer containing 25% horse serum, covering a range of CCL2 serum concentrations from 7.8 to 1000 pg / mL. Briefly, biotinylated anti-CCL2 capture antibody (CNTO0888, CCL2-0004), test samples, and detection reagents (digoxigenylated anti-CCL2 antibody (humanized 11K2, CCL2-0002)) were added stepwise to a 384-well streptavidin-coated microtiter plate and incubated for 1 h, 12–14 min, and 17 min, respectively, on a non-vigorous shaker. For detection of immobilized immune complexes, a polyclonal anti-digoxigenin-POD conjugate was added, and the plate was incubated for 20 min. After each step, the plate was washed three times to remove unbound material. ABTS was added to the plate and incubated at room temperature with shaking. Absorbance was measured at wavelengths of 405 / 490 nm (see Figures 6 and 7). CCL2 concentrations were calculated based on the standard curve response using the analysis software XLfit (IDBS) with dilution factors of 1–4 and 1–40. The ratio of the two sample dilutions analyzed was calculated and is shown in the table below (-=not determined). TIFF0007824236000010.tif60170

[0159] Example 5 Assay according to the invention in 100% serum A cynomolgus monkey CCL2 calibration curve was prepared in 100% horse serum ranging from 4 pg / mL to 1000 pg / mL of CCL2 and analyzed in an ELISA format based on that described in Example 4, but using 100% serum (undiluted) samples. Incubation times of samples on the assay plate varied between 75 seconds and 12 minutes. TIFF0007824236000011.tif87162

[0160] Example 6 The method according to the invention uses a nanoliter-scale microfluidic affinity flow-through format (Gyros assay) with laser-induced fluorescence detection In this example, a Gyrolab™-based method was used. Recombinant cynomolgus monkey CCL2 samples were prepared in assay buffer (PBS, 0.1% (v / v) Tween-20, 1% BSA) and analyzed on a Gyrolab Xplore. A monospecific biotinylated parent anti-CCL2 antibody (CNTO0888, also known as CCL2-004) was used as the capture reagent diluted to 1 μg / mL in assay buffer. For detection, 1 μg / mL of a monospecific dig-labeled anti-CCL2 antibody (humanized 11K2, CCL2-0002) was incubated with 1 μg / mL of mAb in assay buffer for 2 hours. <dig>The samples were preincubated with M-1.71.256-IgG-Alexa 647. All reagents and samples were transferred to a 96-well PCR plate and loaded into the instrument along with a Gyrolab BioAffy 200 nL disk (Gyros Protein Technologies AB). A three-step assay protocol (200-3W-001) was selected. Briefly, the protocol describes the sequential addition of capture reagent, sample, and detection reagent to the designated streptavidin column of the Gyrolab BioAffy 200 disk. Each reagent reaches the column simultaneously after a short spinning step is applied to the disk. After each step, the column was washed with PBS containing 0.05% Tween, and finally, laser-induced fluorescence values ​​were recorded in the instrument. A nonlinear four-parameter curve-fitting function (Wiemer-Rodbard) was applied to the averaged raw data to obtain a calibration curve. See also Figures 8 and 9. TIFF0007824236000012.tif72170 The assay was found to be linear over the selected assay range (312.5 pg / mL to 40,000 pg / mL).

[0161] Example 7 The method according to the invention uses a nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection (Gyros assay) using indirect and direct Alexa labeling of the detection antibodies according to the invention Humanized anti-CCL2 antibody 11K2 (CCL 2-0002) and rabbit anti-CCL2 antibody 1H11 (CCL2-0011) were labeled with Alexa 647 (Molecular Probes, Invitrogen, Cat A20186). A recombinant cynomolgus monkey CCL2 calibration curve in assay buffer (PBS, 0.1% Tween, 1% BSA) was analyzed with different anti-CCL2 capture and detection reagents (1 μg / mL) as described for Example 6. Anti-CCL2 antibody-Bi (biotin-labeled anti-CCL2 antibody CNTO0888) preincubated with digoxigenin-conjugated humanized antibody 11K2 and Alexa 647-conjugated anti-Dig antibody M-1.71.256 (IgG). Anti-CCL2 antibody-Bi (CNTO 0888-Bi), preincubated humanized antibody 11K2 conjugated to Alexa 647 Rabbit anti-CCL2 antibody 2F6 (Bi; CCL2-0014) conjugated to biotin, rabbit anti-CCL2 antibody 1H11 conjugated to Alexa 647 TIFF0007824236000013.tif85170In comparison to pre-incubated detection reagents, increased sensitivity was observed when using detection antibodies directly labeled with Alexa 647 (see table above and Figures 2 and 10).

[0162] A competitive rabbit monoclonal antibody was used to avoid either false-positive results that could arise due to bridging of ADA to constant regions in capture and detection IgGs containing human backbones, or false-negative results due to neutralization of ADA to CDRs of therapeutic molecules that could cross-react with the capture and detection reagents. CCL2 values ​​were back-calculated with the standard curve, and CCL2 recovery (% free) was calculated relative to the unspiked 5 ng / mL CCL2 value (see also Figure 3). The sensitivity for detecting TIFF0007824236000014.tif73170cyCCL2 was comparable between the described human parent capture and detection reagents and the selected competitive rabbit monoclonal anti-CCL2 antibody.

[0163] Four molecules were tested in the POC study: Group 1: CNTO0888-SG1 (IgG1 wild-type) anti-CCL2 antibody (n = 3 animals) (as a control for maximum total CCL2 accumulation); Group 2: CKLO2-SG1 (IgG1 wild-type) biparatopic anti-CCL2 antibody with pH-dependent target binding but no Fc modifications (n ​​= 3); Group 3: CKLO2-SG1100 biparatopic anti-CCL2 antibody with pH-dependent target binding and Fc-pI and additional modifications (n ​​= 4); and Group 4: CKLO2-SG1095 biparatopic anti-CCL2 antibody with pH-dependent target binding, Fc-pI and FcγRII, and additional modifications (n ​​= 4). These four molecules were preincubated at different concentrations in assay buffer containing 5 ng / mL cyCCL2 for 2 hours and then analyzed in the gyros assay with a cyCCL2 calibration curve. The CCL2 values ​​of the samples were back-calculated using the standard curve, and the recovery values ​​(amount of free CCL2) were calculated relative to 5 ng / ml of CCL2. TIFF0007824236000015.tif108170 The data presented in the table above show comparable results for the setup with human parent capture and detection molecules and competing rabbit mAbs.

[0164] Example 8 Determination of free CCL2 in a POC study of CCL2 sweeping efficacy in cynomolgus monkeys Free CCL2 serum samples were analyzed using a qualified, but unvalidated, Gyrolab™ immunoassay performed on a Gyrolab Xplore. A biotinylated anti-CCL2 antibody (M-2F6-IgG) was used as the capture reagent, and an Alexa 647-labeled anti-CCL2 antibody (M-1H11-IgG) was selected for detection. Both reagents were diluted to 1 μg / mL in PBS, 0.1% Tween, and 1% BSA and transferred to a 96-well PCR plate (Fisher Scientific). Cynomolgus monkey CCL2 standard curve samples, QC, and undiluted serum samples were also transferred to the 96-well PCR plate. Both plates, along with Gyrolab BioAffy 200 nL discs (Gyros Protein Technologies AB), were loaded into the instrument. The three-step assay protocol (200-3W-001) was selected. Briefly, the protocol describes the sequential addition of capture reagent, sample, and detection reagent to the designated streptavidin column of a Gyrolab BioAffy 200 disc. Each reagent reaches the column simultaneously after a short spinning step is applied to the disc. After each step, the column is washed with PBS 0.05% Tween, and finally, laser-induced fluorescence values ​​are recorded within the instrument. XL Fit software (IDBS) was used to calculate free cynomolgus CCL2 concentrations based on the calibration curve response.

[0165] To demonstrate assay performance, QC samples (High QC 1820 pg / mL cyCCL2, Mid QC 230 pg / mL cyCCL2, and LQC 30 pg / mL CCL2) were prepared in 1x PBS, 0.1% Tween, and 1% BSA and analyzed in parallel with pooled cynomolgus monkey serum (obtained from Biotrend) in each run. Calibrators ranging from 2430 pg / mL to 10 pg / mL were also prepared in 1x PBS, 0.1% Tween, and 1% BSA. Additionally, pooled cynomolgus monkey serum (CPS) was spiked with CNTO0888 at 7.5 ng / mL and 10 μg / mL, and CKLO2-SG1095 at 15 ng / mL and 10 μg / mL. These samples were also analyzed as QC samples in each assay run (depending on the group). As shown in the table below, the variation in free CCL2 values ​​between assay runs was less than 11% for these free QC samples. Assay QC containing recombinant CCL2 in assay buffer was found to be within + / - 20% of the nominal concentration in all 12 assay runs (data not shown). TIFF0007824236000016.tif106170-:Undecided

[0166] Example 9 Mouse studies To support the studies conducted in the B16-huCCL2 / CCL2 null mouse model, the assay described in Example 8 was performed using recombinant human wild-type CCL2 as a calibrator (see Figure 11 for the assay scheme). Because huCCL2 values ​​in transgenic mice were expected to be higher, as in the cynomolgus monkey studies, the assay range was extended to 21,870 pg / ml as the highest calibrator at the upper end. The linearity of the extended calibration range for two runs is shown in Figure 4 and the table below. TIFF0007824236000017.tif71128

[0167] As a test control, pooled mouse serum (MPS) was spiked with 5 ng / mL recombinant human wild-type CCL2 or 5 ng / mL recombinant human wild-type CCL2 and 5 μg / mL or 50 ng / mL CKLO2-SG1095. Recovery values ​​were calculated relative to a nominal 5 ng / mL. The corresponding data are shown in the table below. TIFF0007824236000018.tif38170

[0168] Example 10 Method according to the invention for determining free C5 in 100% serum samples A Gyrolab® workstation was used. 20 μL of test samples, quality control samples, blank samples, and each positive control standard were transferred to designated wells of a multi-well plate, to which the respective capture and detection reagents were added. The sealed plate was centrifuged at least 3000 g for 10 seconds. Analysis on the Gyrolab® workstation used a three-step method with two washing solutions for needle washing. Samples, blanks, quality controls, and standards were measured in replicates (N=2).

[0169] Interpretation of sample results is based on qualitative interpretation of the sample FU and corresponding quality control (QC). 1% PMT fluorescence raw data was exported as an Excel file using Gyrolab® Evaluator Software. A standard calibration curve was generated by a nonlinear four-parameter fit using the Wiemer-Rodbard function (e.g., using XLfit for MS Excel). Wiemer-Rodbard: [y(x)={(1*A)+((BA) / {1+{(C / x)AD))))]. A and B represent the signal deviation (the approximated start and end of the calibration curve). C and D represent the curve shape. Quantification of results compared to a positive control antibody is performed by back-calculating the mean signal of the sample using the fitted calibration curve. Representative raw data (fluorescence units) from the calibration are shown in the table below. TIFF0007824236000019.tif22170< / dig>

Claims

1. A method for determining free antigens capable of being specifically bound by antibodies in an undiluted serum sample containing free antigens, free antibodies, and antigen-antibody complexes, said method comprising the steps of: a) applying the undiluted serum sample to a solid phase on which a capture antibody is immobilized to form a capture antibody-antigen complex; the capture antibody competes with the antibody for binding to a first epitope on the antigen; b) applying a tracer antibody to the solid phase to form a capture antibody-antigen-tracer antibody complex; the tracer antibody specifically binds to a second epitope on the antigen; the epitope of the tracer antibody does not overlap with the epitope of the capture antibody on the antigen; and c) determining the free antigen of the antibody by determining the tracer antibody in the capture antibody-antigen-tracer antibody complex; Including, the antigen is human CCL2 or human C5; The method.

2. 2. The method of claim 1, wherein in step a), the applying is under conditions such that at most 10% of the antibodies bound to the antigen are displaced by the capture antibodies, and wherein in step a), at most 10% of the antibodies bound to the antigen are displaced.

3. Step a) applying the undiluted serum sample to the solid phase on which a capture antibody is immobilized to form a capture antibody-antigen complex; the capture antibody competes with the antibody for binding to a first epitope on the antigen; the undiluted serum sample is incubated with the solid phase for 240 seconds or less; The method according to claim 1 or 2, wherein

4. The method according to any one of claims 1 to 3, wherein the half-life of a complex between the antigen and the antigen-binding site of the antibody that specifically binds to the first epitope on the antigen is 100 seconds or less.

5. 5. The method of claim 1, wherein the antibody is a bispecific antibody, the bispecific antibody comprising a first antigen-binding site that specifically binds to the first epitope on the antigen and a second, different antigen-binding site that specifically binds to the second epitope on the antigen, and the tracer antibody competes with the bispecific antibody for binding to the second epitope on the antigen.

6. 6. The method of claim 5, wherein the complex between the antigen-binding site of the bispecific antibody that specifically binds to the second epitope on the antigen and the antigen has a half-life of 20 seconds or less.

7. The method of any one of claims 1 to 6, wherein the capture antibody and the tracer antibody are non-human, non-humanized antibodies.

8. The method according to any one of claims 1 to 7, wherein the method is an enzyme-linked immunosorbent assay and the undiluted serum sample is incubated with the solid phase for 180 to 240 seconds.

9. The method of any one of claims 1 to 8, wherein the tracer antibody is incubated with the capture antibody-antigen complex for less than 1200 seconds.

10. 8. The method of any one of claims 1 to 7, wherein the method is a nanoliter-scale microfluidic affinity flow-through format with laser-induced fluorescence detection, and the undiluted serum sample is incubated with the solid phase for 2 seconds or less.

11. The method of any one of claims 1 to 7 and 10, wherein the tracer antibody is incubated with the capture antibody-antigen complex for less than 2 seconds.

12. The method of any one of claims 1 to 11, wherein the antibody is a therapeutic antibody.

13. The method is for determining the amount of free antigen, and step c) comprises: determining the amount of free antigen of the antibody in the undiluted serum sample by determining the amount of the tracer antibody in the capture antibody-antigen-tracer antibody complex. The method according to any one of claims 1 to 12, wherein

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