Methods for elucidating complex, multi-step antibody interactions

Abstract

This provides a novel method for characterizing antibody-FcRn interactions using surface plasmon resonance, taking into account coating density and interaction type. [Solution] This specification reports a method for measuring antibody-FcRn interactions, the method comprising: a step of immobilizing FcRn on a solid surface suitable for surface plasmon resonance measurement (step a); a step of individually coating the solid surface obtained in step a) with solutions containing antibodies of different concentrations and measuring the association rate constant and dissociation rate constant for each concentration (step b); and a step of measuring the KD value of the antibody-FcRn interaction using the rates obtained in step b), wherein the immobilized FcRn is monomeric FcRn, the monomeric FcRn is immobilized using functional (capturing) groups directly bound to the solid surface, the solid surface does not contain branched glucans, and the immobilization is performed at a pH of pH 7 to pH 8.

Description

[Technical Field]

[0001] This invention relates to the field of antibody characterization. More specifically, this specification provides a novel method for characterizing antibody-FcRn interactions using surface plasmon resonance and taking into account coating density and interaction type. This novel method provides an improved determination of antibody affinity to Fc regions and FcRn. [Background technology]

[0002] Background of the Invention The half-life of IgG is influenced by a cellular recycling mechanism that relies on pH-dependent binding to FcRn. While the core interaction site with FcRn is well-established to be located in the CH2-CH3 elbow region, intriguing new data strongly suggest that the Fab arm also contributes to receptor binding. Theoretically, the IgG molecule possesses multiple FcRn binding sites. Experimental data also support that amino acid changes within the variable domain of IgG antibodies can significantly modulate cell transport, FcRn binding, and half-life. Therefore, a complete understanding of the complex, multi-step stoichiometry of IgG-FcRn interactions is necessary.

[0003] Surface plasmon resonance (SPR) is a biosensor-based technology that measures protein-protein interactions in real time. SPR technology has become a standard tool in the research and development of biopharmaceuticals (see, for example, MACooper, Nat. Rev. Drug Dis. 1 (2002) 515-528 (Non-Patent Literature 1); DGMyszka, J. Mol. Recognit. 12 (1999) 390-408 (Non-Patent Literature 2); RLRich and DGMyszka, J. Mol. Recognit. 13 (2000) 388-407 (Non-Patent Literature 3); DGMyszka and RLRich, Pharm. Sci. Technol. Today 3 (2000) 310-317 (Non-Patent Literature 4); R. Karlsson and A. Faelt, J. Immunol. Meth. 200 (1997) 121-133 (Non-Patent Literature 5)), and is frequently used to measure the rate constants of polymer interactions. The ability to measure the association and dissociation dynamics of molecular interactions provides clues to understanding the mechanisms of complex formation (see, for example, TAMorton, DGMyszka, Meth. Enzymol. 295 (1998) 268-294 (Non-Patent Literature 6)). This information is becoming an integral part of the selection and optimization process for monoclonal antibodies and other biopharmaceuticals (see, for example, K. Nagata and H. Handa, in Real-time analysis of biomolecular interactions, Springer, 2000 (Non-Patent Literature 7); RLRich and DGMyszka, Curr. Opin. Biotechnol. 11 (2000) 54-61 (Non-Patent Literature 8); ACMalmborg and CABorrebaeck, J. Immunol. Meth. 183 (1995) 7-13 (Non-Patent Literature 9); W. Huber and F. Mueller, Curr. Pharm. Des. 12 (2006) 3999-4021 (Non-Patent Literature 10)). Furthermore, SPR technology enables, for example, the measurement of the binding activity (binding ability) of antibodies that bind to a target.

[0004] For over a decade, surface plasmon resonance (SPR) has been used to investigate antibody-antigen or antibody-receptor interactions, for example, by measuring the pH-dependent binding affinity of antibodies to human embryonic Fc receptors (FcRn) to understand their contribution to antibody recycling efficiency. Given the complexity of both binding partners, it is impossible to establish a definitive SPR interaction assay that can be evaluated by applying a single 1:1 Langmuir kinetic fit.

[0005] Various strategies have been employed to modulate the serum persistence of therapeutic proteins and endogenous antibodies by utilizing the FcRn salvage pathway, including FcRn-enhancing / inactivating mutations, Fc fusion proteins, and competitive inhibition of FcRn binding. However, therapeutic IgGs may have very different half-lives that do not appear to correlate with huFcRn affinity (Giragossian et al. Curr. Drug Metab. 14 (2013) 764-790 (Non-Patent Literature 11)).

[0006] WO2013 / 181087 (Patent Document 1) reported a multimerized complex with improved in vivo stability, pharmacokinetics, and efficacy.

[0007] U.S. Patent Application Publication No. 2017 / 0037121 (Patent Document 2) reports a polypeptide comprising a first polypeptide and a second polypeptide, each comprising at least a portion of an immunoglobulin hinge region including one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, in the direction from the N-terminus to the C-terminus, wherein i) the first and second polypeptides contain mutations H310A, H433A, and Y436A, or ii) the first and second polypeptides contain mutations L251D, L314D, and L432D, or iii) the first and second polypeptides contain mutations L251S, L314S, and L432S.

[0008] U.S. Patent Application Publication 2020 / 0353078 (Patent Document 3) reports an isolated IL-33 protein, its active fragment, and an antibody against the IL-33 protein, as well as its antigen-binding fragment. Methods for regulating cytokine activity are also provided, for example, for the purpose of treating immunodeficiency and inflammatory disorders.

[0009] Vaughn, DE et al. reported the structural basis of pH-dependent antibody binding by the fetal Fc receptor (Structure 6 (1998) 63-73 (Non-Patent Literature 12)).

[0010] Therefore, antibody-FcRn interactions need to be analyzed using particularly technically advanced methods to address the underlying complexities. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] WO2013 / 181087 [Patent Document 2] U.S. Patent Application Publication No. 2017 / 0037121 [Patent Document 3] U.S. Patent Application Publication No. 2020 / 0353078 [Non-patent literature]

[0012] [Non-Patent Document 1] MACooper, Nat. Rev. Drug Dis. 1 (2002) 515-528 [Non-Patent Document 2] DGMyszka, J.Mol.Recognit.12(1999)390-408 [Non-Patent Document 3] RLRich and DGMyszka, J.Mol.Recognit.13(2000)388-407 [Non-Patent Document 4] D.G.Myszka and R.L.Rich,Pharm.Sci.Technol.Today 3(2000)310-317 [Non-Patent Document 5] R.Karlsson and A.Faelt,J.Immunol.Meth.200(1997)121-133 [Non-Patent Document 6] T.A.Morton,D.G.Myszka,Meth.Enzymol.295(1998)268-294 [Non-Patent Document 7] K.Nagata and H.Handa,in Real-time analysis of biomolecular interactions,Springer,2000 [Non-Patent Document 8] R.L.Rich and D.G.Myszka,Curr.Opin.Biotechnol.11(2000)54-61 [Non-Patent Document 9] A.C.Malmborg and C.A.Borrebaeck,J.Immunol.Meth.183(1995)7-13 [Non-Patent Document 10] W.Huber and F.Mueller,Curr.Pharm.Des.12(2006)3999-4021 [Non-Patent Document 11] Giragossian et al.Curr.Drug Metab.14(2013)764-790 [Non-Patent Document 12] Vaughn,D.E. et al.,Structure 6(1998)63-73 [Summary of the Invention]

[0013] Surface plasmon resonance (SPR) is commonly used to measure the binding of IgG to recombinant fetal Fc receptor (FcRn), but it is not straightforward to interpret the data to obtain reliable binding kinetics. Herein, a novel SPR-based FcRn binding assay for appropriate FcRn binding evaluation is reported. This assay can be combined with suitable visualization to gain a detailed understanding of the contributions of the Fc region and Fab arms of antibodies to FcRn binding kinetics.

[0014] That is, herein, a novel SPR-based antibody-FcRn binding assay is reported that explains the individual Fab-FcRn and Fc region-FcRn interactions for antibody-FcRn binding evaluation. This aspect of the invention is at least partially based on findings regarding the effect of pH-dependent FcRn coating on a solid phase such as an SPR chip.

[0015] Herein, a method for measuring antibody-FcRn interaction is reported, - The FcRn-immobilized surface is an SPR chip, the capture group is directly bound to the solid surface (layer), and the (solid) surface does not contain a dextran matrix / group (is not derivatized with dextran), - The capture reagent is provided in an isolated form, i.e., not dimerized or multimerized, i.e., the capture reagent has only one binding site for the analyte (target molecule), and thus the capture reagent is monomeric, - The running buffer used for immobilization controls the aggregation state of the capture reagent during immobilization, i.e., when maintained in monomeric form or as a dimer / multimer.

[0016] One aspect of the invention is a method for measuring antibody-FcRn interaction, a) immobilizing FcRn on a solid surface suitable for surface plasmon resonance measurement, b) individually applying solutions containing different concentrations of an antibody to the solid surface obtained in step a) and measuring the association rate constant and dissociation rate constant of the antibody-FcRn interaction for each concentration. c) Using the rate obtained in step b), the K of the antibody-FcRn interaction D The process of measuring values Includes, Immobilized FcRn is monomeric FcRn, The monomer FcRn is immobilized (using functional (capturing) groups directly (bonded) to the solid surface), The solid surface does not contain branched glucans, FcRn immobilization is performed at a pH value of pH 7 to pH 8.

[0017] In one embodiment of the above and below embodiments and aspects, the immobilization of FcRn is performed at a pH value of approximately pH 7.4.

[0018] In one embodiment of the above and below embodiments and aspects, FcRn is immobilized at a density of 50 to 150 reaction units (RU). In one preferred embodiment, FcRn is immobilized at a density of 80 to 120 RU.

[0019] In one embodiment of the above and below embodiments and aspects, FcRn is a single-stranded FcRn (scFcRn). In one embodiment, scFcRn is a fusion polypeptide of beta-2-microglobulin and a human FcRn polypeptide conjugated together by a (GGGGS)4-peptide linker, the fusion polypeptide containing a C-terminal His(10)-Avi tag (SEQ ID NO: 07).

[0020] In one embodiment of the above and below embodiments and aspects, FcRn is subjected to amine coupling, resulting in 1 mm 2 The (sc)FcRn is immobilized at a density of approximately 1 pg or more per chip surface, approximately 10 pg or more in certain embodiments, and approximately 50 to 150 pg in certain embodiments. In one preferred embodiment, the FcRn is immobilized at approximately 80 to 120 pg(sc)FcRn / mm³ using amine coupling. 2 It is fixed in place by the density of the chip surface.

[0021] In one embodiment of the above and below embodiments and aspects, FcRn is used with biotin / streptavidin coupling, 1 mm 2 (sc)FcRn is immobilized at a density of approximately 1 pg or more per chip surface, approximately 10 pg or more in certain embodiments, and approximately 50 to 150 pg in certain embodiments.

[0022] In one embodiment of the above and below embodiments and aspects, immobilization is performed using a solution containing FcRn at a concentration of approximately 250 μg / ml in 10 mM HEPES buffer at a pH of approximately 7.4.

[0023] In one embodiment of the above and below embodiments and aspects, the antibody solution applied to the immobilized FcRn in step b) comprises i) 150 mM NaCl, or ii) 400 mM NaCl, or iii) 400 mM NaCl and 20% (w / w) ethylene glycol.

[0024] In one embodiment of the above and below embodiments and aspects, step b) is carried out using i) a solution of antibody containing 150 mM NaCl, and ii) a solution of antibody containing 400 mM NaCl, or / and a solution of antibody containing 400 mM NaCl and 20% (w / w) ethylene glycol. In one embodiment, the solution containing 400 mM NaCl or / and the solution containing 400 mM NaCl and 20% (w / w) ethylene glycol reduces or eliminates Fab-FcRn interactions. In one embodiment, the solution containing 400 mM NaCl or / and the solution containing 400 mM NaCl and 20% (w / w) ethylene glycol reduces or eliminates intermolecular interactions and Fab-FcRn interactions. This enables the measurement of isolated Fc region-FcRn interactions.

[0025] In one embodiment of the above and below embodiments and aspects, the antibody solution applied to the immobilized FcRn in step b) is either 10 mM MES, 150 or 400 mM NaCl, 0.05% (w / v) polysorbate 20 (P-20), and optionally 20% (w / w) ethylene glycol at a pH of 5.8, or 10 mM HEPES, 150 mM or 400 mM NaCl, 0.05% (w / v) P-20, and optionally 20% (w / w) ethylene glycol at a pH of 7.4.

[0026] In one embodiment of the above and below embodiments and aspects, the branched glucan is a complex branched glucan. Glucan is a polysaccharide obtained by the condensation of glucose. In one embodiment, the complex branched glucan is dextran. In one embodiment, the complex branched glucan is a microbial branched poly-α-d-glucoside having mainly C-1 to C-6'' glycosidic bonds. In one embodiment, the dextran has a molecular weight of 3 kDa to 2,000 kDa.

[0027] In one embodiment of the above and below embodiments and aspects, Fab-FcRn interactions and Fc-region-FcRn interactions are separated and visualized using a two-dimensional / three-dimensional diagram in which stability (log kd, off-rate) is shown / corresponding to the x-axis and recognition (log ka, on-rate) is shown / corresponding to the y-axis.

[0028] In one embodiment of the above and the embodiments and aspects described below, the interaction between FcRn and the Fc region of the antibody should be analyzed: - The sensor surface is an SPR chip having a carboxymethylated surface, where the carboxyl groups are directly bonded to the (solid) surface (layer), and the SPR chip does not contain a dextran matrix. - A beta-2-microglobulin-human FcRn fusion polypeptide containing a C-terminal His(10)-Avi tag (these groups are linked by a (GGGGS)4-peptide linker) is immobilized on the sensor surface by amine coupling at a neutral pH (approximately 250 μg / ml in 10 mM HEPES at pH 7.4). - The running buffer is either 10 mM MES, 150 mM NaCl, pH 5.8, 0.05% (w / v) P-20 or 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% (w / v) P-20.

[0029] In one embodiment of the above and below embodiments and aspects, the sensor surface is an SPR chip having a carboxymethylated surface, where the carboxyl groups are directly bonded to the (solid) surface (layer), and the chip is dextran-free. In this embodiment, a beta-2-microglobulin-human FcRn fusion polypeptide containing a C-terminal His(10)-Avi tag (these groups are linked by a (GGGGS)4-peptide linker) is immobilized on the (solid) surface by amine coupling at a neutral pH (approximately 250 μg / ml in 10 mM HEPES at pH 7.4), and the running buffer used is either 10 mM MES, 150 mM NaCl, pH 5.8, 0.05% (w / v) P-20 or 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% (w / v) P-20.

[0030] In one embodiment of the embodiments and aspects described above and below, the method is for selecting an antibody having pH-dependent FcRn-mediated antibody recycling and / or a long in vivo half-life, wherein the selected antibody has a pH-dependent overall antibody-FcRn interaction strength in the range of 100-400 nM at pH 5.5-6.0. The overall antibody-FcRn interaction strength includes Fc-FcRn and Fab-FcRn interactions.

[0031] In one embodiment of the embodiments and aspects described above and below, the method is for selecting an antibody having pH-dependent FcRn-mediated antibody recycling and / or a long in vivo half-life, wherein the selected antibody has a unilateral Fc region-FcRn binding strength of 25 nM or greater at pH values ​​in the range of pH 5.5 to pH 6.5. In one embodiment, the binding strength is 100 nM or greater, or 200 nM or greater, or 300 nM or greater. In one embodiment, if additional Fab-FcRn binding affinity is not present, particularly at pH 7.4, a binding strength of less than 100 (200) nM for unilateral Fc region-FcRn binding affinity is used. This should be related to low or undetectable binding strength at pH 7.4 or greater. In one embodiment, the binding strength is 25 nM or greater, and the antibody dissociates from FcRn at pH 7.4. This can be used to select an antibody variant having improved pharmacokinetic properties.

[0032] In one embodiment of the embodiments and aspects described above and below, the method is for selecting a variant antibody having a modified Fc region, the method comprising performing / carrying out step b) using a parent antibody and at least two variant antibodies having different amino acid sequences in the Fc region, step c) determining the interaction spot pattern, the selection of a variant antibody having an interaction spot pattern similar to / matching the interaction spot pattern of the parent antibody, the spot pattern being a two-dimensional / three-dimensional diagram in which stability (log kd, off-rate) is shown / corresponding on the x-axis and recognition (log ka, on-rate) is shown / corresponding on the y-axis.

[0033] In one embodiment of the above-described embodiment and the embodiments and aspects described later, the above method is for determining the type of Fab-FcRn interaction, that is, for determining whether a charge-based interaction or a hydrophobic interaction exists, and the method first uses an antibody in a solution containing 10 mM MES or HEPES, 150 mM, 0.05% (w / v) P-20 at a pH of 7.4 to obtain a first interaction spot pattern, second uses an antibody in a solution containing 10 mM MES or HEPES, 400 mM, 0.05% (w / v) P-20 at a pH of 7.4 to obtain a second interaction spot pattern, and third uses an antibody in a solution containing 10 mM MES or HEPES, 400 mM, 0.05% (w / v) P-20 at a pH of 7.4 to obtain a third interaction spot pattern The process includes performing / carrying out step b) using an antibody in a solution containing MES or HEPES, 400 mM, 0.05% (w / v) P-20 and 20% (w / w) ethylene glycol, wherein if the first interaction spot pattern and the second interaction spot pattern are different, the type of Fab-FcRn interaction is charge-based; if the first interaction spot pattern and the second interaction spot pattern are similar and the third interaction spot pattern is different, the type of Fab-FcRn interaction is hydrophobic-based; and the interaction spot pattern is a two-dimensional / three-dimensional diagram in which stability (log kd, off-rate) is shown / corresponding on the x-axis and recognition (log ka, on-rate) is shown / corresponding on the y-axis.

[0034] In one embodiment of the above and the embodiments and aspects described below, the antibody is a bispecific antibody.

[0035] In one embodiment of the above and the embodiments and aspects described below, the bispecific antibody is a domain-exchange antibody.

[0036] In one embodiment of the above and the embodiments and aspects described below, the bispecific antibody is a single-arm single-chain antibody.

[0037] In one embodiment of the above and the embodiments and aspects described below, the bispecific antibody is a two-arm single-chain antibody.

[0038] In one embodiment of the above and the embodiments and aspects described below, the bispecific antibody is a general light chain bispecific antibody.

[0039] One aspect of the present invention is a method according to the present invention for selecting an antibody. In one embodiment, the method according to the present invention is carried out using at least two antibodies with different FcRn interactions, thereby selecting an antibody having the highest / higher affinity / strength of (isolated) Fc region-FcRn interactions.

[0040] One aspect of the present invention is a method according to the present invention for antibody manipulation. In one embodiment, the method according to the present invention is carried out using at least two antibodies with different amino acid sequences in the Fc region and / or Fab, thereby selecting the antibody with the largest / greatest ratio of Fc-FcRn interaction to Fab-FcRn interaction.

[0041] One aspect of the present invention is the use of the method according to the present invention for measuring Fab-FcRn and Fc-region-FcRn interactions.

[0042] One aspect of the present invention is the use of a method according to the present invention for measuring the effect of antibody-Fc region mutations on the in vivo half-life of an antibody.

[0043] One aspect of the present invention is the use of a method according to the present invention for selecting antibodies with modified / improved (longer or shorter) in vivo half-lives.

[0044] One aspect of the present invention is the use of the method according to the present invention for measuring Fab-FcRn interactions and Fc region-FcRn interactions of antibodies.

[0045] One aspect of the present invention is the use of the method according to the present invention to describe Fab-FcRn interactions and Fc region-FcRn interactions of antibodies.

[0046] One aspect of the present invention is the use of the method according to the present invention for separately analyzing Fab-FcRn interactions and Fc region-FcRn interactions of antibodies.

[0047] This specification explicitly states that any combination of any aspect and any individual embodiment, or any combination of embodiments, is disclosed, if not in its original form. The aspects reported herein concern individual, independent methods of carrying out the invention, while some embodiments concern specific, dependent methods of carrying out one or more aspects of the invention. [Modes for carrying out the invention]

[0048] Detailed description of the invention This specification reports a novel SPR-based antibody-FcRn binding assay that describes individual Fab-FcRn and Fc-region-FcRn interactions in the evaluation of antibody-FcRn binding. This aspect of the present invention is at least partially based on a pH-dependent FcRn coating on a solid phase, such as an SPR chip.

[0049] This aspect of the present invention has been confirmed by reducing the complexity of the antibody to only the Fc region using just one active FcRn binding site, and then successively adding back further domains to the molecule.

[0050] This invention is at least in part based on the finding that SPR settings for measuring Fc-region-FcRn interactions involve a great deal of variation.

[0051] The present invention is further at least in part based on the finding that information on all antibody-FcRn interactions, namely Fab-FcRn and Fc-region-FcRn interactions, can be obtained by using intentional immobilization of FcRn on the surface of an SPR sensor, that is, by using FcRn capture in combination with intentional buffer settings.

[0052] This invention is at least partially based on the understanding that diverse IgG-FcRn interactions must be interpreted / understood simultaneously. Only after scrutinizing each binding step and binding interaction can the individual molecular interactions contributing to the overall binding be understood. Only based on this scrutiny of interactions can antibodies be successfully manipulated, that is, by manipulating Fc-FcRn and Fab-FcRn interactions separately.

[0053] This invention is at least in part based on the finding that, due to the symmetry of antibody heavy chains, different combinations of binding events occur, and it is important to immobilize a controlled amount, i.e., a specified amount of FcRn, on the surface of the SPR sensor. This is achieved in the method according to the present invention by controlling the dimerization of FcRn, for example, the formation of heterodimers, during the immobilization step. FcRn has been found to dimerize in a pH-dependent manner. By using single-stranded FcRn, a very uniform surface of FcRn on the surface of the SPR sensor can be provided.

[0054] The present invention is at least in part based on the finding that by i) controlling the use of SPR chips, particularly single-stranded FcRn, and immobilization at neutral / physiological pH (i.e., in the range of pH 7 to pH 8), and ii) adjusting buffer conditions, the multi-step binding mechanism between antibody and FcRn can be investigated and used for the selection and screening of engineered antibodies in terms of pharmacokinetic properties. In certain embodiments, the antibody is simplified and / or appropriate visualization using stability on the x-axis (log kd) and recognition on the y-axis (log ka) is used to separate different antibody-FcRn interactions and / or pharmacokinetic properties are pH-dependent FcRn binding.

[0055] The present invention is at least in part based on the finding that the strength of pH-dependent antibody-FcRn interactions must be in the range of 100-400 nM at pH 5.5-6.0 in order to select antibodies that have suitable pH-dependent FcRn-mediated antibody recycling and thereby a long in vivo half-life. In certain embodiments, the strength of antibody-FcRn interactions is the overall strength of antibody-FcRn interactions. This overall strength of antibody-FcRn interactions includes Fc-FcRn and Fab-FcRn interactions. In certain embodiments, the strength of antibody-FcRn interactions is the strength of Fc-FcRn interactions.

[0056] This invention is at least partially based on the finding that the higher the density of immobilized scFcRn (single-stranded FcRn) on an SPR solid surface, i.e., on a chip, the greater the proportion of populations with higher affinity, i.e., spots closer to the origin (lower left corner of a 2D / 3D diagram).

[0057] definition General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acid positions of all constant regions and domains of the heavy and light chains may be numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and referred to herein as "Kabat-compliant numbering." Specifically, the Kabat numbering system (see pp. 647-660) from Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domains CL of copper and lambda isotypes, while the Kabat EU index numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, in this case further clarified by the reference to "numbering according to the Kabat EU index").

[0058] When used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to “a cell” includes multiple such cells and their equivalents known to those skilled in the art. Similarly, the terms “a” (or “an”), “one or more,” and “at least one” may also be used interchangeably herein.

[0059] It should be noted that the terms "comprising," "including," and "having" can also be used interchangeably.

[0060] For those skilled in the art, procedures and methods for converting, for example, the amino acid sequence of a peptide linker or fusion polypeptide to the corresponding coding nucleic acid sequence are well known. Thus, nucleic acids are characterized by a nucleic acid sequence consisting of individual nucleotides, and similarly, by the amino acid sequence of the peptide linker or fusion polypeptide encoded thereby.

[0061] Recombinant DNA technology enables the creation of nucleic acid derivatives. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, alteration, exchange, deletion, or insertion. Modification or derivatization can be carried out, for example, using site-directed mutagenesis. Such modifications can be easily performed by those skilled in the art (see, for example, Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999), Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD, and Higgins, SG, Nucleic acid hybridization - a practical approach (1985), IRL Press, Oxford, England).

[0062] Useful methods and techniques for carrying out the present invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); and Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0063] The term "approximately" represents a range of + / - 20% of the following number. In one embodiment, the term "approximately" represents a range of + / - 10% of the following number. In one embodiment, the term "approximately" represents a range of + / - 5% of the following number.

[0064] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of molecule X for partner Y is generally expressed by the dissociation constant (K). D This dissociation constant can be expressed by the dissociation rate constant and the association rate constant (k, respectively). off and k onIt is the ratio of ). Therefore, as long as the ratio of rate constants remains the same, equivalent affinity may include different rate constants. Affinity can be measured by common methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0065] The term "antibody" as used herein is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies), and antibody fragments, as long as they contain at least an Fc region.

[0066] Antibodies generally consist of two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each heavy chain polypeptide and light chain polypeptide contains a variable domain (variable region) (generally the amino-terminal portion of the polypeptide chain) that contains a binding region capable of interacting with an antigen. Each heavy chain polypeptide and light chain polypeptide also contains a constant region (generally the carboxyl-terminal portion). The constant region of the heavy chain mediates the binding of the antibody to i) cells possessing the Fc gamma receptor (FcγR), such as phagocytes, or ii) cells possessing the embryonic Fc receptor (FcRn), also known as the Brambell receptor. The constant region of the heavy chain also mediates binding to several factors, including classical complement factors such as the (C1q) component. The constant domain of the antibody heavy chain contains the CH1, CH2, and CH3 domains, while the light chain contains only one constant domain CL, which can be either a copper isotype or a lambda isotype.

[0067] The variable domains of the light or heavy chains of immunoglobulins contain different segments, namely four framework regions (FRs) and three hypervariable regions (HVRs).

[0068] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0069] The term "binding (to FcRn)" refers to the binding of an antibody, or at least an antibody Fc region, or an antibody Fc region containing a fusion polypeptide, to a (human) FcRn in an in vitro assay. In one embodiment, binding is measured in a binding assay, in which a (human) FcRn is bound to a solid surface, such as a sensor chip, and the binding of the antibody (or an isolated Fc region or an Fc region constituting a fusion polypeptide) is measured by surface plasmon resonance (SPR).

[0070] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical and / or bind to the same epitope, except for, for example, naturally occurring mutations or variant antibodies that may arise during the preparation of the monoclonal antibody preparation, and such variants are usually present in small amounts. Each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen, in contrast to polyclonal antibody preparations, which usually contain various antibodies against various determinants (epitopes). Therefore, the modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of methods, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0071] As used herein, the term “hypervariable region” or “HVR” refers to each region of an antibody variable domain whose sequence is hypervariable (“complementarity-determining region” or “CDR”) and / or which forms a structurally defined loop (“hypervariable loop”) and / or which contains residues that come into contact with an antigen (“antigen contact”). Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Examples of HVRs used herein include: (a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b) and / or (c) including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).

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

[0073] As used in this application, the term "~valent" indicates the presence of a specific number of binding sites within the (antibody) molecule. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" indicate the presence of two, four, and six binding sites, respectively, within the (antibody) molecule. A bispecific antibody, as reported herein, is "bivalent" in one preferred embodiment.

[0074] The term "binding affinity" describes the strength of the interaction between a single binding site and its respective target. Experimentally, affinity can be measured, for example, by measuring the kinetic constants / rates of association (kA) and dissociation (kd) of the antibody and FcRn in equilibrium.

[0075] The term "binding avidity" represents the sum of the strengths of interactions between multiple binding sites of a single molecule (antibody) and the same target. Therefore, avidity is not the sum of bindings, but the sum of the synergistic strengths of binding affinity. What is required for avidity is the polyvalent nature of the molecule, such as an antibody, or the functional multimer (FcRn) of a single target.

[0076] The Fc association of the complex (monovalent or divalent) is the same for affine and avid bonds. However, the dissociation of the complex for avid bonding depends on the simultaneous dissociation of all involved binding sites. Therefore, the increase in binding strength due to avid bonding (compared to affine bonding) depends on the kinetics of dissociation / stability of the complex: the greater the stability of the complex, the less likely simultaneous dissociation of all involved binding sites is to occur; for very stable complexes, the difference between affine and avid bonding is essentially zero; the less stable the complex, the more likely simultaneous dissociation of all involved binding sites is to occur; and the greater the difference between affine and avid bonding.

[0077] multispecific antibodies In certain embodiments, the antibody used in the method according to the present invention is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites on one antigen or to at least two different antigens. In certain embodiments, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain embodiments, the multispecific antibody may bind to two different epitopes of the same antigen.

[0078] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305(1983) 537-540, WO93 / 08829 and Traunecker, A., et al., EMBO J.10(1991) 3655-3659) and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be produced by manipulating the electrostatic steering effect for producing antibody Fc-heterodimer molecules (WO2009 / 089004).

[0079] The antibody may also be a multispecific antibody, as described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, or WO2010 / 145793.

[0080] The antibody may be a multispecific antibody (also known as "DutaFab"), such as those disclosed in WO2012 / 163520.

[0081] Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two different epitopes on different antigens.

[0082] Various bispecific antibody formats are known.

[0083] Exemplary bispecific antibody formats that may be used in the methods reported herein are as follows:

[0084] - Domain-exchanged antibody (CrossMab format): A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment, a) Only the CH1 and CL domains are substituted for each other (i.e., the light chain of the first Fab fragment contains the VL and CH1 domains, and the heavy chain of the first Fab fragment contains the VH and CL domains); b) Only the VH and VL domains are replaced by each other (i.e., the light chain of the first Fab fragment contains the VH and CL domains, and the heavy chain of the first Fab fragment contains the VL and CH1 domains); or c) The CH1 and CL domains are substituted for each other, and the VH and VL domains are substituted for each other (i.e., the light chain of the first Fab fragment contains the VH and CH1 domains, and the heavy chain of the first Fab fragment contains the VL and CL domains); The second Fab fragment comprises a light chain containing VL and CL domains, and a heavy chain containing VH and CH1 domains; The domain-exchange antibody may comprise a first heavy chain containing a CH3 domain and a second heavy chain containing a CH3 domain, wherein both CH3 domains are manipulated in a complementary manner by their respective amino acid substitutions, for example, as disclosed in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 or WO2013 / 096291 (incorporated herein by reference), and is a multispecific IgG antibody; - Single-arm single-chain antibody (single-arm single-chain format): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - Light chain (variable light chain domain + light chain copper constant domain) - Light chain / heavy chain combination (variable light chain domain + constant light chain domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation) - Heavy chain (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation); - Two-arm single-chain antibody (two-arm single-chain format): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - Light chain / heavy chain 1 combination (variable light chain domain + constant light chain domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation) - Light chain / heavy chain 2 combination (variable light chain domain + constant light chain domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation); - A typical light chain bispecific antibody (typical light chain bispecific antibody format): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - Light chain (variable light chain domain + constant light chain domain) - Heavy chain 1 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with hole mutation) - Heavy chain 2 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with knob mutation).

[0085] In one embodiment, a bispecific antibody is a domain-exchange antibody.

[0086] In one embodiment, the bispecific antibody is a single-arm single-chain antibody.

[0087] In one embodiment, the bispecific antibody is a two-arm single-chain antibody.

[0088] In one embodiment, the bispecific antibody is a general light chain bispecific antibody.

[0089] Surface plasmon resonance spectroscopy The kinetic binding parameters of antibodies against FcRn can be investigated, for example, by surface plasmon resonance using a BIAcore instrument (GE Healthcare Biosciences AB, Uppsala, Sweden).

[0090] Generally, in the case of measuring the affinity of antibodies to a target antigen, anti-IgG antibodies, such as anti-human IgG or anti-mouse IgG antibodies, are immobilized on a sensor chip, such as a CM5 chip, via amine coupling for the capture and presentation of each antibody being analyzed.

[0091] For example, a 10-30 μg / ml anti-IgG antibody with approximately 2,000-12,000 reaction units (RUs) is coupled to several spots on a flow cell of a CM5 sensor tip in a BIAcore B4000 or T200 instrument (e.g., spots 1 and 5 are active and spots 2 and 4 are reference spots, or spots 1 and 2 are reactive and spots 3 and 4 are reference spots) using an amine coupling kit supplied by GE Healthcare.

[0092] Antibody binding to congener antigens can be measured in HBS buffer (HBS-P (10mM HEPES, 150mM NaCl, 0.005% Tween 20, pH 7.4) or HBS-EP+ buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05% v / v Surfactant PS20, pH 7.4) or HBS-ET buffer (10mM HEPES pH 7.4, 150mM NaCl, 3mM EDTA, 0.005% w / v Tween 20)) at 25°C (or alternatively at different temperatures in the range of 12°C to 37°C).

[0093] Therefore, the antibody is injected into each buffer at a concentration ranging from 10 nM to 1 μM for 30 seconds to bind to the reactive spots in each flow cell.

[0094] Subsequently, the corresponding antigen is injected into the solution at various concentrations, such as 144 nM, 48 nM, 16 nM, 5.33 nM, 1.78 nM, 0.59 nM, 0.20 nM, and 0 nM, depending on the affinity of the antibody, and the association is measured at an injection time of 20 seconds to 10 minutes at a flow rate of 10 to 30 μl / min.

[0095] Dissociation is measured by washing the chip surface with each buffer for 3 to 10 minutes.

[0096] K D The values ​​are estimated using a 1:1 Langmuir coupled model with the manufacturer's software and instructions. Negative control data (e.g., buffer curve) is subtracted from the sample curve to correct for system-specific baseline drift and reduce noise signals.

[0097] Specific embodiments of the present invention Due to the structure of the antibody, the binding of FcRn at the molecular level is extremely complex.

[0098] Therefore, antibodies with the same IgG1 Fc region but different Fabs exhibit different behaviors in their FcRn interactions (see Figure 1; SPR sensorgrams of different humanized / chimeric human IgG1 Fc regions containing antibodies (exploratory and verified); sensorgrams were recorded under the same SPR conditions at the same concentration and monomer concentration; the only difference is the antigen-binding site).

[0099] This invention is at least partially based on the finding that FcRn settings include many variations, and that by using FcRn immobilization on the surface of the SPR sensor, i.e., FcRn capture, information on all antibody-FcRn interactions, i.e., information on the Fab and Fc regions, can be obtained.

[0100] This invention is at least partially based on the finding that it is undesirable to interpret diverse IgG-FcRn interactions simultaneously using classical KD interpretations. Only after carefully examining each binding step can the molecular interactions contributing to binding be understood. Only with this understanding can the necessary operations be applied, in the sense of adaptive Fab for Fc manipulation.

[0101] This invention is at least in part based on the finding that, due to the symmetry of antibody heavy chains, different combinations of binding events exist, and therefore, it is important to immobilize a controlled amount, i.e., a specified amount of FcRn, on the SPR sensor surface. This is achieved by controlling the dimerization of FcRn, for example, the formation of heterodimers. FcRn has been found to dimerize in a pH-dependent manner. By using single-stranded FcRn, a highly uniform surface of FcRn on the SPR sensor surface can be provided.

[0102] Using a 2D / 3D diagram where stability (log kd, off-rate) corresponds to the x-axis and recognition (log ka, on-rate) corresponds to the y-axis, antibody-FcRn interactions, i.e., Fab-FcRn interactions and Fc-region-FcRn interactions, can be separated and visualized (see, for example, Figure 5). Any suitable software can be used to generate such 2D / 3D diagrams, such as the Interaction Map (IM) software from Ridgeview Diagnostics AB (Uppsala, Sweden).

[0103] Therefore, theoretically, when analyzing the interaction between Fc regions that each have exactly one binding site to / from FcRn, there should be only one spot.

[0104] Figure 2 shows a three-dimensional diagram of the theoretical 1:1 interaction between an isolated Fab-less Fc region and an FcRn binding site, i.e., an isolated antibody Fc region and a single FcRn binding site obtained by introducing the mutation I253A / H310A / H435A (numbering according to Kabat is used herein) into a single Fc region polypeptide (with stability (log kd) on the x-axis, recognition (log ka) on the y-axis, and intensity on the z-axis). Despite the underlying theory, two spots are present, but it can be seen that one Fc region polypeptide is inactive with respect to FcRn interaction, i.e., unable to bind to FcRn. A portion of the population with higher affinity, i.e., the spot closer to the origin (lower left corner of the diagram), was found to increase as the density of immobilized scFcRn (single-stranded FcRn) on the SPR solid surface, i.e., on the chip, increased.

[0105] Figure 3 shows a two-dimensional diagram of the interaction between a full-length, single-specific anti-digoxigenin antibody and FcRn on a solid surface of SPR. In this case, even three spots can be seen.

[0106] The differences in interactions, as shown in the two examples above, are at least in part due to the mode of interaction, namely whether it is a "complex" interaction or a "simple" interaction between the antibody and FcRn, respectively.

[0107] Therefore, in order to properly elucidate all of these interactions, an improved SPR method must be used.

[0108] Such an improved method is provided in the present invention.

[0109] More specifically, the present invention provides a method for detecting antibody-FcRn interactions, - The immobilized surface is an SPR chip having a surface, with the capture groups directly bonded to the surface layer, and the chip does not have a dextran matrix. - The capture reagent is provided in an isolated form, i.e., in an undimerized or unmultimerized form, meaning the capture reagent has only a single binding site to the analyte (target molecule). - The running buffer used for immobilization controls the aggregation state of the captured reagent during immobilization, i.e., whether it is maintained as a monomer or as a dimer / multimer.

[0110] This method allows for the covalent conjugation of small amounts of capture reagent, i.e., in the range of 50-150 RU, onto a solid surface.

[0111] Therefore, in one aspect of the present invention, the interaction between FcRn and the Fc region / antibody should be analyzed. - The sensor surface is an SPR chip having a carboxymethylated surface, where the carboxyl groups are directly bonded to the surface layer, and the chip does not have a dextran matrix. - A beta-2-microglobulin-human FcRn fusion polypeptide containing a C-terminal His(10)-Avi tag (these groups are linked by a (GGGGS)4-peptide linker) was immobilized on the sensor surface using amine coupling at a neutral pH (approximately 250 μg / ml in 10 mM HEPES at pH 7.4). - The running buffer is either 10 mM MES, 150 mM NaCl, pH 5.8, 0.05% P-20, or HBS-P buffer (10 mM HEPES buffer, pH 7.4, 150 mM NaCl, 0.05% P-20).

[0112] In the method according to the present invention, a small amount of scFcRn, i.e., about 80-120 RU or 50-100 RU, can be covalently conjugated onto a solid surface. Immobilization is performed at pH 7.4, ensuring that the scFcRn is immobilized in monomeric form and does not form aggregates. The effect is shown in Figure 4, and the sensorgram is shown in Figure 5, showing a corresponding two-dimensional diagram (with stability (log kd) on the x-axis and recognition (log ka) on the y-axis) of a 1:1 interaction between a single FcRn binding site obtained by introducing the mutation I253A / H310A / H435A into one Fc region polypeptide and maintaining the corresponding wild-type Fc region polypeptide at a low FcRn immobilization level by amine coupling as the respective other Fc region polypeptides, and an isolated Fab-less antibody Fc region. It can be seen that only a single spot is present.

[0113] In contrast, when scFcRn is immobilized at pH 5.5, dimeric scFcRn is immobilized due to heterodimerization that occurs at this pH value. The signal intensity is twice as high as that of the monomeric scFcRn, indicating that dimeric scFcRn can bind to two Fc regions.

[0114] Figure 6 shows a sensorgram of the complex IgG1 full-length antibody-FcRn interaction. Figures 7 and 8 show two-dimensional diagrams of the complex IgG1 full-length antibody-FcRn interaction at low FcRn immobilization levels using amine coupling and high FcRn immobilization levels using biotin / avidin coupling, respectively. In addition to the Fc region-FcRn interaction, additional spots and their resulting interactions can be observed.

[0115] Therefore, by adding Fab to the Fc region (mutation I253A / H310A / H435A in one Fc region polypeptide and wild-type in each of the other Fc region polypeptides), the effect of isolated Fab-FcRn interactions can be measured.

[0116] In the first example, as shown in Figures 9–11, this is illustrated using anti-digoxigenin Fab attached to an Fc region having a single FcRn binding site (mutant I253A / H310A / H435A in one Fc region polypeptide and wild-type in each of the other Fc region polypeptides; see Figure 42-a for a schematic of the antibody). Depending on the buffer conditions used, the interaction can be strengthened or weakened (low-density FcRn, approximately 80 RU): - 150 mM sodium chloride (Figure 9): Intramolecular interactions (1250 nM; see spot 1 in Figure 16 for a schematic diagram) and intermolecular interactions (20.5 nM; see spot 2 in Figure 16 for a schematic diagram); - 400 mM sodium chloride (Figure 10): Intramolecular interactions (1060 nM; stronger compared to 150 mM sodium chloride) and intermolecular interactions (60 nM); - 400 mM sodium chloride and 20% (w / w) ethylene glycol (MW = 62.07 g / mol) (Figure 11): Intramolecular interactions only (3230 mM; weaker compared to other conditions), and no intermolecular interactions.

[0117] Therefore, by applying the method according to the present invention in combination with a high buffer ionic strength, Fab-FcRn can be reduced or even eliminated. Although not bound by this theory, it is assumed that all intermolecular interactions and Fab-FcRn interactions are eliminated, and the interactions measured under these conditions are Fc-region-FcRn interactions.

[0118] In the second example, the same change in interaction strength is also shown for asymmetric antibodies with mutations I253A / H310A / H435A (eliminating Fc-FcRn interaction) in one Fc region polypeptide and mutations M252Y / S254T / T256E (increasing the strength of Fc-FcRn interaction) in the other Fc region polypeptides (see Figure 42-b for a schematic diagram of the antibody): - 150 mM sodium chloride: intramolecular interactions (184 nM; see spot 1 in Figure 16 for a schematic diagram) and intermolecular interactions (4.7 nM; see spot 2 in Figure 16 for a schematic diagram); - 400 mM sodium chloride: Intramolecular interactions (166 nM; stronger compared to 150 mM sodium chloride) and intermolecular interactions (6.2 nM).

[0119] The same change in interaction strength was also measured for asymmetric antibodies containing mutations I253A / H310A / H435A in one Fc region polypeptide and mutations T307H / N434H in the other Fc region polypeptides (see Figure 42-c for a schematic diagram of the antibodies): - 150 mM sodium chloride: intramolecular interactions (391 nM; see spot 1 in Figure 16 for a schematic diagram) and intermolecular interactions (10.4 nM; see spot 2 in Figure 16 for a schematic diagram); - 400 mM sodium chloride: Intramolecular interactions (325 nM; stronger compared to 150 mM sodium chloride) and intermolecular interactions (4.3 nM and 39 nM).

[0120] Therefore, increasing the salt concentration in the buffer used enhances intramolecular interactions in Fc region asymmetric antibodies. Hence, in one embodiment, when the measurement is of intramolecular interactions of an asymmetric antibody, i.e., a full-length, Y-type, bivalent bispecific antibody, the buffer contains about 400 mM of salt, preferably sodium chloride.

[0121] Using the same anti-digoxigenin Fab attached to the wild-type IgG1 Fc region yields different effects (low-density FcRn, approximately 80RU; Figures 12-14): - 150 mM sodium chloride (Figure 12): Intramolecular bonds 1+2 at 382 nM (see Figure 15 for schematic); intramolecular bonds 1+2+1+2 at 0.16 nM (see Figure 15 for schematic); intermolecular bonds 1+2' at 5.7 nM (see Figure 15 for schematic); intermolecular bonds 1+1' at 126 nM (see Figure 15 for schematic); - 400 mM sodium chloride (Figure 13): Intramolecular bonding 1+2 at 840 nM (see Figure 15 for a schematic diagram), intramolecular bonding 1+2+1+2 at 0.33 nM (see Figure 15 for a schematic diagram); intermolecular bonding 1+2' at 4.8 nM (see Figure 15 for a schematic diagram); intermolecular bonding 1+1' at 100 nM (see Figure 15 for a schematic diagram); compared to 150 mM sodium chloride, intramolecular interactions are weaker and intermolecular interactions are stronger; - 400 mM sodium chloride and 20% (w / w) ethylene glycol (MW = 62.07 g / mol) (Figure 14): Intramolecular bonding 1 + 2 at 1140 nM (see Figure 15 for schematic) and intermolecular bonding 1 + 1' at 75 nM (see Figure 15 for schematic); compared to 150 mM sodium chloride, intramolecular interactions are weaker and intermolecular interactions are stronger (i.e., more dominant compared to other spots).

[0122] The same anti-digoxigenin Fab linked to the IgG1 Fc region, which has mutations M252Y / S254T / T256E in both Fc region polypeptides, produces the same effect (low density FcRn, approximately 80 RU) (see Figure 42-d for a schematic diagram of the antibody): - At 150 mM sodium chloride:92 nM, intramolecular bonds are 1+2 (see Figure 15 for a schematic diagram) and 1+2+1+2 (see Figure 15 for a schematic diagram); - 400 mM sodium chloride: At 92 nM, intramolecular bonds are 1+2 (see Figure 15 for a schematic); at 1.6 nM, intramolecular bonds are 1+2+1+2 (see Figure 15 for a schematic).

[0123] As seen at high Fc binding strengths, when the Fc region is manipulated for very strong FcRn binding, the balance between spots driven by Fc-FcRn interactions and Fab-Fc-mediated avidity spots shifts to Fc-FcRn interactions only.

[0124] The same anti-digoxigenin Fab linked to the IgG1 Fc region containing the mutation T307H / N434H in both Fc region polypeptides yields the same effect (low density FcRn, approximately 80 RU) (see Figure 42-e for a schematic diagram of the antibody): - 150 mM sodium chloride: Intramolecular bonds 1+2 at 177 nM (see Figure 15 for a schematic); intramolecular bonds 1+2+1+2 at 0.12 nM (see Figure 15 for a schematic); intermolecular bonds 1+2' at 3 nM (see Figure 15 for a schematic); intermolecular bonds 1+1' at 71 nM (see Figure 15 for a schematic); - 400 mM sodium chloride: Intramolecular bonds 1+2 at 156 nM (see Figure 15 for a schematic); intramolecular bonds 1+2+1+2 at 0.13 nM (see Figure 15 for a schematic); intermolecular bonds 1+1' at 25 nM (see Figure 15 for a schematic).

[0125] It was found that a dramatic increase in antibody-FcRn interactions achieved by manipulating antibodies to enhance pH-dependent FcRn interactions does not necessarily result in similarly improved pharmacokinetic properties. TIFF2026102664000002.tif27128

[0126] Furthermore, for example, when introducing a YTE mutation, the antibody may have modified thermal stability, and it has been found that it is preferable to have an interaction spot pattern that matches the parent antibody rather than the shift pattern in the case of pharmacokinetic manipulation of the antibody (see Figure 36).

[0127] The complex, multi-step antibody-FcRn binding mechanism is a multivariate mechanism that includes the following: - pH-dependent affinity: FcRn binding cannot be explained by a simple 1:1 interaction. - pH-dependent avidity: Both Fc domain heavy chains are involved in Fc-FcRn interactions; - Fab's contribution: Due to additional and simultaneous Fab-FcRn interactions, several interactions combine to create a heterogeneous binding pattern.

[0128] Therefore, the bifurcated binding mechanism of IgG to the fetal Fc receptor controls the complex stability and serum half-life of IgG. This complexity is visualized in Figures 15 and 16.

[0129] The present invention is at least in part based on the finding that a multi-step binding mechanism between antibody and FcRn can be used for the selection and screening of engineered antibodies with respect to pharmacokinetic properties by i) controlling the use of SPR chips, particularly single-stranded FcRn, and immobilization at neutral / physiological pH (i.e., in the range of pH 7 to pH 8), and ii) adjusting buffer conditions. In certain embodiments, the antibody is simplified and / or appropriate visualization using stability on the x-axis (log kd) and recognition on the y-axis (log ka) is used to separate different antibody-FcRn interactions and / or pharmacokinetic properties are pH-dependent FcRn binding.

[0130] Therefore, measurements performed using the method according to the present invention are carried out using monomer-immobilized FcRn. This makes it possible to clarify the following: - Influence of bond / interaction valence - Influence of Fab charge - Influence of FcRn density on the chip surface - Influence of buffer composition.

[0131] First, by using amine coupling or biotin / avidin coupling to immobilize (sc)FcRn on the surface of the SPR chip, the coating density is controlled to a low level (see FIGS. 19 to 26), that is, the FcRn density on the chip surface is reduced compared with other methods. Thereby, the sensitivity of the method is improved, and various interactions can be visualized in an individualized form. By using a two-dimensional or three-dimensional diagram having stability (log kd) on the x-axis and recognition (log ka) on the y-axis, that is, by appropriate visualization, the influence of Fc manipulation on the one hand and the influence of Fab-FcRn interaction on the other hand on the entire antibody-FcRn interaction can be visualized. In particular, the correlation between the Fc-FcRn binding strength and the Fab-FcRn binding strength can be separated (see FIGS. 17 and 18).

[0132] To generate such a two-dimensional or three-dimensional diagram, any suitable software such as, for example, Interaction Map (IM) software of Ridgeview Diagnostics AB (Uppsala, Sweden) can be used.

[0133] In FIGS. 21 and 22, the partitions obtained by using biotin / avidin coupling at a coating density of about 1700 RU are shown. In FIGS. 23 and 24, the partitions obtained by using biotin / avidin coupling at a coating density of about 80 RU are shown. In FIGS. 25 and 26, the partitions obtained by using biotin / avidin coupling at a coating density of about 80 RU are shown.

[0134] In a preferred embodiment, the coating density using amine coupling in the method according to the invention is about 80 to 115 pg (sc)FcRn / mm 2 of the chip surface (corresponding to 80 to 115 RU).

[0135] In one preferred embodiment, the coating density using biotin / avidin coupling in the method according to the present invention is approximately 1700 pg(sc)FcRn / mm². 2 This is the chip surface (corresponding to 1700RU).

[0136] Secondly, controlled immobilization of scFcRn monomers is achieved by performing SPR tip coating in a pH-dependent manner. Therefore, immobilization is carried out at pH 7.4 to ensure that scFcRn is immobilized in monomeric form and does not form dimers or polymers during the immobilization process. This allows a small amount of scFcRn, i.e., about 80-120 RU, to be covalently conjugated onto the solid surface. In contrast, if scFcRn immobilization is carried out at pH 5.5, dimeric scFcRn is immobilized due to heterodimerization that occurs at this pH value.

[0137] In one specific embodiment, the sensor surface is an SPR chip having a carboxymethylated surface, where the carboxyl groups are directly bonded to the surface layer, and the chip does not contain a dextran matrix. In this embodiment, a beta-2-microglobulin-human FcRn fusion polypeptide containing a C-terminal His(10)-Avi tag (these groups are linked by a (GGGGS)4-peptide linker) is immobilized on a solid surface using amine coupling at a neutral pH (approximately 250 μg / ml in 10 mM HEPES at pH 7.4). This covalently conjugates approximately 80 RU of FcRn onto the solid surface. The running buffer used is either 10 mM MES, 150 mM NaCl, pH 5.8, 0.05% P-20, or HBS-P buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P-20).

[0138] The method according to the present invention can be used to analyze the effects of Fab-FcRn interactions and Fc-domain-FcRn interactions. Figure 27 shows two-dimensional diagrams of five Fab charge variants of the same parental anti-CD44 antibody. It can be seen that the Fab-FcRn interaction changes depending on the type of modification. Figure 28 shows two-dimensional diagrams (upper left figure) of four Fc-domain variants of the same parental anti-CD44 antibody. It can be seen that the Fc-domain-FcRn interaction changes depending on the type of modification.

[0139] Therefore, the effects described in this invention can be illustrated using a two-dimensional or three-dimensional diagram in which the dissociation constant (stability; log kd) is shown on the x-axis and the association constant (recognition; (log ka)) is shown on the y-axis. By using such a two-dimensional or three-dimensional diagram, the influence of Fc manipulation on the overall antibody-FcRn interaction and the influence of Fab-FcRn interaction on the other can be visualized. In particular, the interrelationship between Fc-FcRn binding strength and Fab-FcRn binding strength can be separated (see Figures 17 and 18).

[0140] To generate such two-dimensional or three-dimensional diagrams, any suitable software can be used, such as the Interaction Map (IM) software from Ridgeview Diagnostics AB (Uppsala, Sweden).

[0141] By simplifying the antibody, it is possible to monitor the effect of a single modification within the antibody on the overall antibody-FcRn interaction (see Figures 29-31).

[0142] The present invention is at least in part based on the finding that the pH-dependent overall antibody-FcRn interaction strength must be in the range of 100-400 nM at pH 5.5-6.0 in order to select antibodies that have a long in vivo half-life by having suitable pH-dependent FcRn-mediated antibody recycling. The overall antibody-FcRn interaction strength includes Fc-FcRn and Fab-FcRn interactions.

[0143] High unilateral Fc binding strength (e.g., ≥200 nM) in the pH 5.5–6.5 range should be related to low or undetectable binding strength above pH 7.4. This can be used to select antibody variants with improved pharmacokinetic properties.

[0144] The proportion of all interactions arising from Fab-FcRn interactions can be derived from additional spots observed in a 2D or 3D diagram and / or by separately analyzing the interactions in the Fc region (e.g., after cleaving the Fab fragment). The greater the number of additional spots / affected spots, the greater the presence of Fab-FcRn interactions.

[0145] The aforementioned Fab-FcRn interaction can be reduced, for example, by mutating residues within Fab, or the pH-dependent Fc region-FcRn interaction can be increased, for example, by Fc manipulation. In best cases, both manipulation techniques are combined.

[0146] For example, in Figures 31 and 32, antibodies mAb-1, mAb-2, and mAb-3 exhibit silent FcRn binding in one Fc region polypeptide by introducing the mutation I253A / H310A / H435A, and increased FcRn affinity in the other Fc region polypeptides by introducing the mutations M252Y / S254T / T256E and M252Y / S254T / T256E / T307Q / N434Y, respectively. It can be seen that Fab-FcRn interaction is reduced and Fc region-FcRn affinity is increased. As a result, antibody-FcRn interaction becomes dependent solely on Fc region-FcRn interaction, and the contribution / distortion caused by Fab-FcRn interaction is almost eliminated.

[0147] The upper limit for improving / increasing Fc-FcRn interactions is the complete elimination of pH-dependent binding. Such elimination can be achieved, for example, by introducing mutant MST-HN into the human IgG1 wt-Fc region (e.g., Patel et al., J.Immunol.187(2011)1015-1022) (Met252 to Tyr, Ser254 to Thr, Thr256 to Glu, His433 to Lys, Asn434 to Phe).

[0148] The type of interaction between Fab and FcRn, i.e., whether it is charge-based or hydrophobic, can be revealed in SPR analysis using different buffer compositions. For example, if the Fc-FcRn / Fab-FcRn interaction ratio increases when ethylene glycol is added to the SPR buffer, the presence of hydrophobic Fab-FcRn interactions can be observed. Similarly, the presence of ionic / charge-driven Fab-FcRn interactions can be observed when the Fc-FcRn / Fab-FcRn interaction ratio increases when salt is added to the SPR buffer (see Figures 9-14 and 29-32).

[0149] This is summarized in Figure 33.

[0150] The method according to the present invention allows for the separate analysis of different antibody-FcRn interactions (see Figure 34).

[0151] Based on the separation of different interactions, the method according to the present invention can be used for multiple applications in antibody development and selection / screening.

[0152] One embodiment is a method according to the present invention for antibody screening. In antibody screening, the affinity / strength of the Fc-FcRn interaction is the selection criterion. Intramolecular avidity can be measured / attenuated by increasing / adding salt. Intermolecular avidity can be attenuated by the solution density. This is schematically shown in Figure 35.

[0153] One embodiment is a method according to the present invention for antibody manipulation. In antibody manipulation, an increase in the ratio of Fc-FcRn interaction to Fab-FcRn interaction is the criterion for targeting. For example, different Fc region manipulations, i.e., the introduction of different mutations that affect FcRn binding, result in different patterns (see Figure 36).

[0154] To demonstrate the effect of Fc region manipulation on antibody-FcRn interactions, the antibodies briakinumab (Ozespa®) and ustekinumab (Stelara®) were used as model systems. Both briakinumab and ustekinumab are fully human monoclonal IgG1 antibodies. They bind to the same human p40 subunit of interleukin-12 (IL-12) and interleukin-23 (IL-23) and are not cross-reactive to the corresponding mouse IL-12 and IL-23. Briakinumab and ustekinumab are IgG1κ antibodies with variable heavy and light chain domains for the VH5 and Vκ1D germline families, respectively, and IgG1λ antibodies with variable heavy and light chain domains for the VH3 and Vλ1 germline families, respectively. In addition to their different variable domains, briakinumab and ustekinumab exhibit several allotype-specific amino acid differences in their constant domains (see alignments in Figures 40 and 41; light and heavy chain sequence alignments of briakinumab and ustekinumab - VH and VL regions are italicized; CDR is marked with an asterisk (*)).

[0155] However, the differences in these amino acids are located outside the (congeneral) FcRn binding region and therefore may not play a role in FcRn-dependent pharmacokinetics (see, for example, Ropeenian, DC and Akilesh, S., Nat. Rev. Immunol. 7 (2007) 715-725). Interestingly, ustekinumab has a (reported) median terminal half-life of 22 days (Zhu, Y., et al., J. Clin. Pharmacol. 49 (2009) 162-175), while briakinumab has a terminal half-life of only 8-9 days (see Gandhi, M., et al., Semin. Cutan. Med. Surg. 29 (2010) 48-52; Lima, XT, et al. Expert. Opin. Biol. Ther. 9 (2009) 1107-1113; Weger, W., Br. J. Pharmacol. 160 (2010) 810-820).

[0156] The amino acid sequences of the antibody briakinumab are reported in WO2013 / 087911 (SEQ ID NO: 01 and 02), the amino acid sequences of the antibody ustekinumab are reported in WO2013 / 087911 (SEQ ID NO: 03 and 04), and the amino acid sequences of the antibody bevacizumab are reported in Drug Bank entry DB00112.

[0157] Figure 37 shows a pH-dependent two-dimensional diagram of the interaction between ustekinumab with the YTE mutation and FcRn, which extends the in vivo half-life. This interaction is strong at low pH values ​​and weak at physiological pH values. This results in efficient pH-dependent FcRn-mediated recycling, and therefore a long in vivo half-life.

[0158] pH-dependent two-dimensional diagrams of the interaction between briakinumab with a YTE mutation that extends the in vivo half-life and briakinumab are shown in Figures 38 and 39, respectively. This interaction is observed to be strong at low and physiological pH values. This impairs pH-dependent FcRn-mediated recycling, resulting in a shorter in vivo half-life. Manipulation of the Fc region in the case of briakinumab also appears to increase the Fab-FcRn interaction.

[0159] Furthermore, the shift of spots in a two-dimensional or three-dimensional diagram is an indicator of the distortion in the Fc region resulting from the manipulation of the wt-Fc region.

[0160] Therefore, by using two-dimensional or three-dimensional visualization of antibody-FcRn interactions, in vivo effects can be analyzed, for example, in comparison to FcRn column chromatography.

[0161] Abstract Antibody half-life is influenced by FcRn. The underlying recycling mechanism is based on pH-dependent binding of antibodies to FcRn. The bifurcated binding mechanism of antibody-FcRn interaction has been previously described (Jensen et al., Mol. Cell Proteom. 16(2017) 451-456). This mechanism was elucidated by hydrogen-deuterium exchange (HDX).

[0162] The antibody Fc region contains two heavy chains. All assay settings utilizing surface-bound FcRn are hampered by the problem that kinetic behavior is a combination of 2:1 and 1:1 interactions. Depending on the density of the applied FcRn coating, the Fc region can interact with both or just one binding site.

[0163] Classical coupling chemistry typically only allows for random occupation of SPR chips. Lower FcRn concentrations on the chip can only lead to the probability of localized high FcRn densities.

[0164] The method according to the present invention demonstrated that FcRn also exhibits pH-dependent self-interactions. This interaction should be considered in assay settings to provide more detailed information regarding the mechanism.

[0165] Therefore, this aspect of the present invention is a novel SPR-based Fc-FcRn binding assay that describes individual interactions in the evaluation of Fc-FcRn binding.

[0166] This aspect of the present invention is at least partially based on a pH-dependent FcRn coating on a solid phase, such as an SPR chip.

[0167] This aspect of the present invention has been confirmed by reducing the complexity of the antibody to only the Fc region using just one active FcRn binding site, and then successively adding back further domains to the molecule.

[0168] Complex kinetics can be elucidated using software called Interaction Map. This allows for the characterization and isolation of simultaneous antibody-FcRn interactions.

[0169] By comparing the data obtained using the method according to the present invention with the binding profiles of each wild-type antibody, the predictive ability of the in vitro assay according to the present invention for antibody transport and recycling (of higher complexity), such as in vivo pharmacokinetics, was demonstrated in the human epithelial recycling assay (HERA) and human FcRn transgenic mice.

[0170] In the case of anti-Dig antibodies, the contribution of Fab-FcRn binding is very strong, and it was demonstrated that no clear difference could be observed in vivo compared to symmetric YTE-modified antibodies in a human transgenic mouse model.

[0171] The following examples and figures are provided to aid in understanding the present invention, and the true scope of the invention is set forth in the appended claims. It is understood that modifications to the procedures shown can be made without departing from the spirit of the invention. [Brief explanation of the drawing]

[0172] [Figure 1] SPR sensorgrams of different humanized / chimeric human IgG1 Fc regions containing antibodies (exploratory and validated); sensorgrams were recorded under the same SPR conditions at the same concentration and monomer concentration; the only difference was the antigen-binding site. [Figure 2] A three-dimensional diagram of the theoretical 1:1 interaction between an isolated Fab-less Fc region and an FcRn interaction, i.e., an isolated antibody Fc region and a single FcRn binding site obtained by introducing the mutation I253A / H310A / H435A (numbering according to Kabat is used herein) into a single Fc region polypeptide (with stability (log kd) on the x-axis, recognition (log ka) on the y-axis, and intensity on the z-axis). [Figure 3] Two-dimensional diagram of the interaction between a full-length, single-specific anti-digoxigenin antibody and FcRn on a solid surface of SPR. [Figure 4] Sensorgrams of a simple Fc region-FcRn interaction, i.e., a single FcRn binding site obtained by introducing the mutation I253A / H310A / H435A into one Fc region polypeptide and maintaining the corresponding wild-type Fc region polypeptide at a low FcRn immobilization level by amine coupling with the other Fc region polypeptides, and an isolated Fab-less antibody Fc region. [Figure 5] A two-dimensional diagram (with stability (log kd) on the x-axis and recognition (log ka) on the y-axis) of a 1:1 interaction between a single FcRn binding site obtained by introducing the mutation I253A / H310A / H435A into one Fc region polypeptide and maintaining the corresponding wild-type Fc region polypeptide at a low FcRn immobilization level by amine coupling as the other Fc region polypeptides, and an isolated Fab-less antibody Fc region, illustrating a simple Fc region-FcRn interaction, i.e., introducing the mutation I253A / H310A / H435A into one Fc region polypeptide and maintaining the corresponding wild-type Fc region polypeptide at a low FcRn immobilization level by amine coupling, and an isolated Fab-less antibody Fc region. [Figure 6] A sensor gram of complex IgG1 full-length antibody-FcRn interaction. [Figure 7] Two-dimensional diagram of complex IgG1 full-length antibody-FcRn interactions at low FcRn immobilization levels using amine coupling. [Figure 8] Two-dimensional diagram of complex IgG1 full-length antibody-FcRn interactions at high FcRn immobilization levels using biotin / avidin coupling. [Figure 9] Effect of isolated Fab-FcRn interaction of anti-digoxigenin Fab attached to an Fc region having a single FcRn binding site (mutation I253A / H310A / H435A in one Fc region polypeptide and wild-type in each of the other Fc region polypeptides), measured with 150 mM sodium chloride. [Figure 10]Effect of isolated Fab-FcRn interaction of anti-digoxigenin Fab attached to an Fc region having a single FcRn binding site (mutation I253A / H310A / H435A in one Fc region polypeptide and wild-type in each of the other Fc region polypeptides), measured with 400 mM sodium chloride. [Figure 11] Effect of isolated Fab-FcRn interaction of anti-digoxigenin Fab attached to an Fc region having a single FcRn binding site (mutant I253A / H310A / H435A in one Fc region polypeptide and wild-type in each of the other Fc region polypeptides), measured with 400 mM sodium chloride and 20% (w / w) ethylene glycol (MW=62.07 g / mol). [Figure 12] Effect of isolated Fab-FcRn interaction of anti-digoxigenin Fab attached to the wild-type IgG1 Fc region, measured with 150 mM sodium chloride. [Figure 13] Effect of isolated Fab-FcRn interaction of anti-digoxigenin-Fab attached to the wild-type IgG1 Fc region, measured with 400 mM sodium chloride. [Figure 14] Effect of isolated Fab-FcRn interaction of anti-digoxigenin-Fab attached to the wild-type IgG1 Fc region, measured with 400 mM sodium chloride and 20% (w / w) ethylene glycol (MW = 62.07 g / mol). [Figure 15] A schematic diagram illustrating different intramolecular Fab-FcRn and Fc domain-FcRn interactions. [Figure 16] A schematic diagram illustrating intramolecular and intermolecular antibody-FcRn interactions. [Figure 17] The interrelationship between the binding strengths of Fc-FcRn and Fab-FcRn can be separated into distinct sensorgrams. [Figure 18] A two-dimensional diagram showing the interrelationship between Fc-FcRn and Fab-FcRn bond strengths, separated from each other. [Figure 19]Biotin / avidin coupling density for immobilizing (sc)FcRn on the surface of the SPR tip. [Figure 20] Amine coupling for immobilizing (sc)FcRn on the surface of an SPR tip to control the coating density to a low level. [Figure 21] A two-dimensional diagram showing the binding strength of Fc-FcRn and Fab-FcRn of an anti-digoxigenin antibody containing a wild-type IgG1 Fc region, using a chip with approximately 1700 RU of (sc)FcRn captured by biotin / avidin coupling. [Figure 22] A two-dimensional diagram showing the binding strength of Fc-FcRn and Fab-FcRn of an anti-digoxigenin antibody containing a symmetric M252Y / S254T / T256E mutation in the IgG1 Fc region to a (sc)FcRn of approximately 1700 RU captured by biotin / avidin coupling. [Figure 23] A two-dimensional diagram showing the binding strength of Fc-FcRn and Fab-FcRn of an anti-digoxigenin antibody containing a wild-type IgG1 Fc region, using a chip with approximately 80 RU of (sc)FcRn captured by biotin / avidin coupling. [Figure 24] A two-dimensional diagram showing the binding strength of Fc-FcRn and Fab-FcRn of an anti-digoxigenin antibody containing a symmetric M252Y / S254T / T256E mutation in the IgG1 Fc region to a (sc)FcRn of approximately 80 RU captured by biotin / avidin coupling. [Figure 25] A two-dimensional diagram showing the binding strength of Fc-FcRn and Fab-FcRn of an anti-digoxigenin antibody containing a wild-type IgG1 Fc region, using a chip with approximately 80 RU of (sc)FcRn captured by amine coupling. [Figure 26]A two-dimensional diagram showing the binding strength of Fc-FcRn and Fab-FcRn of an anti-digoxigenin antibody containing an IgG1 Fc region with a symmetric M252Y / S254T / T256E mutation to a (sc)FcRn of approximately 80 RU captured by amine coupling. [Figure 27] Two-dimensional diagrams of Fab-FcRn and Fc-region-FcRn interactions for five Fab charge variants of the parent and the same parental anti-CD44 antibody. [Figure 28] A two-dimensional diagram (top left diagram) of the Fab-FcRn interaction and the Fab-FcRn and Fc-region-FcRn interactions of four Fc-region variants of the same parental anti-CD44 antibody is shown. [Figure 29] Monitoring the effect of introducing M252Y / S254T / T256E mutations, i.e., single modifications, into the overall antibody-FcRn interaction using a single-arm Fab-Fc region fusion. [Figure 30] Monitoring the effect of introducing a single variant, the V308P / Y436H mutation, into the antibody on the overall antibody-FcRn interaction using a single-arm Fab-Fc region fusion. [Figure 31] Monitoring the effects of introducing I253A / H310A / H435A mutations, i.e., single modifications, to the overall antibody-FcRn interaction using a single-arm Fab-Fc region fusion, and the removal of Fab within the antibody (vs. mAb-2 in Figure 29). [Figure 32] Monitoring the effect of introducing T307Q / N434A and even V308P / Y436H mutations, i.e., two single modifications, into the antibody on the overall antibody-FcRn interaction using a single-arm Fab-Fc region fusion. [Figure 33] Delinearization of hydrophobic and charge-driven antibody-FcRn interactions. [Figure 34] Analysis of various antibody-FcRn interactions. [Figure 35] A two-dimensional scheme showing the measurement / weakening of intramolecular avidity by increasing / adding salt and the measurement / weakening of intermolecular avidity by solution density. [Figure 36] For example, when introducing a YTE mutation, the antibody may have modified thermal stability, and therefore, the interaction spot pattern is preferably one that matches the interaction spot pattern of the parent antibody rather than the shift spot pattern in the case of pharmacokinetic manipulation of the antibody. [Figure 37] pH-dependent two-dimensional diagram of the interaction between ustekinumab with the YTE mutation and FcRn. [Figure 38] pH-dependent 2D diagram of the interaction between briakinumab and FcRn. [Figure 39] pH-dependent 2D diagram of the interaction between briakinumab with YTE mutation and FcRn. [Figure 40] Light chain amino acid sequence alignment of ustekinumab and briakinumab; CDRs are marked with an asterisk (*). [Figure 41] Alignment of the heavy chain amino acid sequences of ustekinumab and briakinumab; CDRs are marked with an asterisk (*). [Figure 42] A schematic diagram of the antibodies used in the examples.

[0173] material Manufacturer's information: Sensor chip C1 has a carboxymethylated flat surface. It provides the same functionality as sensor chip CM5 but lacks a dextran matrix (carboxyl groups are directly bonded to the surface layer). Due to the absence of a surface matrix, sensor chip C1 is less hydrophilic than sensor chip CM5. The experimental protocol follows the same principles for both sensor chip C1 and sensor chip CM5. The absence of a surface matrix results in an immobilization yield that is approximately 10% of the immobilization yield obtained on sensor chip CM5 under equivalent conditions.

[0174] Amine coupling utilizes the ε-amino groups of the N-terminus and lysine residues of the ligand.

[0175] Immobilization procedure Generally, the immobilization procedure consists of the following three distinct parts: Activation: Priming of a sensor chip so that it can form covalent bonds with other molecules. Coupling: Injection of a ligand that forms a covalent bond with the sensor surface. Inactivation: Injection of low molecular weight reactive groups to quench the remaining active surface groups.

[0176] Activation: In the case of covalent amine bonding chemistry on dextran-based sensor chips, carboxyl groups are activated with a mixture of NHS (N-hydroxysuccinimide) and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to produce N-hydroxysuccinimide esters. By varying the activation time, more or fewer carboxyl groups are activated. Furthermore, the amount of activated carboxyl groups can be controlled by varying the concentration of the NHS / EDC mixture. The amount of activated groups measures how much of the ligand can bind to the sensor surface. The standard activation time for BIACORE's CM5 sensor chip is 7 minutes when using 0.05M NHS / 0.2M EDC at a flow rate of 5 μl / min.

[0177] Coupling: The reactivity of the ligand at the selected pH measures the rate at which the ligand binds to the activated surface. The rate of preconcentration is directly related to the ligand concentration and pH of the immobilization solution. If the ligand concentration is too high, the preconcentration response of the ligand will be high, but it will also become difficult to immobilize an appropriate amount of ligand. The relationship between the time the ligand is in contact with the activated surface and the amount of ligand bound is not linear because the sensor tip will reach saturation.

[0178] Amount of ligand to immobilize: The amount of ligand immobilized depends on the application.

[0179] For measuring specificity, almost any ligand density is acceptable as long as it provides a good signal.

[0180] Concentration measurement requires the highest ligand density to facilitate limiting mass transfer. In total mass transfer controlled experiments, binding depends on the analyte concentration and not on the binding kinetics between the ligand and the analyte.

[0181] Affinity ranking can be performed using low-to-medium density sensor chips. It is important that the analyte saturates the ligand within an appropriate time frame.

[0182] Kinetics should be performed at the lowest ligand density that still provides a good response without being hindered by secondary factors such as mass transfer or steric hindrance.

[0183] Low molecular weight binding should be performed using a high-density sensor chip to bind as many analytes as possible to obtain a suitable signal.

[0184] Generally, in kinetic measurements, when an analyte is injected (1),(2), a total analyte response of at most 100 RU is desirable (see mass transport). With this value (Rmax) in mind, the amount of ligand to be immobilized (reaction units) can be calculated using: Rmax response / ligand response.

[0185] Inactivation: The inactivation solution blocks all remaining active sites with an excess of reagent, and due to its high ionic strength and high pH, ​​the solution washes away most of the electrostatically bound ligand. Amine coupling procedures are typically blocked with ethanolamine, but BSA or casein can also be used. If a high salt concentration is detrimental to the ligand, the experimenter can wait until all active sites decay and revert to carboxyl groups. The purpose of blocking is to remove the activated groups and inactivate the surface as much as possible.

[0186] When analyzing positively charged analytes, the surface can be blocked with ethylenediamine to reduce the negative charge on the sensor surface, and thus reduce the possibility of nonspecific binding.

[0187] References: (1) Karlsson, R. et al Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. Journal of Immunological Methods 229-240; (1991). (2)Myszka, DGSurvey of the 1998 optical biosensor literature.J.Mol.Recognit.12:390-408;(1999).

[0188] Amine coupling Amine coupling utilizes the ε-amino groups of the N-terminus and lysine residues of the ligand. The numbered points refer to different steps in the immobilization procedure.

[0189] 1) Baseline of the unmodified sensor chip surface by continuous flow (5 μl / min).

[0190] 2) Inject 35 μl of NHS / EDC to activate the surface by modifying the carboxymethyl group with N-hydroxysuccinimide ester.

[0191] 3) Baseline after activation. Surface activation has only a very slight effect on the SPR signal (100-200 RU).

[0192] 4) Injection of ligand (10-200 μg / ml) results in electrostatic attraction and coupling to the surface matrix. At this point, the ligand solution is still in contact with the sensor surface, and the response includes both immobilized ligand and non-covalently bound ligand. N-hydroxysuccinimide ester spontaneously reacts with the amine on the ligand to form a covalent bond (1).

[0193] 5) Immobilized ligand before inactivation. The ligand passes through the sensor surface, and most of the non-covalently bound proteins are eluted.

[0194] 6) Inactivation of unreacted NHS esters using 35 μl of 1 M ethanolamine hydrochloride adjusted to pH 8.5 with NaOH. The increase in the SPR signal is due to a change in the bulk refractive index. The inactivation process also removes any remaining electrostatically bound ligands.

[0195] 7) Point 7-3 provides the amount of immobilized ligand after inactivation.

[0196] Amine coupling is the first choice for coupling new molecules. However, immobilizing acidic ligands (pI < 3.5) is difficult. Furthermore, when free amine groups are present in the biologically active site, one of the other chemical approaches must be considered.

[0197] References (1)Johnsson, B. et al Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors.Analytical Biochemistry 198:268-277;(1991). [Examples]

[0198] Example 1 General SPR method for measuring Fc-FcRn interaction All measurements were performed at 25°C using a BIAcore T200 instrument (GE Healthcare). Biotinylated single-stranded human FcRn was used in all interaction studies.

[0199] FcRn was immobilized using two different methods: low-density immobilization and high-density immobilization.

[0200] For low-density immobilization, FcRn was immobilized on the C1 tip using standard amine coupling. Therefore, the protein was diluted to a concentration of 0.245 mg / ml in buffer (10 mM HEPES; pH 7.4) and injected onto the tip surface for 60 seconds. This immobilization resulted in an immobilization level of approximately 80 RU.

[0201] For high-density immobilization, FcRn was immobilized by biotin capture. First, neutraavidin (ThermoScientific) was immobilized on the C1 tip using standard amine coupling. Neutraavidin was diluted to a concentration of 0.1 mg / ml in 10 mM sodium acetate buffer at pH 4.5 and injected onto the tip surface for 6 minutes. This immobilization resulted in approximately 1000 RU. After neutraavidin immobilization, FcRn was captured by injecting biotinylated protein at a concentration of 0.24 mg / ml onto the tip for 5 minutes. This capture resulted in an immobilization level of approximately 1700 RU.

[0202] To measure interactions with different antibodies, a buffer consisting of 10 mM MES, 150 mM NaCl, and 0.05% P-20 at pH 5.8 was used. Antibody-FcRn interactions were analyzed using single-cycle or multi-cycle kinetics and 2x or 3x dilution series. Recorded sensorgrams were double references, subtracted using a reference flow cell and blank injection. The resulting sensorgrams were evaluated using TraceDrawer software (Ridgeview Instruments AB).

[0203] Sequence information SEQUENCE LISTING <110> F. Hoffmann-La Roche AG <120> Method for resolving complex, multistep antibody interactions <150> EP 21157850.5 <151> 2021-02-18 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain variable region of briakinumab (CJ-695, ABT-874). <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the light chain variable region of briakinumab (J-695, ABT-874). <400> 2 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Tyr Thr 85 90 95 His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain variable region of ustekinumab (CTNO-1275). <400> 3 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile 35 40 45 Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the light chain variable region of ustekinumab (CTNO-1275). <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> G4S-linker <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Avi-Tag <400> 6 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 7 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> His(10)-Avi-Tag <400> 7 His His His His His His His His His His Gly Leu Asn Asp Ile Phe 1 5 10 15 Glu Ala Gln Lys Ile Glu Trp His Glu 20 25

Claims

1. A method for measuring antibody-FcRn interactions, a) A process for immobilizing FcRn on a solid surface suitable for surface plasmon resonance measurement, b) A step of individually coating the solid surface obtained in step a) with solutions containing antibodies of different concentrations, and measuring the association rate constant and dissociation rate constant for each concentration. c) Using the rate obtained in step b), the K of the antibody-FcRn interaction Ｄ The process of measuring values Includes, The immobilized FcRn is monomeric FcRn, The monomer FcRn is immobilized using functional (capture) groups directly bonded to the solid surface. The solid surface does not contain branched glucans, and The immobilization of FcRn is performed at a pH value of pH 7 to pH 8. method.

2. The method according to claim 1, wherein the immobilization is performed at a pH value of approximately pH 7.

4.

3. The method according to any one of claims 1 or 2, wherein the FcRn is immobilized at a density of 50 to 150 RU.

4. The method according to any one of claims 1 to 3, wherein the FcRn is a single-stranded FcRn (scFcRn).

5. The aforementioned scFcRn is (GGGGS). ４ The method according to claim 4, wherein the fusion polypeptide is a fusion polypeptide of beta-2-microglobulin and a human FcRn fusion polypeptide conjugated together by a peptide linker, the fusion polypeptide comprising a C-terminal Avi tag.

6. The method according to any one of claims 1 to 5, wherein the FcRn is immobilized using amine coupling or biotin / streptavidin coupling.

7. The FcRn is approximately 50 to 150 pg / mm³. ２ The method according to any one of claims 1 to 6, wherein the chip is fixed by the density of the chip surface.

8. The method according to any one of claims 1 to 7, wherein the immobilization is carried out using a solution containing FcRn at a concentration of approximately 250 μg / ml in 10 mM HEPES buffer at a pH of 7.

4.

9. The method according to any one of claims 1 to 8, wherein the solution of the antibody applied to the immobilized FcRn in step b) comprises 150 mM NaCl, or 400 mM NaCl, or 400 mM NaCl and 20% (w / w) ethylene glycol.

10. The method according to claim 9, wherein the solution of the antibody applied to the immobilized FcRn in step b) contains either 10 mM MES, 150 or 400 mM NaCl, 0.05% P-20, and optionally 20% (w / w) ethylene glycol at a pH of 5.8, or 10 mM HEPES, 150 mM or 400 mM NaCl, 0.05% P-20, and optionally 20% (w / w) ethylene glycol at a pH of 7.

4.

11. The method according to any one of claims 1 to 10, wherein the branched glucan is dextran.

12. A 2D / 3D diagram in which stability (log kd, off-rate) is shown / corresponding to the x-axis, and recognition (log ka, on-rate) is shown / corresponding to the y-axis. The method according to any one of claims 1 to 11, wherein Fab-FcRn interactions and Fc-region-FcRn interactions are separated and visualized using [a specific method / tool].

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