Methods of modifying plasma retentivity and immunogenicity of antigen-binding molecules

Abstract

To provide an Fc region in which the pharmacokinetics of antigen-binding molecules is improved and the immune response of antigen-binding molecules is reduced, by modification of Fc region.SOLUTION: We found that a pharmacokinetics of antigen-binding molecules is improved and an immune response of antigen-binding molecules is reduced, by modifying an Fc region of an antigen-binding molecule into an Fc region that does not form a hetero complex comprising the four, i.e., an antigen-binding molecule, two molecules of FcRn and an active Fcγ receptor, in conditions of pH neutral range. We found an antigen-binding molecule having the above properties and a production method thereof and, at the same time, we succeeded in finding an excellent characteristics compared with conventional antigen-binding molecules the characteristics being that pharmacokinetics is improved and the immune response by an administered organism is reduced, when such an antigen-binding molecule or a pharmaceutical composition containing as an active ingredient an antigen-binding molecule produced by the production method according to the present invention is administered.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for improving the pharmacokinetics of an antigen-binding molecule in a living body to which the molecule is administered or a method for reducing an immune response to the antigen-binding molecule, by modifying the Fc region of the antigen-binding molecule, which comprises an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and an Fc region that has FcRn-binding activity under a neutral pH condition. The present invention also relates to antigen-binding molecules that have improved pharmacokinetics or reduced immune responses by the living body when administered to the living body. The present invention further relates to methods for producing the antigen-binding molecules, and pharmaceutical compositions containing the antigen-binding molecules as an active ingredient. [Background technology]

[0002] Antibodies have attracted attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Many IgG-type antibody drugs are currently on the market, and numerous antibody drugs are currently being developed (Non-Patent Documents 1 and 2). Meanwhile, various technologies applicable to second-generation antibody drugs have been developed, including those that improve effector function, antigen-binding ability, pharmacokinetics, and stability, or reduce the risk of immunogenicity (Non-Patent Document 3). Because antibody drugs generally require very high dosages, challenges include the difficulty of preparing subcutaneous formulations and high manufacturing costs. Potential methods for reducing the dosage of antibody drugs include improving the pharmacokinetics of antibodies and improving the affinity between antibodies and antigens.

[0003] Artificial amino acid substitution in the constant region has been reported as a method for improving the pharmacokinetics of antibodies (Non-Patent Documents 4 and 5). Affinity maturation technology (Non-Patent Document 6) has been reported as a technique for enhancing antigen-binding ability and antigen-neutralizing ability, and it is possible to enhance antigen-binding activity by introducing mutations into amino acids in the CDR region of the variable region, etc. Enhanced antigen-binding ability can improve in vitro biological activity or reduce dosage, and can also improve in vivo drug efficacy (Non-Patent Document 7).

[0004] On the other hand, the amount of antigen that can be neutralized per antibody molecule depends on affinity. By increasing affinity, it is possible to neutralize the antigen with a small amount of antibody. Various methods can increase antibody affinity (Non-Patent Document 6). Furthermore, if an antibody can be covalently bound to an antigen and affinity can be increased infinitely, it would be possible to neutralize one antigen molecule (two antigens in the case of a bivalent antibody). However, previous methods were limited to a stoichiometric neutralization reaction of one antigen molecule (two antigens in the case of a bivalent antibody) with one antibody molecule, making it impossible to completely neutralize an antigen with an antibody amount less than the amount of antigen. In other words, there was a limit to the effectiveness of increasing affinity (Non-Patent Document 9). In the case of a neutralizing antibody, in order to maintain its neutralizing effect for a certain period of time, an antibody amount greater than the amount of antigen produced in the body during that period must be administered. Therefore, there was a limit to reducing the required antibody dosage by simply improving the pharmacokinetics of the antibody or affinity maturation technology described above. Therefore, in order to maintain the antigen neutralization effect for a desired period of time with an antibody amount equal to or less than the antigen amount, it is necessary to neutralize multiple antigens with a single antibody. As a new method for achieving this, an antibody that binds to an antigen in a pH-dependent manner has recently been reported (Patent Document 1). A pH-dependent antigen-binding antibody binds strongly to an antigen under the neutral conditions of plasma and dissociates from the antigen under the acidic conditions of endosomes, and is capable of dissociating from the antigen within the endosome. After dissociating from the antigen, a pH-dependent antigen-binding antibody can re-bind to the antigen when recycled into plasma by FcRn, allowing a single pH-dependent antigen-binding antibody to repeatedly bind to multiple antigens.

[0005] Furthermore, the plasma retention time of antigens is very short compared to antibodies that bind to FcRn and are recycled. When such an antibody with a long plasma retention time binds to the antigen, the plasma retention time of the antibody-antigen complex becomes as long as that of the antibody. Therefore, by binding to the antibody, the antigen actually retains its plasma retention time longer, and the plasma antigen concentration increases.

[0006] IgG antibodies maintain long plasma retention by binding to FcRn. Binding of IgG to FcRn is only observed under acidic conditions (pH 6.0) and is almost nonexistent under neutral conditions (pH 7.4). IgG antibodies are nonspecifically internalized by cells, but return to the cell surface by binding to FcRn in endosomes under acidic conditions in the endosome. They then dissociate from FcRn under neutral plasma conditions. Introducing mutations into the Fc region of IgG to abolish FcRn binding under acidic conditions significantly impairs plasma retention, as the antibody is no longer recycled from endosomes to plasma. A method for improving plasma retention of IgG antibodies has been reported that enhances FcRn binding under acidic conditions. Introducing amino acid substitutions into the Fc region of IgG antibodies to improve FcRn binding under acidic conditions increases the recycling efficiency from endosomes to plasma, thereby improving plasma retention. When introducing amino acid substitutions, it is important not to increase FcRn binding under neutral conditions. If an IgG antibody binds to FcRn under neutral conditions, even if it returns to the cell surface by binding to FcRn under the acidic conditions in the endosome, it will not be recycled into plasma unless it dissociates from FcRn in plasma under neutral conditions, and therefore its plasma retention will be impaired. For example, it has been reported that an antibody that has been made capable of binding to mouse FcRn under neutral conditions (pH 7.4) by introducing amino acid substitutions into IgG1 was shown to have a reduced plasma retention when administered to mice (Non-Patent Document 10). It has also been reported that introducing amino acid substitutions into IgG1 improves human FcRn binding under acidic conditions (pH 6.0), but at the same time, when an antibody that also binds to human FcRn under neutral conditions (pH 7.4) is administered to cynomolgus monkeys, the plasma retention of the antibody is not improved and no change in plasma retention was observed (Non-Patent Documents 10, 11, and 12).Therefore, antibody engineering techniques for improving antibody function have focused solely on improving antibody plasma retention by increasing human FcRn binding under acidic conditions without increasing human FcRn binding under neutral conditions (pH 7.4), and no benefits have been reported to date for increasing human FcRn binding under neutral conditions (pH 7.4) by introducing amino acid substitutions into the Fc region of an IgG antibody. Even if the affinity of an antibody for an antigen is improved, it is not possible to promote the elimination of the antigen from plasma. It has been reported that the above-mentioned pH-dependent antigen-binding antibodies are also effective as a method for promoting the elimination of antigens from plasma compared to conventional antibodies (Patent Document 1).

[0007] Thus, pH-dependent antigen-binding antibodies bind to multiple antigens with a single antibody and can accelerate antigen elimination from plasma compared to conventional antibodies, thereby exhibiting effects not possible with conventional antibodies. However, no antibody engineering techniques have been reported to date that further improve the effects of pH-dependent antigen-binding antibodies, such as the ability to repeatedly bind to antigens and the acceleration of antigen elimination from plasma.

[0008] On the other hand, the immunogenicity of antibody drugs is extremely important in terms of plasma retention, efficacy, and safety when administered to humans. It has been reported that the production of antibodies against administered antibody drugs in the human body can cause undesirable events such as accelerated elimination of the antibody drug from plasma, reduced efficacy, and hypersensitivity reactions that affect safety (Non-Patent Document 13).

[0009] When considering the immunogenicity of antibody drugs, it is first necessary to understand the functions of natural antibodies in vivo. First, most antibody drugs are antibodies belonging to the IgG class, and the existence of Fcγ receptors (hereinafter also referred to as FcγRs) is known as Fc receptors that act by binding to the Fc region of IgG antibodies. FcγRs are expressed on the cell membrane of dendritic cells, NK cells, macrophages, neutrophils, adipocytes, etc., and are known to transmit activating or inhibitory intracellular signals to immune cells upon binding to the Fc region of IgG. The human FcγR protein family has been reported to include isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and their respective allotypes have also been reported (Non-Patent Document 14). Two human FcγRIIa allotypes have been reported, with Arg (hFcγRIIa(R)) and His (hFcγRIIa(H)) at position 131. Two human FcγRIIIa allotypes have been reported, with Val (hFcγRIIIa(V)) and Phe (hFcγRIIIa(F)) at position 158. Furthermore, the mouse FcγR protein family has been reported to include FcγRI, FcγRIIb, FcγRIII, and FcγRIV (Non-Patent Document 15).

[0010] Human FcγRs are classified into activating receptors FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and inhibitory receptor FcγRIIb. Similarly, mouse FcγRs are classified into activating receptors FcγRI, FcγRIII, and FcγRIV, and inhibitory receptor FcγRIIb.

[0011] When activating FcγR is crosslinked with immune complexes, it induces phosphorylation of immunoreceptor tyrosine-based activating motifs (ITAMs) contained in the intracellular domain or the FcR common γ-chain, which is its interacting partner, and activates the signal transduction factor SYK, initiating an activation signal cascade and causing an inflammatory immune response (Non-Patent Document 15).

[0012] It has been shown that the binding between the Fc region and FcγR is important due to several amino acid residues in the hinge region and CH2 domain of an antibody and to the sugar chain attached to Asn at EU numbering position 297 attached to the CH2 domain (Non-Patent Documents 15, 16, and 17). Mutants with various FcγR-binding properties have been studied, primarily focusing on antibodies with mutations introduced at these sites, and Fc region mutants with higher affinity for activating FcγR have been obtained (Patent Documents 2, 3, 4, and 5).

[0013] On the other hand, FcγRIIb, an inhibitory FcγR, is the only FcγR expressed on B cells (Non-Patent Document 18). It has been reported that interaction of the Fc region of an antibody with FcγRIIb suppresses B cell priming (Non-Patent Document 19). It has also been reported that cross-linking of FcγRIIb on B cells with the B cell receptor (BCR) via immune complexes in the blood suppresses B cell activation and antibody production by B cells (Non-Patent Document 20). The immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb is required for the transmission of immunosuppressive signals via BCR and FcγRIIb (Non-Patent Document 21, Non-Patent Document 22). This immunosuppressive effect occurs via phosphorylation of ITIM. As a result of phosphorylation, SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, which inhibits the transmission of signal cascades of other activating FcγRs and suppresses inflammatory immune responses (Non-Patent Document 23).

[0014] Due to this property, FcγRIIb is expected to be a method for directly reducing the immunogenicity of antibody drugs. When mouse IgG1 is fused to Exendin-4 (Ex4), a foreign protein for mice, a molecule (Ex4 / Fc) is administered to mice, no antibodies are produced. However, when Ex4 / Fc is modified so that it does not bind to FcγRIIb on B cells (Ex4 / Fc mut), antibodies against Ex4 are produced (Non-Patent Document 24). These results suggest that Ex4 / Fc binds to FcγRIIb on B cells, thereby suppressing the production of mouse antibodies against Ex4 by B cells.

[0015] FcγRIIb is also expressed in dendritic cells, macrophages, activated neutrophils, mast cells, and basophils. In these cells, FcγRIIb inhibits activating FcγR functions, such as phagocytosis and the release of inflammatory cytokines, thereby suppressing inflammatory immune responses (Non-Patent Document 25).

[0016] The importance of the immunosuppressive function of FcγRIIb has been elucidated in studies using FcγRIIb knockout mice. It has been reported that FcγRIIb knockout mice exhibit improper humoral immunity control (Non-Patent Document 26), increased susceptibility to collagen-induced arthritis (CIA) (Non-Patent Document 27), and exhibit lupus-like symptoms and Goodpasture syndrome-like symptoms (Non-Patent Document 28).

[0017] Dysregulation of FcγRIIb has also been reported to be associated with human autoimmune diseases. For example, genetic polymorphisms in the FcγRIIb promoter region or transmembrane domain have been associated with the incidence of systemic lupus erythematosus (SLE) (Non-Patent Documents 29, 30, 31, 32, and 33), and reduced expression of FcγRIIb on the surface of B cells in SLE patients has been reported (Non-Patent Documents 34 and 35).

[0018] Based on these mouse model and clinical findings, FcγRIIb is thought to play a role in controlling autoimmune and inflammatory diseases, primarily through its involvement with B cells, and is a promising target molecule for controlling autoimmune and inflammatory diseases.

[0019] IgG1, which is primarily used as a commercially available antibody pharmaceutical, is known to bind strongly not only to FcγRIIb but also to activating FcγR (Non-Patent Document 36). By utilizing an Fc region with enhanced binding to FcγRIIb or improved selectivity for binding to FcγRIIb compared to activating FcγR, it may be possible to develop antibody pharmaceuticals with immunosuppressive properties compared to IgG1. For example, it has been suggested that B cell activation can be inhibited by utilizing an antibody having a variable region that binds to BCR and an Fc with enhanced binding to FcγRIIb (Non-Patent Document 37).

[0020] However, it is known that FcγRIIb shares 93% sequence identity with FcγRIIa, an activating FcγR, in the extracellular domain, resulting in a highly similar structure. Furthermore, FcγRIIa has two genetic polymorphisms: an H type in which the 131st amino acid in the second Ig domain is His, and an R type in which the 131st amino acid is Arg, and these types exhibit different interactions with antibodies (Non-Patent Document 38). Therefore, the most challenging task in creating an Fc region that specifically binds to FcγRIIb is to confer to the antibody Fc region a property that selectively improves FcγRIIb-binding activity, i.e., to increase FcγRIIb-binding activity while maintaining or decreasing the binding activity to each FcγRIIa genetic polymorphism.

[0021] Previously, there have been reports of increasing the specificity of FcγRIIb binding by introducing amino acid modifications into the Fc region (Non-Patent Document 39). In this document, mutants were prepared that maintained better binding to FcγRIIb than to FcγRIIa at both gene polymorphisms, compared to IgG1. However, all of the mutants reported in this document that were said to have improved FcγRIIb specificity had reduced FcγRIIb binding compared to native IgG1. Therefore, it is considered difficult for these mutants to actually induce an FcγRIIb-mediated immunosuppressive response greater than that of IgG1.

[0022] It has also been reported that FcγRIIb binding was enhanced (Non-Patent Document 37). In this document, FcγRIIb binding was enhanced by introducing alterations such as S267E / L328F, G236D / S267E, and S239D / S267E into the Fc region of an antibody. Among these, an antibody into which the S267E / L328F mutation was introduced showed the strongest binding to FcγRIIb, but this mutant maintained binding to FcγRIa and the H type of FcγRIIa at a level comparable to that of native IgG1. Even if FcγRIIb binding is enhanced compared to IgG1, it is thought that for cells such as platelets that express FcγRIIa but not FcγRIIb (Non-Patent Document 25), only the effect of enhanced FcγRIIa binding, not enhanced FcγRIIb binding, would have an effect. For example, it has been reported that in systemic lupus erythematosus, platelets are activated by an FcγRIIa-dependent mechanism, and that platelet activation correlates with the severity of the disease (Non-Patent Document 40). Furthermore, according to another report, this alteration enhances FcγRIIa binding to the R-type by several hundred times, to the same extent as binding to FcγRIIb, but the R-type does not improve the selectivity of binding to FcγRIIb over FcγRIIa (Patent Document 17). Furthermore, for cell types that express both FcγRIIa and FcγRIIb, such as dendritic cells and macrophages, selectivity of binding to FcγRIIb over FcγRIIa is required for the transmission of inhibitory signals, but this is not achieved in the R-type.

[0023] The H and R types of FcγRIIa are observed at approximately the same frequency in Caucasians and African Americans (Non-Patent Documents 41 and 42). These findings suggest that there are certain limitations to the use of antibodies with enhanced binding to the R type of FcγRIIa in the treatment of autoimmune diseases. Even if the binding to FcγRIIb is enhanced compared to activating FcγR, the fact that the binding to any of the genetic polymorphisms of FcγRIIa is enhanced cannot be overlooked from the perspective of use as a therapeutic agent for autoimmune diseases.

[0024] In creating antibody drugs for the treatment of autoimmune diseases using FcγRIIb, it is important that, compared to native IgG, Fc-mediated binding is not increased or preferably decreased for any genetic polymorphism of FcγRIIa, and that binding to FcγRIIb is enhanced. However, no mutants with such properties have been reported to date, and their development has been desired.

[0025] Prior art documents relating to the present invention are listed below. [Prior art documents] [Patent documents]

[0026] [Patent Document 1] WO2009 / 125825 [Patent Document 2] WO2000 / 042072 [Patent Document 3] WO2006 / 019447 [Patent Document 4] WO2004 / 099249 [Patent Document 5] WO2004 / 029207 [Non-patent literature]

[0027] [Non-Patent Document 1] Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic., Nat. Biotechnol. (2005) 23, 1073 - 1078 [Non-patent document 2] Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. (2005) 59 (3), 389-396 [Non-patent document 3] Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. (2005) 20 (1), 17-29 [Non-patent document 4] Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J. Immunol. (2006) 176 (1), 346-356 [Non-patent document 5] Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat. Biotechnol. (1997) 15 (7), 637-640 [Non-patent document 6] Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc. Natl. Acad. Sci. USA (2005) 102 (24), 8466-8471 [Non-Patent Document 7] [ PMC free article ] [ PubMed ] Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA. Biol. (2007) 368, 652–665

Outdoor Track 8

Outdoor Tools9

Outdoor Tools 10

Outdoor Content11

Outdoor Tools22

Optional Trademark23

Optional Trademark24

Direct Entries 25

Non-Patent Document 29

Non-Patent Document 30

Non-Patent Document 31

Direct Environment 32

Direct Entries 33

Direct Entries 34

Direct Entries 35

Direct Entries 36

Direct Entries 37

Direct Entries 38

[0028] In addition to the involvement of activating FcγRs mentioned above, a process called antigen presentation is also crucial for eliciting an immune response to an administered antibody drug. Antigen presentation is an immune mechanism in which antigen-presenting cells, such as macrophages and dendritic cells, internalize and degrade exogenous and endogenous antigens, such as those from bacteria, and then present a portion of the antigen on their cell surface. The presented antigen is recognized by T cells and activates cellular and humoral immunity.

[0029] Dendritic cells present antigens via a pathway in which antigens taken up as immune complexes (complexes formed from multivalent antibodies and antigens) are degraded in lysosomes, and antigen-derived peptides are presented on MHC class II. FcRn plays an important role in this pathway, and it has been reported that antigen presentation and the resulting T cell activation do not occur when FcRn-deficient dendritic cells or immune complexes that do not bind to FcRn are used (Non-Patent Document 43).

[0030] When a foreign antigen protein is administered to a normal animal, antibodies against the administered antigen protein are frequently produced. For example, when a soluble human IL-6 receptor, a foreign protein, is administered to a mouse, mouse antibodies against the soluble human IL-6 receptor are produced. However, when a human IgG1 antibody, a foreign protein, is administered to a mouse, mouse antibodies against the human IgG1 antibody are hardly produced. This difference is thought to be influenced by the rate at which the administered foreign protein is eliminated from the plasma.

[0031] As shown in Reference Example 4, human IgG1 antibodies have the ability to bind to mouse FcRn under acidic conditions, and thus human IgG1 antibodies taken up into endosomes are recycled via mouse FcRn, just like mouse antibodies. Therefore, when human IgG1 antibodies are administered to normal mice, their elimination from plasma is extremely slow. On the other hand, soluble human IL-6 receptors are not recycled via mouse FcRn and are therefore rapidly eliminated after administration. Meanwhile, as shown in Reference Example 4, mouse antibodies against soluble human IL-6R antibodies are produced in normal mice administered with soluble human IL-6 receptors, whereas mouse antibodies against human IgG1 antibodies are not produced in normal mice administered with human IgG1 antibodies. In other words, in mice, soluble human IL-6 receptors, which are rapidly eliminated, are more immunogenic than human IgG1 antibodies, which are slowly eliminated.

[0032] It is thought that part of the pathway for the elimination of these foreign proteins (soluble human IL-6 receptor or human IgG1 antibody) from plasma involves uptake by antigen-presenting cells. After being internalized by antigen-presenting cells, the foreign proteins associate with MHC class II molecules and are transported to the cell membrane. When antigens are presented to antigen-specific T cells (e.g., T cells that specifically respond to soluble human IL-6 receptor or human IgG1 antibody), they are activated. Therefore, it is thought that foreign proteins that are slowly eliminated from plasma are less likely to be processed by antigen-presenting cells, resulting in less antigen presentation to antigen-specific T cells.

[0033] It is known that binding to FcRn under neutral conditions reduces the plasma retention of antibodies. Once an IgG antibody binds to FcRn under neutral conditions, even if it returns to the cell surface by binding to FcRn under the acidic conditions in the endosome, it will not be recycled into plasma unless it dissociates from FcRn in plasma under neutral conditions, resulting in impaired plasma retention. For example, it has been reported that when an antibody that binds to mouse FcRn under neutral conditions (pH 7.4) by introducing amino acid substitutions into IgG1 is administered to mice, the plasma retention of the antibody is impaired (Non-Patent Document 10). However, it has also been reported that when an antibody that binds to human FcRn under neutral conditions (pH 7.4) is administered to cynomolgus monkeys, the plasma retention of the antibody is not improved, and no change in plasma retention was observed (Non-Patent Documents 10, 11, and 12). If the plasma retention of an antigen-binding molecule is impaired by enhancing its FcRn binding under neutral conditions (pH 7.4), the antigen-binding molecule may be eliminated more quickly, potentially resulting in increased immunogenicity.

[0034] Furthermore, FcRn has been reported to be expressed on antigen-presenting cells and to be involved in antigen presentation. Although it is not an antigen-binding molecule, a report evaluating the immunogenicity of a protein (hereinafter referred to as MBP-Fc) composed of myelin basic protein (MBP) fused to the Fc region of mouse IgG1 showed that T cells specifically reacting with MBP-Fc were activated and proliferated when cultured in the presence of MBP-Fc. It is known that modification of the Fc region of MBP-Fc to enhance FcRn binding enhances in vitro T cell activation by increasing uptake into antigen-presenting cells via FcRn expressed on the antigen-presenting cells. However, it has been reported that modification to enhance FcRn binding accelerates clearance from plasma, but in vivo T cell activation is attenuated (Non-Patent Document 44). Therefore, enhanced FcRn binding and accelerated clearance do not necessarily result in increased immunogenicity.

[0035] For these reasons, the effects of enhancing the FcRn binding of an antigen-binding molecule having an FcRn-binding domain under neutral conditions (pH 7.4) on the plasma retention and immunogenicity of the antigen-binding molecule have not been fully studied. Therefore, no methods have been reported to date for improving the plasma retention and immunogenicity of antigen-binding molecules that have FcRn-binding activity under neutral conditions (pH 7.4).

[0036] It has been found that antigen elimination from plasma can be promoted by using an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, and an antigen-binding molecule containing an Fc region that has FcRn-binding activity under neutral pH conditions. However, the effects of enhancing the FcRn-binding activity of the Fc region under neutral pH conditions on the plasma retention and immunogenicity of antigen-binding molecules have not been fully studied. In the course of their research, the present inventors discovered the problem that enhancing the FcRn-binding activity of the Fc region under neutral pH conditions reduces the plasma retention of antigen-binding molecules (worsening pharmacokinetics) and increases the immunogenicity of antigen-binding molecules (exacerbating the immune response to the antigen-binding molecule).

[0037] The present invention was made in light of these circumstances and aims to provide a method for improving the pharmacokinetics of an antigen-binding molecule in a living body by modifying the Fc region of an antigen-binding molecule, which comprises an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and an Fc region that has FcRn-binding activity under neutral pH conditions. Another aim of the present invention is to provide a method for reducing the immune response of an antigen-binding molecule by modifying the Fc region of an antigen-binding molecule, which comprises an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and an Fc region that has FcRn-binding activity under neutral pH conditions. Another aim of the present invention is to provide an antigen-binding molecule that has improved pharmacokinetics or reduced immune response by the living body when administered to the living body. Furthermore, another aim of the present invention is to provide a method for producing the antigen-binding molecule, as well as a pharmaceutical composition containing the antigen-binding molecule as an active ingredient. [Means for solving the problem]

[0038] The present inventors conducted extensive research to achieve the above-mentioned objectives and found that antigen-binding molecules containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and an Fc region that has FcRn-binding activity under neutral pH conditions form a tetrameric heterocomplex consisting of the antigen-binding molecule, two FcRn molecules, and an activating Fcγ receptor (Figure 48), and that the formation of this tetrameric complex adversely affects pharmacokinetics and immune responses. By modifying the Fc region of such antigen-binding molecules to one that does not form a tetrameric heterocomplex consisting of two FcRn molecules and an activating Fcγ receptor under neutral pH conditions, the inventors found that modifying the Fc region to one that does not form a tetrameric complex with two FcRn molecules and an activating Fcγ receptor under neutral pH conditions improves the pharmacokinetics of the antigen-binding molecule. They also found that the immune response of an organism to which the antigen-binding molecule is administered can be modified. Furthermore, the inventors have found that the immune response of an antigen-binding molecule is reduced by modifying the Fc region so that it does not form a heterocomplex containing two FcRn molecules and an activating Fcγ receptor under neutral pH conditions. Furthermore, the inventors have discovered antigen-binding molecules with the above properties and a method for producing them, and have also found that when such antigen-binding molecules or pharmaceutical compositions containing antigen-binding molecules produced by the production methods of the present invention are administered as active ingredients, they have improved pharmacokinetics and reduced immune responses in the recipient organism, demonstrating superior properties compared to conventional antigen-binding molecules, thereby completing the present invention.

[0039] That is, the present invention provides the following. [1] any of the following methods, comprising modifying the Fc region of an antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and an Fc region that has FcRn-binding activity under a neutral pH range into an Fc region that does not form a heterocomplex containing two FcRn molecules and one activating Fcγ receptor molecule under a neutral pH range; (a) a method for improving the pharmacokinetics of an antigen-binding molecule, or (b) a method for reducing the immunogenicity of an antigen-binding molecule; [2] The method of [1], wherein modifying the Fc region to one that does not form heterocomplexes comprises modifying the Fc region to one whose binding activity to an activating Fcγ receptor is lower than the binding activity of the Fc region of native human IgG to the activating Fcγ receptor. [3] The method of [1] or [2], wherein the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F). [4] The method of any one of [1] to [3], comprising substituting one or more amino acids at positions 235, 237, 238, 239, 270, 298, 325, and 329 (EU numbering) among the amino acids in the Fc region. [5] Amino acids represented by EU numbering in the Fc region: the amino acid at position 234 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, or Trp; the amino acid at position 235 is Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg; the amino acid at position 236 is selected from Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, and Tyr; the amino acid at position 237 is Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg; the amino acid at position 238 is Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg; the amino acid at position 239 is either Gln, His, Lys, Phe, Pro, Trp, Tyr or Arg; the amino acid at position 265 is Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; The amino acid at position 266 is either Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr; The amino acid at position 267 is selected from Arg, His, Lys, Phe, Pro, Trp, and Tyr; the amino acid at position 269 is Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; the amino acid at position 270 is Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; The amino acid at position 271 is either Arg, His, Phe, Ser, Thr, Trp, or Tyr; The amino acid at position 295 is selected from Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, and Tyr; The amino acid at position 296 is selected from Arg, Gly, Lys, and Pro; The amino acid at position 297 is Ala, The amino acid at position 298 is selected from Arg, Gly, Lys, Pro, Trp, and Tyr; The amino acid at position 300 is either Arg, Lys, or Pro. The amino acid at position 324 is either Lys or Pro. The amino acid at position 325 is Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, TrpTyr, or Val; the amino acid at position 327 is selected from Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; The amino acid at position 328 is selected from Arg, Asn, Gly, His, Lys, and Pro; the amino acid at position 329 is selected from Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, and Arg; The amino acid at position 330 is either Pro or Ser, The amino acid at position 331 is either Arg, Gly, or Lys, or The amino acid at position 332 is either Arg, Lys, or Pro; The method according to [4], comprising substituting any one or more of: [6] The method of [1], wherein modifying the Fc region so that it does not form heterocomplexes comprises modifying the Fc region so that its binding activity to inhibitory Fcγ receptors is higher than its binding activity to activating Fcγ receptors. [7] The method of [6], wherein the inhibitory Fcγ receptor is human FcγRIIb. [8] The method of [6] or [7], wherein the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F). [9] The method according to any one of [6] to [8], comprising substituting the amino acid at position 238 or 328 according to EU numbering.

[10] The method according to [9], which comprises substituting amino acid 238 (EU numbering) with Asp or amino acid 328 (EU numbering) with Glu.

[11] an amino acid represented by EU numbering; The amino acid at position 233 is Asp, The amino acid at position 234 is either Trp or Tyr. the amino acid at position 237 is Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr; The amino acid at position 239 is Asp, the amino acid at position 267 is either Ala, Gln or Val; The amino acid at position 268 is either Asn, Asp, or Glu; The amino acid at position 271 is Gly, the amino acid at position 326 is Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr; The amino acid at position 330 can be either Arg, Lys, or Met. the amino acid at position 323 is Ile, Leu, or Met; The amino acid at position 296 is Asp, The method according to [9] or

[10] , wherein the substitution is at least one of:

[12] The method of any one of [1] to

[11] , wherein the Fc region contains one or more amino acids different from those of a native Fc region, including any one of 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering).

[13] Amino acids represented by EU numbering in the Fc region: The amino acid at position 237 is Met; The amino acid at position 248 is Ile; The amino acid at position 250 is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; The amino acid at position 252 is either Phe, Trp, or Tyr; The amino acid at position 254 is Thr; The amino acid at position 255 is Glu; the amino acid at position 256 is Asp, Asn, Glu, or Gln; the amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The amino acid at position 258 is His; The amino acid at position 265 is Ala, The amino acid at position 286 is either Ala or Glu, The amino acid at position 289 is His; The amino acid at position 297 is Ala, The amino acid at position 298 is Gly; The amino acid at position 303 is Ala, The amino acid at position 305 is Ala, the amino acid at position 307 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The amino acid at position 309 is Ala, Asp, Glu, Pro, or Arg; the amino acid at position 311 is Ala, His, or Ile; The amino acid at position 312 is either Ala or His; The amino acid at position 314 is either Lys or Arg; the amino acid at position 315 is Ala, Asp, or His; The amino acid at position 317 is Ala, The amino acid at position 332 is Val; The amino acid at position 334 is Leu, The amino acid at position 360 is His, The amino acid at position 376 is Ala, The amino acid at position 380 is Ala, The amino acid at position 382 is Ala, The amino acid at position 384 is Ala, The amino acid at position 385 is either Asp or His; The amino acid at position 386 is Pro, The amino acid at position 387 is Glu; The amino acid at position 389 is either Ala or Ser, The amino acid at position 424 is Ala, the amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 433 is Lys; The amino acid at position 434 is Ala, Phe, His, Ser, Trp, or Tyr, or The amino acid at position 436 is His, Ile, Leu, Phe, Thr, or Val; The method according to

[12] , wherein the method is a combination of any one or more of the following:

[14] The method of any one of [1] to

[13] , wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity changes depending on calcium ion concentration conditions.

[15] The method of

[14] , wherein the antigen-binding domain is an antigen-binding domain whose binding activity is changed so that its antigen-binding activity under a low calcium ion concentration is lower than that under a high calcium ion concentration.

[16] The method of any one of [1] to

[13] , wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity changes depending on pH conditions.

[17] The method of

[16] , wherein the antigen-binding domain is an antigen-binding domain whose binding activity changes so that its antigen-binding activity in an acidic pH range is lower than its antigen-binding activity in a neutral pH range.

[18] The method of any one of [1] to

[17] , wherein the antigen-binding domain is an antibody variable region.

[19] The method of any one of [1] to

[18] , wherein the antigen-binding molecule is an antibody.

[20] The method of [1], wherein modifying the Fc region to one that does not form a heterocomplex comprises modifying the Fc region so that one of the two polypeptides constituting the Fc region has FcRn-binding activity in a neutral pH range, and the other does not have FcRn-binding activity in a neutral pH range.

[21] The method of

[20] , comprising substituting one or more amino acids at 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the amino acid sequence of one of the two polypeptides forming the Fc domain.

[22] Amino acids represented by EU numbering in the Fc region: The amino acid at position 237 is Met, The amino acid at position 248 is Ile, The amino acid at position 250 may be Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; Amino acid at position 252 is replaced by Phe, Trp, or Tyr; The amino acid at position 254 is changed to Thr. The amino acid at position 255 is replaced with Glu. The amino acid at position 256 may be Asp, Asn, Glu, or Gln; The amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The amino acid at position 258 is His, The amino acid at position 265 is Ala, The amino acid at position 286 is Ala or Glu, The amino acid at position 289 is His, The amino acid at position 297 is Ala, The amino acid at position 298 is Gly, The amino acid at position 303 is Ala, The amino acid at position 305 is Ala, Amino acid at position 307 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; The amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The amino acid at position 309 may be Ala, Asp, Glu, Pro, or Arg; Amino acid at position 311 is replaced by Ala, His, or Ile; The amino acid at position 312 is replaced with Ala or His. Amino acid at position 314 is replaced with Lys or Arg, The amino acid at position 315 is replaced with Ala, Asp, or His. The amino acid at position 317 is Ala, The amino acid at position 332 is Val, The amino acid at position 334 is replaced with Leu. The amino acid at position 360 is His, The amino acid at position 376 is Ala, The amino acid at position 380 is Ala, The amino acid at position 382 is Ala, The amino acid at position 384 is Ala, The amino acid at position 385 is replaced with Asp or His. The amino acid at position 386 is Pro, The amino acid at position 387 is replaced with Glu. Amino acid at position 389 is replaced with Ala or Ser; The amino acid at position 424 is Ala, The amino acid at position 428 may be Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 433 is Lys, The amino acid at position 434 is Ala, Phe, His, Ser, Trp, or Tyr, or Amino acid at position 436 is His, Ile, Leu, Phe, Thr, or Val The method according to

[21] , comprising substituting any one or more of:

[23] The method of any one of

[20] to

[22] , wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity changes depending on calcium ion concentration conditions.

[24] The method of

[23] , wherein the antigen-binding domain is an antigen-binding domain whose binding activity is changed so that its antigen-binding activity under a low calcium ion concentration is lower than that under a high calcium ion concentration.

[25] The method of any one of

[20] to

[22] , wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity changes depending on pH conditions.

[26] The method of

[25] , wherein the antigen-binding domain is an antigen-binding domain whose binding activity changes so that its antigen-binding activity in an acidic pH range is lower than its antigen-binding activity in a neutral pH range.

[27] The method of any one of

[20] to

[26] , wherein the antigen-binding domain is an antibody variable region.

[28] The method of any one of

[20] to

[27] , wherein the antigen-binding molecule is an antibody.

[29] an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, and amino acids represented by EU numbering in an Fc region that has FcRn-binding activity in a neutral pH range; The amino acid at position 234 is Ala, the amino acid at position 235 is either Ala, Lys, or Arg; The amino acid at position 236 is Arg, the amino acid at position 238 is Arg, The amino acid at position 239 is Lys; The amino acid at position 270 is Phe, The amino acid at position 297 is Ala, The amino acid at position 298 is Gly; The amino acid at position 325 is Gly; The amino acid at position 328 is Arg, or the amino acid at position 329 is Lys or Arg, An antigen-binding molecule comprising an Fc region containing one or more amino acids selected from the following:

[30] Amino acids represented by EU numbering in the Fc region: The amino acid at position 237 is either Lys or Arg; The amino acid at position 238 is Lys The amino acid at position 239 is Arg, or The amino acid at position 329 is either Lys or Arg; The antigen-binding molecule of

[29] , comprising one or more amino acids selected from the following:

[31] An antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, and an Fc domain in which one of two polypeptides comprising the Fc domain has FcRn-binding activity in a neutral pH range, and the other polypeptide does not have FcRn-binding activity in a neutral pH range.

[32] The antigen-binding molecule of any one of

[29] to

[31] , wherein the Fc region is an Fc region in which any one or more amino acids, 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering), differ from the amino acids of a native Fc region.

[33] Amino acids represented by EU numbering in the Fc region: The amino acid at position 237 is Met; The amino acid at position 248 is Ile; The amino acid at position 250 is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; Amino acid at position 252 is Phe, Trp, or Tyr; The amino acid at position 254 is Thr; The amino acid at position 255 is Glu; The amino acid at position 256 is Asp, Asn, Glu, or Gln; The amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The amino acid at position 258 is His; The amino acid at position 265 is Ala, The amino acid at position 286 is Ala or Glu, The amino acid at position 289 is His; The amino acid at position 297 is Ala, The amino acid at position 303 is Ala, The amino acid at position 305 is Ala, the amino acid at position 307 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The amino acid at position 309 is Ala, Asp, Glu, Pro, or Arg; The amino acid at position 311 is Ala, His, or Ile; The amino acid at position 312 is Ala or His, The amino acid at position 314 is Lys or Arg; The amino acid at position 315 is Ala, Asp, or His; The amino acid at position 317 is Ala, The amino acid at position 332 is Val; The amino acid at position 334 is Leu, The amino acid at position 360 is His, The amino acid at position 376 is Ala, The amino acid at position 380 is Ala, The amino acid at position 382 is Ala, The amino acid at position 384 is Ala, The amino acid at position 385 is Asp or His, The amino acid at position 386 is Pro, The amino acid at position 387 is Glu; The amino acid at position 389 is Ala or Ser; The amino acid at position 424 is Ala, The amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 433 is Lys. The amino acid at position 434 is Ala, Phe, His, Ser, Trp, or Tyr, or The amino acid at position 436 is His, Ile, Leu, Phe, Thr, or Val; The antigen-binding molecule of

[32] ,

[34] The antigen-binding molecule of any one of

[29] to

[33] , wherein the antigen-binding activity of the antigen-binding domain changes depending on calcium ion concentration.

[35] The antigen-binding molecule of

[34] , wherein the antigen-binding domain is an antigen-binding domain whose binding activity is changed such that its antigen-binding activity under a low calcium ion concentration is lower than that under a high calcium ion concentration.

[36] The antigen-binding molecule of any one of

[29] to

[33] , wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity changes depending on pH conditions.

[37] The antigen-binding molecule of

[36] , wherein the antigen-binding domain is an antigen-binding domain whose binding activity changes so that its antigen-binding activity in an acidic pH range is lower than that in a neutral pH range.

[38] The antigen-binding molecule of any one of

[29] to

[37] , wherein the antigen-binding domain is an antibody variable region.

[39] The antigen-binding molecule of any one of

[29] to

[38] , wherein the antigen-binding molecule is an antibody.

[40] A polynucleotide encoding the antigen-binding molecule of any one of

[29] to

[39] .

[41] A vector to which the polynucleotide according to

[40] is operably linked.

[42] A cell into which the vector according to

[41] has been introduced.

[43] A method for producing the antigen-binding molecule of any one of

[29] to

[39] , comprising the step of recovering the antigen-binding molecule from a culture medium of the cell of

[42] .

[44] A pharmaceutical composition comprising, as an active ingredient, the antigen-binding molecule of any one of

[29] to

[39] , or the antigen-binding molecule obtained by the production method of

[43] .

[0040] The present invention also relates to a kit for use in the method of the present invention, which comprises the antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention. The present invention also relates to an agent for improving the pharmacokinetics of an antigen-binding molecule or an agent for reducing the immunogenicity of an antigen-binding molecule, which comprises as an active ingredient the antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention. The present invention also relates to a method for treating an immunoinflammatory disease, which comprises administering to a subject the antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention. The present invention also relates to the use of the antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention in the production of an agent for improving the pharmacokinetics of an antigen-binding molecule or an agent for reducing the immunogenicity of an antigen-binding molecule. The present invention also relates to the antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention, for use in the method of the present invention. [Effects of the Invention]

[0041] The present invention provides a method for improving the pharmacokinetics of an antigen-binding molecule or a method for reducing the immunogenicity of an antigen-binding molecule. The present invention enables antibody-based therapy without causing undesirable events in the body, as compared to conventional antibodies. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 shows the effects on soluble antigens of existing neutralizing antibodies and pH-dependent antigen-binding antibodies with enhanced FcRn binding under neutral conditions. [Figure 2] FIG. 1 shows the time course of plasma concentration of Fv4-IgG1 or Fv4-IgG1-F1 when administered intravenously or subcutaneously to normal mice. [Figure 3] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to human FcγRIa. [Figure 4] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to human FcγRIIa (R). [Figure 5] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to human FcγRIIa (H). [Figure 6] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to human FcγRIIb. [Figure 7] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to human FcγRIIIa (F). [Figure 8] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to mouse FcγRI. [Figure 9] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to mouse FcγRIIb. [Figure 10] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to mouse FcγRIII. [Figure 11] This figure shows that Fv4-IgG1-F157 bound to human FcRn binds to mouse FcγRIV. [Figure 12] This figure shows that Fv4-IgG1-F20 bound to mouse FcRn binds to mouse FcγRI, mouse FcγRIIb, mouse FcγRIII, and mouse FcγRIV. [Figure 13] FIG. 10 shows that mPM1-mIgG1-mF3 bound to mouse FcRn binds to mouse FcγRIIb and mouse FcγRIII. [Figure 14] Fig. 14 shows the time courses of plasma concentrations of Fv4-IgG1-F21, Fv4-IgG1-F140, Fv4-IgG1-F157, and Fv4-IgG1-F424 in human FcRn transgenic mice. [Figure 15]Fig. 1 shows the time courses of plasma concentrations of Fv4-IgG1 and Fv4-IgG1-F760 in human FcRn transgenic mice. [Figure 16] FIG. 1 shows the time course of plasma concentrations of Fv4-IgG1-F11, Fv4-IgG1-F890, Fv4-IgG1-F947, Fv4-IgG1-F821, Fv4-IgG1-F939, and Fv4-IgG1-F1009 in human FcRn transgenic mice. [Figure 17] FIG. 10 is a graph showing the time courses of plasma concentrations of mPM1-mIgG1-mF14, mPM1-mIgG1-mF38, mPM1-mIgG1-mF39, and mPM1-mIgG1-mF40 in normal mice. [Figure 18] Fig. 1 shows the results of immunogenicity evaluation using Fv4-IgG1-F21 and Fv4-IgG1-F140. [Figure 19] Fig. 1 shows the results of immunogenicity evaluation using hA33-IgG1-F21 and hA33-IgG1-F140. [Figure 20] Fig. 1 shows the results of immunogenicity evaluation using hA33-IgG1-F698 and hA33-IgG1-F699. [Figure 21] Fig. 1 shows the results of immunogenicity evaluation using hA33-IgG1-F698 and hA33-IgG1-F763. [Figure 22] FIG. 10 shows the antibody titers of mouse antibodies produced against Fv4-IgG1-F11 3, 7, 14, 21, and 28 days after administration to human FcRn transgenic mice. [Figure 23] FIG. 10 shows the antibody titers of mouse antibodies produced against Fv4-IgG1-F821 3, 7, 14, 21, and 28 days after administration to human FcRn transgenic mice. [Figure 24] Figure 1 shows the antibody titers of mouse antibodies produced against Fv4-IgG1-F890 3, 7, 14, 21, and 28 days after administration to human FcRn transgenic mice. B is an enlarged version of A. [Figure 25] FIG. 10 shows the antibody titers of mouse antibodies produced against Fv4-IgG1-F939 3, 7, 14, 21, and 28 days after administration to human FcRn transgenic mice. [Figure 26] Fig. 10 shows the antibody titers of mouse antibodies produced against Fv4-IgG1-F947 3, 7, 14, 21, and 28 days after administration to human FcRn transgenic mice. [Figure 27] FIG. 10 shows the antibody titers of mouse antibodies produced against Fv4-IgG1-F1009 3, 7, 14, 21, and 28 days after administration to human FcRn transgenic mice. [Figure 28] FIG. 1 shows the antibody titers of mouse antibodies produced against mPM1-IgG1-mF14 14, 21, and 28 days after administration to normal mice. [Figure 29] FIG. 1 shows the antibody titers of mouse antibodies produced against mPM1-IgG1-mF39 14, 21, and 28 days after administration to normal mice. [Figure 30] FIG. 1 shows the antibody titers of mouse antibodies produced against mPM1-IgG1-mF38 14, 21, and 28 days after administration to normal mice. [Figure 31] FIG. 1 shows the antibody titers of mouse antibodies produced against mPM1-IgG1-mF40 14, 21, and 28 days after administration to normal mice. [Figure 32] Fig. 1 shows the antibody concentrations of Fv4-IgG1-F947 and Fv4-IgG1-FA6a / FB4a in plasma 15 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, and 7 days after administration to human FcRn transgenic mice. [Figure 33] FIG. 1 shows the distribution of FcγRIIb binding and FcγRIa binding for each B3 mutant. [Figure 34]FIG. 10 shows the distribution of FcγRIIb binding and FcγRIIa(H) binding for each B3 mutant. [Figure 35] FIG. 10 shows the distribution of FcγRIIb binding and FcγRIIa(R) binding for each B3 mutant. [Figure 36] FIG. 1 shows the distribution of FcγRIIb binding and FcγRIIIa binding for each B3 mutant. [Figure 37] FIG. 1 shows the plasma kinetics of soluble human IL-6 receptor in normal mice and the antibody titer of mouse antibodies against soluble human IL-6 receptor in mouse plasma. [Figure 38] FIG. 1 shows the plasma kinetics of soluble human IL-6 receptor in normal mice administered with anti-mouse CD4 antibody, and the antibody titer of mouse antibody against soluble human IL-6 receptor in mouse plasma. [Figure 39] FIG. 1 shows the plasma kinetics of anti-IL-6 receptor antibodies in normal mice. [Figure 40] FIG. 1 shows the time course of soluble human IL-6 receptor concentration when soluble human IL-6 receptor and anti-IL-6 receptor antibody were simultaneously administered to human FcRn transgenic mice. [Figure 41] FIG. 1 shows the structure of the heavy chain CDR3 of the Fab fragment of the 6RL#9 antibody, determined by X-ray crystal structure analysis. [Figure 42] FIG. 1 shows the time course of plasma antibody concentrations of H54 / L28-IgG1, 6RL#9-IgG1, and FH4-IgG1 in normal mice. [Figure 43] FIG. 1 shows the time course of soluble human IL-6 receptor concentration in plasma of normal mice administered with H54 / L28-IgG1, 6RL#9-IgG1, or FH4-IgG1. [Figure 44] FIG. 1 shows the time course of antibody concentrations in plasma of H54 / L28-N434W, 6RL#9-N434W, and FH4-N434W in normal mice. [Figure 45]FIG. 1 shows the time course of soluble human IL-6 receptor concentration in plasma of normal mice administered with H54 / L28-N434W, 6RL#9-N434W, or FH4-N434W. [Figure 46] 1 shows ion-exchange chromatograms of an antibody comprising the human Vk5-2 sequence and an antibody comprising the hVk5-2_L65 sequence, in which the glycosylation sequence in the human Vk5-2 sequence has been altered. The solid line represents the chromatogram of the antibody comprising the human Vk5-2 sequence (heavy chain: CIM_H (SEQ ID NO: 108) and light chain: hVk5-2 (SEQ ID NO: 4)), and the dashed line represents the chromatogram of the antibody having the hVk5-2_L65 sequence (heavy chain: CIM_H (SEQ ID NO: 108), light chain: hVk5-2_L65 (SEQ ID NO: 107)). [Figure 47] FIG. 1 shows the alignment of the constant region sequences of IgG1, IgG2, IgG3, and IgG4 with EU numbering. [Figure 48] FIG. 1 shows a schematic diagram of the formation of a quaternary complex consisting of one molecule of an Fc region having FcRn-binding activity under neutral pH conditions, two molecules of FcRn, and one molecule of FcγR. [Figure 49] Fig. 1 is a schematic diagram showing the interaction of an Fc region that has FcRn-binding activity in the neutral pH range and whose binding activity to activating FcγR is lower than that of a native Fc region, with two FcRn molecules and one FcγR molecule. [Figure 50] Fig. 1 is a schematic diagram showing the actions of an Fc region that has FcRn-binding activity and selective binding activity to inhibitory FcγR under neutral pH conditions, together with two FcRn molecules and one FcγR molecule. [Figure 51] Fig. 1 is a schematic diagram showing the interactions of an Fc domain, in which one of the two polypeptides comprising the FcRn-binding domain has FcRn-binding activity in the neutral pH range, and the other has no FcRn-binding activity in the neutral pH range, with two FcRn molecules and one FcγR molecule. [Figure 52]This graph shows the relationship between the amino acid distribution (labeled Library) and the designed amino acid distribution (labeled Design) of the sequence information of 290 clones isolated from Escherichia coli into which a Ca-dependent antigen-binding antibody gene library was introduced. The horizontal axis shows the amino acid position represented by Kabat numbering, and the vertical axis shows the amino acid distribution ratio. [Figure 53] This graph shows the relationship between the amino acid distribution (labeled Library) and the designed amino acid distribution (labeled Design) of the sequence information of 132 clones isolated from E. coli into which a pH-dependent antigen-binding antibody gene library has been introduced. The horizontal axis shows the amino acid position represented by Kabat numbering, and the vertical axis shows the amino acid distribution ratio. [Figure 54] Fig. 10 is a graph showing the time courses of Fv4-IgG1-F947 and Fv4-IgG1-F1326 concentrations in mouse plasma when Fv4-IgG1-F947 and Fv4-IgG1-F1326 were administered to human FcRn transgenic mice. [Figure 55] The horizontal axis represents the relative binding activity of each PD variant to FcγRIIb, and the vertical axis represents the relative binding activity of each PD variant to FcγRIIa R type. The binding amount of each PD variant to each FcγR was divided by the binding amount of the control antibody IL6R-F652 (an altered Fc in which Pro at position 238 (EU numbering) was replaced with Asp), and the resulting value was multiplied by 100 to obtain the relative binding activity of each PD variant to each FcγR. The plot labeled F652 in the figure shows the value for IL6R-F652. [Figure 56]The vertical axis shows the relative FcγRIIb-binding activity of variants obtained by introducing each alteration into GpH7-B3, which does not have the P238D alteration, and the horizontal axis shows the relative FcγRIIb-binding activity of variants obtained by introducing each alteration into IL6R-F652, which has the P238D alteration. The value for the amount of FcγRIIb binding of each variant was divided by the value for the amount of FcγRIIb binding of the antibody before the alteration was introduced, and then multiplied by 100 to obtain the relative binding activity. Region A includes alterations that exhibit an FcγRIIb-binding-enhancing effect when introduced into GpH7-B3 that does not have P238D and when introduced into IL6R-F652 that has P238D, while Region B includes alterations that exhibit an FcγRIIb-binding-enhancing effect when introduced into GpH7-B3 that does not have P238D but do not exhibit an FcγRIIb-binding-enhancing effect when introduced into IL6R-F652 that has P238D. [Figure 57] 1 shows the crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex. [Figure 58] Figure 1 shows a diagram of the crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex and the model structure of the Fc(WT) / FcγRIIb extracellular region complex superimposed by the least-squares method based on the Cα interatomic distances for the FcγRIIb extracellular region and Fc CH2 domain A. [Figure 59] The crystal structure of the Fc(P238D) / FcγRIIb extracellular region complex and the model structure of the Fc(WT) / FcγRIIb extracellular region complex were superimposed using the least-squares method based on the Cα interatomic distances for the Fc CH2 domain A and the Fc CH2 domain B alone, and the detailed structure around P238D was compared. [Figure 60]

[0046] FIG. 10 shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, a hydrogen bond is observed between the main chain of Gly at position 237 (EU numbering) in Fc CH2 domain A and Tyr at position 160 in FcγRIIb. [Figure 61]FIG. 10 shows that in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, electrostatic interaction is observed between Asp at position 270 (EU numbering) in Fc CH2 domain B and Arg at position 131 in FcγRIIb. [Figure 62] The horizontal axis shows the relative binding activity of each 2B variant to FcγRIIb, and the vertical axis shows the relative binding activity of each 2B variant to FcγRIIa type R. The binding amount of each 2B variant to each FcγR was divided by the binding amount of the control antibody before alteration (altered Fc in which Pro at position 238 (EU numbering) was replaced with Asp), and the resulting value was multiplied by 100 to obtain the relative binding activity of each 2B variant to each FcγR. [Figure 63] FIG. 10 shows Glu at position 233 (EU numbering) of Fc Chain A in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, and surrounding residues in the extracellular region of FcγRIIb. [Figure 64] FIG. 10 shows the Ala at position 330 (EU numbering) of Fc Chain A in the crystal structure of the Fc (P238D) / FcγRIIb extracellular region complex, and surrounding residues in the extracellular region of FcγRIIb. [Figure 65] Figure 1 shows the structure of Pro at position 271 (EU numbering) of Fc Chain B, obtained by superimposing the crystal structures of the Fc(P238D) / FcγRIIb extracellular region complex and the Fc(WT) / FcγRIIIa extracellular region complex using the least-squares method based on the Cα interatomic distances relative to Fc Chain B. DETAILED DESCRIPTION OF THE INVENTION

[0043] The following definitions and detailed description are provided to facilitate understanding of the invention described herein. amino acid As used herein, amino acids are represented by one-letter or three-letter codes, or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, and Val / V.

[0044] antigen As used herein, the term "antigen" is not limited to a specific structure as long as it contains an epitope to which an antigen-binding domain binds. In another sense, an antigen can be inorganic or organic. Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / E phB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalcohol In, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, Tegrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressed Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, virus Rux antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81 CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa,Examples include factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and receptors for hormones and growth factors.

[0045] An epitope, meaning an antigenic determinant present in an antigen, refers to a site on an antigen to which an antigen-binding domain in an antigen-binding molecule disclosed herein binds. Thus, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of an antigen-binding molecule that recognizes the epitope to the antigen. When the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute the epitope. Furthermore, when the epitope is a sugar chain, the epitope can also be identified by a specific sugar chain structure.

[0046] A linear epitope is one in which the primary amino acid sequence comprises a recognized epitope, typically comprising at least three, and most usually at least five, e.g., about 8 to about 10, 6 to 20 amino acids in a unique sequence.

[0047] Conformational epitopes, in contrast to linear epitopes, are epitopes in which the primary sequence of amino acids comprising the epitope is not the single, defined component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes may encompass an increased number of amino acids relative to linear epitopes. In recognizing conformational epitopes, antibodies recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0048] Binding activity Methods for confirming epitope binding by test antigen-binding molecules containing an antigen-binding domain for IL-6R are exemplified below; however, methods for confirming epitope binding by test antigen-binding molecules containing antigen-binding domains for antigens other than IL-6R can also be appropriately carried out in accordance with the examples below.

[0049] For example, whether a test antigen-binding molecule containing an IL-6R antigen-binding domain recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region of IL-6R cDNA encoding the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain to the test antigen-binding molecule containing the IL-6R antigen-binding domain is assessed. For example, the binding activity of the antigen-binding molecule to the peptide can be assessed by ELISA using an immobilized linear peptide as the antigen. Alternatively, the binding activity of the antigen-binding molecule to the linear peptide can be determined based on the level of inhibition by the linear peptide of binding of the antigen-binding molecule to IL-6R-expressing cells. These tests can determine the binding activity of the antigen-binding molecule to the linear peptide.

[0050] Furthermore, whether a test antigen-binding molecule containing an IL-6R antigen-binding domain recognizes a conformational epitope can be confirmed as follows. For this purpose, IL-6R-expressing cells are prepared. Examples of such confirmation include when a test antigen-binding molecule containing an IL-6R antigen-binding domain binds strongly to IL-6R-expressing cells upon contact with the cells, but does not substantially bind to a linear peptide consisting of the amino acid sequence forming the extracellular domain of immobilized IL-6R. Here, "not substantially binding" refers to a binding activity that is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity toward human IL-6R-expressing cells.

[0051] Methods for measuring the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain toward IL-6R-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, the binding activity can be assessed by ELISA or fluorescence activated cell sorting (FACS) using IL-6R-expressing cells as antigens.

[0052] In the ELISA format, the binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain toward IL-6R-expressing cells is quantitatively assessed by comparing the signal levels generated by the enzymatic reaction. Specifically, a test polypeptide complex is added to an ELISA plate on which IL-6R-expressing cells have been immobilized, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody-binding titer toward IL-6R-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule toward IL-6R-expressing cells to be compared.

[0053] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following: FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are trade names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)

[0054] For example, one suitable method for measuring the antigen-binding activity of a test antigen-binding molecule containing an IL-6R antigen-binding domain is as follows: First, the test antigen-binding molecule is reacted with cells expressing IL-6R and stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is diluted with an appropriate buffer solution to prepare the desired concentration. For example, the antigen-binding molecule can be used at any concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antibody binding to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, the Geometric Mean value allows the binding activity of the test antigen-binding molecule, represented by the amount of binding of the test antigen-binding molecule, to be measured.

[0055] Whether a test antigen-binding molecule containing an IL-6R antigen-binding domain shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. For example, competitive ELISA assays are preferred cross-blocking assays.

[0056] Specifically, in a cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competing antigen-binding molecule, and then a test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to IL-6R protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to wells coated with IL-6R protein.

[0057] The amount of test antigen-binding molecules bound to the wells via the IL-6R protein can be easily measured by labeling the antigen-binding molecules in advance. For example, biotin-labeled antigen-binding molecules can be measured using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly known as competitive ELISA assays. Antigen-binding molecules can also be labeled with other detectable or measurable labeling substances. Specific examples include radiolabels and fluorescent labels.

[0058] If a competitor antigen-binding molecule can block the binding of a test antigen-binding molecule comprising an antigen-binding domain to IL-6R by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of a candidate competitor antigen-binding molecule, the test antigen-binding molecule is an antigen-binding molecule that binds to substantially the same epitope as the competitor antigen-binding molecule or competes for binding to the same epitope.

[0059] When the structure of the epitope to which a test antigen-binding molecule containing an IL-6R antigen-binding domain binds has been identified, whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activity of both antigen-binding molecules toward peptides in which amino acid mutations have been introduced into the peptide constituting the epitope.

[0060] For example, such binding activity can be measured by comparing the binding activity of test and control antigen-binding molecules to a mutated linear peptide in the ELISA format described above. Alternatively, binding activity to the mutant peptide bound to a column can be measured by flowing the test and control antigen-binding molecules down the column and then quantifying the antigen-binding molecules eluted in the eluate. Methods for adsorbing mutant peptides to a column, for example, as fusion peptides with GST, are known.

[0061] Furthermore, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share a common epitope can be assessed by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. These cells are suspended in an appropriate buffer, such as PBS, and the test and control antigen-binding molecules are added to the cell suspension. Next, an FITC-labeled antibody that can recognize the test and control antigen-binding molecules is added to the cell suspension after washing with an appropriate buffer. The fluorescence intensity and cell count of cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test and control antigen-binding molecules are diluted with a suitable buffer to the desired concentration and used. For example, they are used at a concentration between 10 μg / ml and 10 ng / ml. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the Geometric Mean value, the binding activity of the test and control antigen-binding molecules, represented by the amount of bound labeled antibody, can be measured.

[0062] In this method, "substantially no binding to mutant IL-6R-expressing cells" can be determined, for example, by the following method. First, test and control antigen-binding molecules bound to mutant IL-6R-expressing cells are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used for flow cytometry to detect fluorescence, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The percentage increase in fluorescence intensity due to antigen-binding molecule binding can be determined by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the polypeptide complex using the following formula:

[0063] ΔGeo-Mean = Geo-Mean (in the presence of polypeptide complex) / Geo-Mean (in the absence of polypeptide complex)

[0064] The Geometric Mean comparison value (mutant IL-6R molecule ΔGeo-Mean value) obtained by analysis, which reflects the binding amount of the test antigen-binding molecule to mutant IL-6R-expressing cells, is compared with the ΔGeo-Mean comparison value, which reflects the binding amount of the test antigen-binding molecule to IL-6R-expressing cells. In this case, it is particularly preferred that the test antigen-binding molecules used to determine the ΔGeo-Mean comparison values ​​for mutant IL-6R-expressing cells and IL-6R-expressing cells are prepared at the same or substantially the same concentrations. An antigen-binding molecule previously confirmed to recognize an epitope in IL-6R is used as a control antigen-binding molecule.

[0065] A test antigen-binding molecule is deemed to "not substantially bind to mutant IL-6R-expressing cells" if the ΔGeo-Mean comparison value for the test antigen-binding molecule for the mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value for the test antigen-binding molecule for the IL-6R-expressing cells. The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). When the comparison values ​​are substantially equivalent, the epitopes of the test and control antigen-binding molecules can be determined to be identical.

[0066] antigen-binding domain As used herein, the term "antigen-binding domain" may refer to any domain of any structure as long as it binds to a target antigen. Examples of such domains include the variable regions of the heavy and light chains of an antibody, a module called an A domain of approximately 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (WO2004 / 044011, WO2005 / 040229), Adnectin (WO2002 / 032925) containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on cell membranes, Affibody (WO1995 / 001937) using an IgG-binding domain composed of a 58-amino acid three-helix bundle of Protein A as a scaffold, and DARPins (Designed Ankyrin Repeats), which are regions exposed on the molecular surface of ankyrin repeats (AR) with a structure in which a 33-amino acid turn, two antiparallel helices, and a loop subunit are repeatedly stacked. Preferred examples of the antigen-binding domain of the present invention include an anticalin molecule (WO 2002 / 020565), which is a four-loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules of the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish, are repeatedly stacked (WO 2008 / 016854). Preferred examples of the antigen-binding domain of the present invention include antigen-binding domains comprising the variable regions of the heavy and light chains of antibodies.Suitable examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2."

[0067] The antigen-binding domains in the antigen-binding molecules of the present invention can bind to the same epitope. Here, the same epitope can exist, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. It can also exist in a protein consisting of amino acids 20 to 365 of the amino acid sequence set forth in SEQ ID NO: 1. Alternatively, the antigen-binding domains in the antigen-binding molecules of the present invention can bind to different epitopes. Here, the different epitopes can exist, for example, in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. It can also exist in a protein consisting of amino acids 20 to 365 of the amino acid sequence set forth in SEQ ID NO: 1.

[0068] specific "Specific" refers to a state in which one of the specifically binding molecules does not exhibit any significant binding to any molecules other than the one or more molecules to which it binds. The term also applies when an antigen-binding domain is specific to a specific epitope among multiple epitopes contained in an antigen. Furthermore, when the epitope to which the antigen-binding domain binds is contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing that epitope.

[0069] antibody As used herein, an antibody refers to a natural or partially or fully synthetically produced immunoglobulin. Antibodies can be isolated from natural sources such as plasma or serum where they occur, or from the culture supernatant of antibody-producing hybridoma cells, or can be partially or fully synthesized using techniques such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes and their isotype subclasses. Nine known classes (isotypes) of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4.

[0070] Methods for producing antibodies with desired binding activity are known to those skilled in the art. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be produced appropriately according to the following examples.

[0071] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Monoclonal antibodies derived from mammals are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques. The monoclonal antibodies of the present invention also include "humanized antibodies" and "chimeric antibodies."

[0072] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Next, monoclonal antibody-producing cells can be screened using conventional screening methods to select hybridomas that produce anti-IL-6R antibodies.

[0073] Specifically, monoclonal antibodies can be produced, for example, as follows. First, the IL-6R protein represented by SEQ ID NO: 1, which is used as a sensitizing antigen for antibody production, can be obtained by expressing the IL-6R gene, whose nucleotide sequence is disclosed in SEQ ID NO: 2. Specifically, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1, which is a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968), is expressed in place of the IL-6R protein represented by SEQ ID NO: 1. Purified native IL-6R protein can also be used as a sensitizing antigen.

[0074] The purified IL-6R protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. Alternatively, it can be obtained by incorporating a portion of the IL-6R gene into an expression vector and expressing it. It can also be obtained by degrading the IL-6R protein using a protease. However, the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. A preferred region can be any sequence selected from the amino acid sequence corresponding to amino acids 20-357 in the amino acid sequence of SEQ ID NO: 1. The number of amino acids constituting the peptide used as a sensitizing antigen is preferably at least 5 or more, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0075] Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook, J et al., Molecular Cloning, 2nd ed., pp. 9:47-9:58 (1989) Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R to be used as a sensitizing antigen and immunization methods using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0076] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0077] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, a common method for immunization is to administer the sensitizing antigen to a mammal by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, and optionally mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. A suitable carrier can also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0078] Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the immunized animal's body, thereby conferring immune stimulation. Compared to general immunization methods in which a protein antigen is administered to the immunized animal, DNA immunization is expected to have the following advantages: -Maintaining the structure of membrane proteins such as IL-6R can provide immune stimulation -No need to purify the immunogen

[0079] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing the IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. Commercially available expression vectors, such as pcDNA3.1, can be suitably used as the expression vector. Commonly used methods can be used to administer the vector to a living body. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of an animal to be immunized using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0080] After a mammal is immunized in this manner and an increase in the serum titer of an antibody that binds to IL-6R is confirmed, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells.

[0081] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells deficient in HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue DNA synthesis by utilizing the salvage pathway of normal cells, allowing them to grow even in HAT selective medium.

[0082] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in selective media. Another selectable marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines suitable for cell fusion are known.

[0083] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), and S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be suitably used.

[0084] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, the cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), with the addition of an adjuvant such as dimethyl sulfoxide, if desired, to further enhance the fusion efficiency.

[0085] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, the ratio of immune cells to myeloma cells is preferably 1 to 10. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium suitable for growing the myeloma cell line, and may further be suitably supplemented with serum supplements such as fetal calf serum (FCS).

[0086] For cell fusion, predetermined amounts of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., an average molecular weight of approximately 1000 to 6000) preheated to approximately 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, an appropriate culture medium such as those listed above is successively added, and the mixture is centrifuged and the supernatant is removed. This procedure is repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

[0087] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued using the HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0088] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. Culture in the above HAT medium is continued for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody can then be screened and single-cell cloned by the conventional limiting dilution method.

[0089] Screening and monocloning of the desired antibody can be suitably carried out by known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that analyzes cells contacted with a fluorescent antibody using laser light and measures the fluorescence emitted by individual cells, thereby enabling measurement of antibody binding to the cell surface.

[0090] To screen for hybridomas producing the monoclonal antibodies of the present invention by FACS, first, cells expressing IL-6R are prepared. Preferred cells for screening are mammalian cells overexpressing IL-6R. By using non-transformed mammalian cells as a control host cell, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, hybridomas producing IL-6R monoclonal antibodies can be obtained by selecting hybridomas producing antibodies that do not bind to host cells but bind to cells overexpressing IL-6R.

[0091] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody capable of binding to the antigen and are selected by these screening methods can be cloned by limiting dilution or other methods.

[0092] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a conventional culture medium and can be stored for a long period of time in liquid nitrogen.

[0093] The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be isolated from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be isolated from the ascites. The former method is suitable for obtaining highly purified antibodies.

[0094] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody genes are incorporated into an appropriate vector and introduced into a host, whereby the antibodies encoded by the genes are expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0095] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells that produce the anti-IL-6R antibody. To do this, total RNA is usually first extracted from the hybridoma. The following methods can be used to extract mRNA from cells. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0096] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or similar. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are commercially available. Using such kits, mRNA can be isolated from hybridomas. cDNA encoding antibody V regions can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or similar. Alternatively, the SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002; Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be used appropriately for cDNA synthesis and amplification. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites, as described below, can be introduced at both ends of the cDNA.

[0097] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0098] A convenient way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers specifically designed for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

[0099] The resulting 5'-RACE cDNA library is used as a template for PCR amplification of antibody genes. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is recommended that the subclass be determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0100] Specifically, for example, when the goal is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains can be used. To amplify IgG variable region genes, the 3' primer generally anneals to a region corresponding to the constant region close to the variable region. Meanwhile, the 5' primer used is a primer included in the 5' RACE cDNA library construction kit.

[0101] The PCR products thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The desired antibodies can be screened using the binding activity of the reconstituted immunoglobulins to IL-6R as an indicator. For example, when the goal is to obtain antibodies against IL-6R, it is more preferable that the antibodies bind to IL-6R specifically. Antibodies that bind to IL-6R can be screened, for example, as follows: (1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with an IL-6R-expressing cell; (2) detecting the binding of the antibody to the IL-6R-expressing cells; and (3) A step of selecting an antibody that binds to IL-6R-expressing cells.

[0102] Methods for detecting the binding of an antibody to IL-6R-expressing cells are known. Specifically, the binding of an antibody to IL-6R-expressing cells can be detected by techniques such as the above-mentioned FACS. Fixed specimens of IL-6R-expressing cells can be used as appropriate to evaluate the binding activity of an antibody.

[0103] Panning methods using phage vectors are also suitable for screening antibodies using binding activity as an index. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening methods using phage vectors are advantageous. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form single-chain Fvs (scFvs). Phages expressing scFvs on their surface can be obtained by inserting a gene encoding an scFv into a phage vector. After contacting this phage with a desired antigen, DNA encoding an scFv with the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this procedure as necessary, scFvs with the desired binding activity can be enriched.

[0104] After obtaining cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear infrequently in the nucleotide sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, it is preferable to insert a restriction enzyme that generates cohesive ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by fusing a gene encoding the antibody constant region (C region) with a gene encoding the V region in frame. Here, a chimeric antibody refers to an antibody in which the constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusion in-frame of both DNAs digested with the same combination of restriction enzymes.

[0105] To produce an anti-IL-6R monoclonal antibody, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. In the Examples described below, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Next, appropriate host cells are transformed with this expression vector to obtain recombinant cells expressing DNA encoding the anti-IL-6R antibody.

[0106] For antibody gene expression, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) are incorporated into separate expression vectors. By co-transfecting the same host cells with vectors incorporating the H chain and L chain, antibody molecules comprising both H and L chains can be expressed. Alternatively, host cells can be transformed by incorporating DNA encoding the H chain and L chain into a single expression vector (see International Publication WO 1994 / 011523).

[0107] Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into a suitable host. All of these expression systems can be applied to isolating the antigen-binding domains of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specific examples of animal cells include the following: (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, HEK (human embryonic kidney) 293, etc. (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0108] Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. Callus cultured cells can be appropriately used for transformation of plant cells.

[0109] Furthermore, the following fungal cells can be used: - Yeast: Saccharomyces genus such as Saccharomyces cerevisiae, Pichia genus such as Pichia pastoris -Filamentous fungi: Aspergillus genus, such as Aspergillus niger

[0110] Expression systems for antibody genes using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be used as appropriate. An expression vector containing the antibody gene of interest is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody can be obtained from the culture of the transformed cells.

[0111] In addition to the host cells described above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein specifically produced in milk. Examples of proteins secreted into milk include goat beta-casein. A DNA fragment containing a fusion gene with an antibody gene inserted therein is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The transgenic goat (or its offspring) born to the goat that received the embryo produces milk from which the desired antibody can be obtained as a fusion protein with a milk protein. Furthermore, hormones can be administered to transgenic goats to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0112] When the polypeptide complexes described herein are administered to humans, the antigen-binding domain in the complex can be appropriately derived from an artificially modified recombinant antibody with the aim of reducing heterologous antigenicity to humans. Examples of recombinant antibodies include humanized antibodies. These modified antibodies can be appropriately produced using known methods.

[0113] The antibody variable regions used to prepare the antigen-binding domains of the polypeptide complexes described herein typically consist of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs essentially determine the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. However, the amino acid sequences comprising FRs often show high identity even among antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of one antibody can be transferred to another antibody by CDR grafting.

[0114] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies in which CDRs from non-human animals, such as mouse antibodies, are grafted onto human antibodies are well known. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting mouse antibody CDRs onto human FRs. In overlap extension PCR, a nucleotide sequence encoding the mouse antibody CDR to be grafted is added to a primer for synthesizing the human antibody FR. Primers are prepared for each of the four FRs. In general, when grafting mouse CDRs onto human FRs, selecting human FRs that are highly identical to the mouse FRs is considered advantageous in terms of maintaining CDR function. In other words, it is generally preferable to use human FRs whose amino acid sequences are highly identical to the amino acid sequences of the FRs adjacent to the mouse CDR to be grafted.

[0115] The nucleotide sequences to be linked are designed to be connected in frame with each other. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is added to each FR. The nucleotide sequences encoding the mouse CDRs of each product are designed to overlap with each other. Next, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to each other to perform complementary strand synthesis. This reaction links the human FRs via the mouse CDR sequences.

[0116] The V region gene, in which three CDRs and four FRs are finally linked, is amplified in its entirety using primers that anneal to the 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A humanized antibody expression vector can be constructed by inserting the DNA obtained as described above and DNA encoding a human antibody C region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, resulting in the production of the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400 and International Publication WO 1996 / 002576).

[0117] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, it is possible to suitably select FRs of a human antibody that form a good antigen-binding site with the CDRs when linked via the CDRs. If necessary, amino acid residues in the FRs can be substituted so that the CDRs of a reshaped human antibody form a suitable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FRs by applying the PCR method used to graft mouse CDRs onto human FRs. Specifically, partial nucleotide sequence mutations can be introduced into primers annealing to the FRs. Nucleotide sequence mutations are introduced into the FRs synthesized using such primers. By measuring and evaluating the antigen-binding activity of mutant antibodies with amino acid substitutions using the above method, mutant FR sequences with desired properties can be selected (Cancer Res., (1993) 53, 851-856).

[0118] Alternatively, transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) can be used as immunized animals to obtain desired human antibodies by DNA immunization.

[0119] Furthermore, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv) by phage display. Phages expressing scFvs that bind to an antigen can be selected. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the genes of the selected phage. After determining the DNA sequence of the scFv that binds to the antigen, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell such as those listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0120] In addition to the above, methods for obtaining antibody genes include B cell cloning techniques (such as identification and cloning of the coding sequence of each antibody, isolation thereof, and use to construct expression vectors for producing each antibody (particularly IgG1, IgG2, IgG3, or IgG4)) as described in Bernasconi et al. (Science (2002) 298, 2199-2202) or WO2008 / 081008.

[0121] EU numbering and Kabat numbering According to the method used in the present invention, the amino acid positions assigned to the CDRs and FRs of an antibody are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). Herein, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids in the variable regions are represented according to the Kabat numbering, and the amino acids in the constant regions are represented according to the EU numbering based on the Kabat amino acid positions.

[0122] Ion concentration conditions Metal ion concentration conditions In one embodiment of the present invention, the ion concentration refers to the metal ion concentration. "Metal ions" refer to ions of elements belonging to Group I, such as alkali metals and copper group elements excluding hydrogen, Group II, such as alkaline earth metals and zinc group elements, Group III, such as boron, Group IV, such as carbon and silicon, Group VIII, such as iron group elements and platinum group elements, and each of the A subgroups of Groups V, VI, and VII, as well as metal elements such as antimony, bismuth, and polonium. Metal atoms have the property of releasing valence electrons to become cations, which is called ionization tendency. Metals with a high ionization tendency are considered to be chemically active.

[0123] An example of a suitable metal ion in the present invention is calcium ion. Calcium ions are involved in the regulation of many biological phenomena, including muscle contraction (e.g., skeletal muscle, smooth muscle, cardiac muscle), activation of leukocytes (e.g., motility and phagocytosis), activation of platelets (e.g., deformation and secretion), activation of lymphocytes, activation of mast cells (e.g., histamine secretion), cell responses mediated by catecholamine α receptors and acetylcholine receptors, exocytosis, release of transmitters from neuronal terminals, and axonal flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and myosin light chain, which have multiple calcium ion-binding sites and are thought to have originated from a common molecular evolutionary origin, and many of their binding motifs are also known. Well-known examples include the cadherin domain, the EF hand found in calmodulin, the C2 domain found in protein kinase C, the Gla domain found in the blood coagulation protein Factor IX, the C-type lectins found in the asialoglycoprotein receptor and mannose-binding receptor, the A domain found in the LDL receptor, annexin, the thrombospondin type 3 domain, and the EGF-like domain.

[0124] In the present invention, when the metal ion is a calcium ion, calcium ion concentration conditions include low and high calcium ion concentrations. "The binding activity changes depending on the calcium ion concentration" refers to the change in antigen-binding activity of an antigen-binding molecule due to the difference between low and high calcium ion concentrations. For example, this may be the case where the antigen-binding activity of an antigen-binding molecule is higher under high calcium ion concentrations than under low calcium ion concentrations. Another example is where the antigen-binding activity of an antigen-binding molecule is higher under low calcium ion concentrations than under high calcium ion concentrations.

[0125] As used herein, a high calcium ion concentration is not limited to a specific numerical value, but may preferably be a concentration selected from the range of 100 μM to 10 mM. In another embodiment, it may be a concentration selected from the range of 200 μM to 5 mM. In a different embodiment, it may be a concentration selected from the range of 400 μM to 3 mM, and in another embodiment, it may be a concentration selected from the range of 200 μM to 2 mM. It may also be a concentration selected from the range of 400 μM to 1 mM. In particular, a concentration selected from the range of 500 μM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood) in vivo, is preferred.

[0126] As used herein, a low calcium ion concentration is not limited to a specific numerical value, but may preferably be a concentration selected from the range of 0.1 μM to 30 μM. In another embodiment, it may be a concentration selected from the range of 0.2 μM to 20 μM. In a different embodiment, it may be a concentration selected from the range of 0.5 μM to 10 μM, and in another embodiment, it may be a concentration selected from the range of 1 μM to 5 μM. Furthermore, it may be a concentration selected from the range of 2 μM to 4 μM. In particular, a concentration selected from the range of 1 μM to 5 μM, which is close to the ionized calcium concentration in early endosomes in vivo, is preferred.

[0127] In the present invention, "the antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration" means that the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 0.1 μM to 30 μM is weaker than that at a calcium ion concentration selected from the range of 100 μM to 10 mM. Preferably, the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 0.5 μM to 10 μM is weaker than that at a calcium ion concentration selected from the range of 200 μM to 5 mM. Particularly preferably, the antigen-binding activity at a calcium ion concentration in early endosomes in vivo is weaker than that at a calcium ion concentration in plasma in vivo. Specifically, the antigen-binding activity of an antigen-binding molecule at a calcium ion concentration selected from the range of 1 μM to 5 μM is weaker than that at a calcium ion concentration selected from the range of 500 μM to 2.5 mM.

[0128] Whether or not the antigen-binding activity of an antigen-binding molecule changes depending on the metal ion concentration can be determined by using known measurement methods, such as those described above in the section on binding activity. For example, to confirm that the antigen-binding activity of an antigen-binding molecule changes more significantly under high calcium ion concentrations than under low calcium ion concentrations, the antigen-binding activities of the antigen-binding molecule under low and high calcium ion concentrations are compared.

[0129] Furthermore, in the present invention, the expression "the antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration" can also be expressed as "the antigen-binding activity of an antigen-binding molecule at a high calcium ion concentration is higher than that at a low calcium ion concentration." In the present invention, "the antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration" can also be expressed as "the antigen-binding ability at a low calcium ion concentration is weaker than that at a high calcium ion concentration," and "the antigen-binding activity at a low calcium ion concentration is reduced compared to that at a high calcium ion concentration" can also be expressed as "the antigen-binding ability at a low calcium ion concentration is weakened compared to that at a high calcium ion concentration."

[0130] Conditions other than calcium ion concentration when measuring antigen-binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, measurement can be performed under conditions of HEPES buffer and 37°C. For example, measurement can be performed using Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by passing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane-type antigen, the binding activity to the membrane-type antigen can be evaluated by passing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized.

[0131] In the antigen-binding molecules of the present invention, the ratio of antigen-binding activity under low calcium ion concentrations to that under high calcium ion concentrations is not particularly limited, as long as the antigen-binding activity under low calcium ion concentrations is weaker than that under high calcium ion concentrations, but preferably the ratio of the KD (Dissociation constant) for the antigen under low calcium ion concentrations to the KD under high calcium ion concentrations, KD (3 μM Ca) / KD (2 mM Ca), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of KD (3 μM Ca) / KD (2 mM Ca) is not particularly limited, and may be any value, such as 400, 1,000, or 10,000, as long as it can be produced by those skilled in the art.

[0132] As a value of antigen-binding activity, KD (dissociation constant) can be used when the antigen is a soluble antigen, but apparent KD (apparent dissociation constant) can be used when the antigen is a membrane-type antigen. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0133] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an indicator of the ratio between the antigen-binding activity of an antigen-binding molecule of the present invention under low calcium concentration conditions and under high calcium concentration conditions. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of the binding activity ratio, the ratio of kd (dissociation rate constant) for the antigen under low calcium concentration conditions to kd (dissociation rate constant) under high calcium concentration conditions, kd (low calcium concentration condition) / kd (high calcium concentration condition), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. There are no particular upper limits to the value of Kd (low calcium concentration condition) / kd (high calcium concentration condition), and any value, such as 50, 100, or 200, may be used as long as it can be produced within the technical common sense of a person skilled in the art.

[0134] When the antigen is a soluble antigen, kd (dissociation rate constant) can be used as the value of antigen-binding activity, whereas when the antigen is a membrane-type antigen, apparent kd (apparent dissociation rate constant) can be used. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare) or a flow cytometer. In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different calcium ion concentrations, it is preferable to keep all conditions other than the calcium concentration the same.

[0135] For example, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening for antigen-binding domains or antibodies, comprising the following steps (a) to (c): (a) determining the antigen-binding activity of an antigen-binding domain or antibody under a low calcium concentration condition; (b) determining the antigen-binding activity of the antigen-binding domain or antibody under high calcium concentration conditions; (c) selecting an antigen-binding domain or antibody whose antigen-binding activity at a low calcium concentration is lower than that at a high calcium concentration.

[0136] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies or a library thereof, comprising the following steps (a) to (c): (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under a high calcium concentration condition; (b) placing the antigen-binding domain or antibody bound to the antigen in step (a) under low calcium concentration conditions; (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0137] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under low calcium concentration conditions; (b) selecting antigen-binding domains or antibodies that do not bind to the antigen in step (a); (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to an antigen under high calcium concentration conditions; (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0138] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains or antibodies with a column onto which an antigen has been immobilized under high calcium concentration conditions; (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under low calcium concentration conditions; (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0139] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) passing a library of antigen-binding domains or antibodies through a column onto which an antigen is immobilized under low calcium concentration conditions; (b) recovering the antigen-binding domain or antibody that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain or antibody recovered in step (b) to bind to an antigen under high calcium concentration conditions; (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0140] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration is lower than that at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under high calcium concentration conditions; (b) obtaining the antigen-binding domain or antibody that bound to the antigen in step (a); (c) placing the antigen-binding domain or antibody obtained in step (b) under low calcium concentration conditions; (d) isolating antigen-binding domains or antibodies whose antigen-binding activity in step (c) is weaker than the criterion selected in step (b).

[0141] The above steps may be repeated two or more times. Thus, the present invention provides antigen-binding domains or antibodies whose antigen-binding activity at low calcium ion concentrations is lower than that at high calcium ion concentrations, obtained by the above-mentioned screening methods, which further include repeating steps (a) to (c) or (a) to (d) two or more times. The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is typically within 10 times.

[0142] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody under low calcium concentration conditions is not particularly limited, as long as it is an antigen-binding activity at an ionized calcium concentration of 0.1 μM to 30 μM, with a preferred ionized calcium concentration being 0.5 μM to 10 μM. A more preferred ionized calcium concentration is the ionized calcium concentration in early endosomes in vivo, specifically 1 μM to 5 μM. Furthermore, the antigen-binding activity of an antigen-binding domain or antibody under high calcium concentration conditions is not particularly limited, as long as it is an antigen-binding activity at an ionized calcium concentration of 100 μM to 10 mM, with a preferred ionized calcium concentration being 200 μM to 5 mM. A more preferred ionized calcium concentration is the ionized calcium concentration in plasma in vivo, specifically 0.5 mM to 2.5 mM.

[0143] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art, and conditions other than ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), kd (Dissociation rate constant), or apparent kd (Apparent dissociation rate constant), etc. These can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plots, FACS, etc.

[0144] In the present invention, the step of selecting an antigen-binding domain or antibody whose antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition is the same as the step of selecting an antigen-binding domain or antibody whose antigen-binding activity under a low calcium concentration condition is lower than that under a high calcium concentration condition.

[0145] As long as the antigen-binding activity under a high calcium concentration is higher than that under a low calcium concentration, the difference between the antigen-binding activity under a high calcium concentration and that under a low calcium concentration is not particularly limited; however, the antigen-binding activity under a high calcium concentration is preferably at least 2 times, more preferably at least 10 times, and even more preferably at least 40 times that under a low calcium concentration.

[0146] The antigen-binding domains or antibodies of the present invention to be screened by the above-mentioned screening methods may be any antigen-binding domains or antibodies, and it is possible to screen, for example, the antigen-binding domains or antibodies described above. For example, antigen-binding domains or antibodies having native sequences may be screened, or antigen-binding domains or antibodies with substituted amino acid sequences may be screened.

[0147] Library According to one embodiment, the antigen-binding domains or antibodies of the present invention can be obtained from a library mainly consisting of multiple antigen-binding molecules with different sequences, each of which contains at least one amino acid residue in its antigen-binding domain that changes the antigen-binding activity of the antigen-binding molecule depending on the ion concentration. Preferred examples of ion concentrations include metal ion concentration and hydrogen ion concentration.

[0148] As used herein, the term "library" refers to multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules, or nucleic acids or polynucleotides encoding these sequences. The sequences of the multiple antigen-binding molecules or multiple fusion polypeptides containing antigen-binding molecules contained in a library are not a single sequence, but rather are antigen-binding molecules or fusion polypeptides containing antigen-binding molecules with sequences that differ from one another.

[0149] As used herein, the term "different sequences" in the description of multiple antigen-binding molecules with different sequences means that the sequences of the individual antigen-binding molecules in the library are different from each other. In other words, the number of different sequences in the library reflects the number of independent clones with different sequences in the library, and is sometimes referred to as the "library size." In a typical phage display library, 10 6 From 10 12 By applying known techniques such as ribosome display, the library size can be increased to 10 14However, the actual number of phage particles used in panning selection of a phage library is usually 10 to 10,000 times larger than the library size. This excess, also called the "library equivalent number," indicates that there may be 10 to 10,000 individual clones with the same amino acid sequence. Therefore, the term "different in sequence from each other" in the present invention means that the sequences of the individual antigen-binding molecules in the library, excluding the library equivalent number, are different from each other, more specifically, there may be 10 or more antigen-binding molecules with different sequences from each other. 6 From 10 14 molecules, preferably 10 7 From 10 12 molecules, more preferably 10 8 From 10 11 , particularly preferably 10 8 From 10 10 It means to exist.

[0150] Furthermore, the term "plurality" in the description of a library of the present invention consisting essentially of a plurality of antigen-binding molecules generally refers to a collection of two or more types of the substance, for example, the antigen-binding molecules, fusion polypeptides, polynucleotide molecules, vectors, or viruses of the present invention. For example, if two or more substances differ from each other in a specific trait, this indicates that there are two or more types of the substance. An example would be variant amino acids observed at specific amino acid positions in an amino acid sequence. For example, if there are two or more antigen-binding molecules of the present invention that have substantially the same, preferably identical, sequences other than flexible residues or specific variant amino acids at highly diverse, surface-exposed amino acid positions, then there are plural antigen-binding molecules of the present invention. In another example, if there are two or more polynucleotide molecules of the present invention that have substantially the same, preferably identical sequences other than bases encoding flexible residues or bases encoding specific variant amino acids at highly diverse, surface-exposed amino acid positions, then there are plural polynucleotide molecules of the present invention.

[0151] Furthermore, the term "mainly consisting of" in the description of a library of the present invention consisting mainly of multiple antigen-binding molecules reflects the number of antigen-binding molecules whose antigen-binding activity varies depending on the ion concentration condition among the number of independent clones with different sequences in the library. Specifically, the term "mainly consisting of" refers to the number of antigen-binding molecules whose antigen-binding activity varies depending on the ion concentration condition among the number of independent clones with different sequences in the library. Specifically, the term "mainly consisting of" refers to the number of antigen-binding molecules whose antigen-binding activity varies depending on the ion concentration condition among the independent clones with different sequences in the library. 4 It is preferable that the antigen-binding domain of the present invention has at least 10 antigen-binding molecules that exhibit such binding activity. 5 More preferably, the antigen-binding domain of the present invention can be obtained from a library containing at least 10 antigen-binding molecules that exhibit such binding activity. 6 Particularly preferably, the antigen-binding domain of the present invention can be obtained from a library containing at least 10 antigen-binding molecules that exhibit such binding activity. 7 Preferably, the antigen-binding domain of the present invention can be obtained from a library containing at least 10 antigen-binding molecules that exhibit such binding activity. 8 The antigen-binding domain of the present invention can be obtained from a library containing antigen-binding molecules present in the library. Alternatively, it can be suitably expressed as the proportion of antigen-binding molecules whose antigen-binding activity varies depending on the ion concentration conditions, among the number of independent clones with different sequences in the library. Specifically, the antigen-binding domain of the present invention can be obtained from a library in which antigen-binding molecules exhibiting such binding activity account for 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, even more preferably 2% to 20%, and particularly preferably 4% to 10% of the number of independent clones with different sequences in the library. Fusion polypeptides, polynucleotide molecules, or vectors can also be expressed as the number of molecules or the proportion of all molecules, as described above. Viruses can also be expressed as the number of virus individuals or the proportion of all individuals, as described above.

[0152] Amino acids that change the binding activity of antigen-binding domains to antigens depending on calcium ion concentration conditions The antigen-binding domains or antibodies of the present invention to be screened by the above-described screening methods may be prepared in any manner. For example, when the metal ion is calcium ion concentration, it is possible to use pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, or antibodies or libraries in which amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations have been introduced into these antibodies or libraries (libraries with an increased content of amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acids, libraries in which amino acids capable of chelating calcium (e.g., aspartic acid or glutamic acid) or unnatural amino acid mutations have been introduced at specific sites, etc.).

[0153] As described above, when the metal ion is a calcium ion, examples of amino acids that change the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions include any amino acid that forms a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al. (Cell (2000) 102, 275-277), Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490), Moncrief et al. (J. Mol. Evol. (1990) 30, 522-562), Chauvaux et al. (Biochem. J. (1990) 265, 261-265), Bairoch and Cox (FEBS Lett. (1990) 269, 454-456), Davis (New Biol. (1990) 2, 410-419), Schaefer et al. (Genomics (1995) 25, 638-643), Economou et al. (EMBO J. (1990) 9, 349-354), Wurzburg et al. (Structure. (2006) 14, 6, 1049-1058)). That is, any known calcium-binding motif, such as that of ASGPR, CD23, MBR, or C-type lectins such as DC-SIGN, can be contained in the antigen-binding molecules of the present invention. In addition to the above, preferred examples of such calcium-binding motifs include the calcium-binding motif contained in the antigen-binding domain of SEQ ID NO: 4.

[0154] Furthermore, amino acids that change the antigen-binding activity of antigen-binding molecules depending on calcium ion concentration conditions can also be suitably used, including amino acids with metal chelating activity, such as serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)), and glutamic acid (Glu(E)).

[0155] The position of the antigen-binding domain containing the amino acid is not limited to a specific position and can be any position in the heavy chain variable region or light chain variable region that forms the antigen-binding domain, as long as the antigen-binding activity of the antigen-binding molecule is changed depending on the calcium ion concentration. That is, the antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain antigen-binding domain contains an amino acid that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain CDR3 contains the amino acid. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, in which the heavy chain CDR3 contains the amino acid at positions 95, 96, 100a, and / or 101, as defined by the Kabat numbering system, in the heavy chain CDR3.

[0156] Furthermore, in one embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains an amino acid in its light chain antigen-binding domain that changes the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration. In another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains the amino acid in its light chain CDR1. In other embodiments, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences, each of which contains the amino acid at positions 30, 31, and / or 32, as defined by the Kabat numbering system, in light chain CDR1.

[0157] In another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences that contain the amino acid residue in light chain CDR2. In another embodiment, a library is provided that mainly consists of antigen-binding molecules with different sequences that contain the amino acid residue at position 50 (Kabat numbering) in light chain CDR2.

[0158] In yet another embodiment, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences containing the amino acid residue at position 92 (Kabat numbering) in the light chain CDR3.

[0159] In another embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in two or three CDRs selected from the above-described light chain CDR1, CDR2, and CDR3. Furthermore, antigen-binding domains of the present invention can be obtained from a library mainly composed of antigen-binding molecules with different sequences in which the amino acid residue is contained in any one or more of positions 30, 31, 32, 50, and / or 92 according to the Kabat numbering system in the light chain.

[0160] In a particularly preferred embodiment, it is desirable that the framework sequences of the light chain and / or heavy chain variable regions of the antigen-binding molecule have human germline framework sequences. Therefore, in one aspect of the present invention, if the framework sequences are completely human sequences, the antigen-binding molecules of the present invention are expected to induce little or no immunogenic response when administered to humans (e.g., for the treatment of a disease). In this sense, "comprising a germline sequence" of the present invention means that a portion of the framework sequence of the present invention is identical to a portion of any human germline framework sequence. For example, an antigen-binding molecule of the present invention "comprising a germline sequence" is also an antigen-binding molecule of the present invention in which the heavy chain FR2 sequence is a combination of heavy chain FR2 sequences from multiple different human germline framework sequences.

[0161] Suitable examples of frameworks include currently known fully human framework region sequences included on websites such as V-Base (http: / / vbase.mrc-cpe.cam.ac.uk / ). These framework region sequences can be appropriately used as germline sequences contained in the antigen-binding molecules of the present invention. Germline sequences can be classified based on their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798), Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461), and Cox et al. (Nat. Genetics (1994) 7, 162-168)). Suitable germline sequences can be appropriately selected from Vκ, which are classified into seven subgroups, Vλ, which are classified into ten subgroups, and VH, which are classified into seven subgroups.

[0162] Fully human VH sequences include, but are not limited to, sequences from the VH1 subgroup (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1-69), the VH2 subgroup (e.g., VH2-5, VH2-26, VH2-70), the VH3 subgroup (e.g., VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-3), and the VH4 subgroup (e.g., VH4-5, VH4-6, VH4-70, VH4-8, VH4-9, VH4-10, VH4-11, VH4-13, VH4-15, VH4-16, VH4-20, VH4-21, VH4-23, VH4-3). Suitable examples of VH sequences include those of the VH4 subgroup (VH4-0, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, VH3-74), the VH4 subgroup (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, VH4-61), the VH5 subgroup (VH5-51), the VH6 subgroup (VH6-1), and the VH7 subgroup (VH7-4, VH7-81). These are also described in known literature (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)), and those skilled in the art can appropriately design antigen-binding molecules of the present invention based on this sequence information. Completely human frameworks or framework subregions other than these can also be suitably used.

[0163] Fully human Vk sequences include, but are not limited to, A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, and O18, which are classified into the Vk1 subgroup, and A1, A2, A3, A5, A7, A17, and A19, which are classified into the Vk2 subgroup. Preferred examples thereof include 18, A19, A23, O1, O11, A11, A27, L2, L6, L10, L16, L20, and L25, which are classified into the Vk3 subgroup, B3, which is classified into the Vk4 subgroup, B2, which is classified into the Vk5 subgroup (also referred to as Vk5-2 in this specification), and A10, A14, and A26, which are classified into the Vk6 subgroup (Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028), Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022), and Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

[0164] Completely human VL sequences include, but are not limited to, V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, and V1-22, which are classified into the VL1 subgroup; V2-1, V2-6, V2-7, and V2-8, which are classified into the VL1 subgroup; Preferred examples thereof include V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19; ​​V3-2, V3-3, and V3-4, which are classified into the VL3 subgroup; V4-1, V4-2, V4-3, V4-4, and V4-6, which are classified into the VL4 subgroup; and V5-1, V5-2, V5-4, and V5-6, which are classified into the VL5 subgroup (Kawasaki et al., Genome Res. (1997) 7, 250-261).

[0165] Typically, these framework sequences differ from each other by one or more amino acid residues. These framework sequences can be used together with "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions" of the present invention. Other examples of fully human frameworks that can be used together with "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on ion concentration conditions" of the present invention include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY, POM, etc. (e.g., Kabat et al. (1991) and Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

[0166] Although the present invention is not bound by any particular theory, it is believed that one reason the use of germline sequences is expected to eliminate adverse immune responses in most individuals is that somatic mutations frequently occur in the variable regions of immunoglobulins as a result of the affinity maturation step that occurs during a normal immune response. These mutations occur primarily around the CDRs, whose sequences are hypervariable, but also affect residues in the framework regions. These framework mutations are absent from germline genes and are unlikely to be immunogenic in patients. On the other hand, the normal human population is exposed to the majority of framework sequences expressed by germline genes, and as a result of immune tolerance, these germline frameworks are expected to be less immunogenic or non-immunogenic in patients. To maximize the likelihood of immune tolerance, genes encoding the variable regions can be selected from a commonly present set of functional germline genes.

[0167] To prepare antigen-binding molecules of the present invention in which the framework sequence contains amino acids that change the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration conditions, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used.

[0168] For example, a library containing a plurality of antigen-binding molecules of the present invention with different sequences can be prepared by combining a light chain variable region selected as a framework sequence that previously contains at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration with a heavy chain variable region prepared as a randomized variable region sequence library. A non-limiting example of such a library, when the ion concentration is calcium ion concentration, is a library that combines the light chain variable region sequence set forth in SEQ ID NO: 4 (Vk5-2) with a heavy chain variable region prepared as a randomized variable region sequence library.

[0169] Furthermore, the light chain variable region sequence selected as a framework sequence already containing at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration can be designed to contain various amino acids as residues other than the amino acid residue. In the present invention, such residues are referred to as flexible residues. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the ion concentration, the number and position of the flexible residues are not limited to a particular embodiment. That is, one or more flexible residues may be contained in the CDR sequence and / or FR sequence of the heavy chain and / or light chain. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence of SEQ ID NO: 4 (Vk5-2) include the amino acid residues listed in Table 1 or Table 2.

[0170] [Table 1]

[0171] [Table 2]

[0172] As used herein, "flexible residues" refers to amino acid residue variations present at positions in the light and heavy chain variable regions where amino acids are highly diverse when comparing the amino acid sequences of known and / or natural antibodies or antigen-binding domains, with several different amino acids present at that position. Highly diverse positions are typically present in the CDR regions. In one embodiment, data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md.) (1987 and 1991) are useful for determining highly diverse positions in known and / or natural antibodies. Additionally, several databases on the Internet (http: / / vbase.mrc-cpe.cam.ac.uk / , http: / / www.bioinf.org.uk / abs / index.html) provide collected sequences and their arrangements of numerous human light and heavy chains. Information on these sequences and their arrangements is useful for determining highly diverse positions in the present invention. According to the present invention, an amino acid position is said to be highly diverse if it has a diversity of preferably about 2 to about 20, preferably about 3 to about 19, preferably about 4 to about 18, preferably 5 to 17, preferably 6 to 16, preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, preferably 10 to 12 possible different amino acid residues at that position. In some embodiments, an amino acid position may have a diversity of preferably at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, preferably about 12 possible different amino acid residues.

[0173] Furthermore, a library containing a plurality of antigen-binding molecules of the present invention with different sequences can also be prepared by combining a light chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the ion concentration conditions has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library. A non-limiting example of such a library, when the ion concentration is calcium ion concentration, is a library that combines a light chain variable region sequence in which a specific germline residue, such as SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), or SEQ ID NO: 8 (Vk4), has been substituted with at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the calcium ion concentration, with a heavy chain variable region prepared as a randomized variable region sequence library. Non-limiting examples of such amino acid residues include amino acid residues contained in the light chain CDR1. Other non-limiting examples of such amino acid residues include amino acid residues contained in the light chain CDR2. Further, other non-limiting examples of such amino acid residues include amino acid residues contained in the CDR3 of the light chain.

[0174] As described above, non-limiting examples of amino acid residues contained in the light chain CDR1 include the amino acid residues at positions 30, 31, and / or 32 according to EU numbering in the CDR1 of the light chain variable region. A non-limiting example of an amino acid residue contained in the light chain CDR2 includes the amino acid residue at position 50 according to Kabat numbering in the CDR2 of the light chain variable region. A non-limiting example of an amino acid residue contained in the light chain CDR3 includes the amino acid residue at position 92 according to Kabat numbering in the CDR3 of the light chain variable region. These amino acid residues may be contained alone or in combination, as long as they form a calcium-binding motif and / or the antigen-binding activity of the antigen-binding molecule changes depending on calcium ion concentration. Troponin C, calmodulin, parvalbumin, myosin light chain, and the like are known to have multiple calcium ion-binding sites and are thought to have originated from a common origin in molecular evolution, and it is also possible to design light chain CDR1, CDR2, and / or CDR3 to contain these binding motifs. For example, for the above purpose, a cadherin domain, an EF hand contained in calmodulin, a C2 domain contained in protein kinase C, a Gla domain contained in the blood coagulation protein Factor IX, C-type lectins contained in asialoglycoprotein receptors and mannose-binding receptors, an A domain contained in LDL receptors, annexins, thrombospondin type 3 domains, and EGF-like domains can be appropriately used.

[0175] Even when a light chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the ion concentration conditions is introduced is combined with a heavy chain variable region prepared as a randomized variable region sequence library, flexible residues can be designed to be included in the sequence of the light chain variable region, as described above. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the ion concentration conditions, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues can be included in the CDR sequences and / or FR sequences of the heavy and / or light chains. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence include the amino acid residues listed in Table 1 or Table 2.

[0176] A suitable example of a heavy chain variable region to be combined is a randomized variable region library. A randomized variable region library can be prepared by appropriately combining known methods. In a non-limiting aspect of the present invention, an immune library constructed from antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infectious diseases or humans with increased blood antibody titers after vaccination, cancer patients, or patients with autoimmune diseases can be suitably used as a randomized variable region library.

[0177] In a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of V genes in genomic DNA or reconstructed functional V genes are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of appropriate length can also be used as a randomized variable region library. In this case, since diversity in the gene sequences of heavy chain CDR3 is observed, it is also possible to replace only the CDR3 sequence. The criterion for generating amino acid diversity in the variable regions of antigen-binding molecules is to provide diversity to amino acid residues at surface-exposed positions of the antigen-binding molecule. A surface-exposed position refers to a position that is determined to be surface-exposed and / or capable of contacting an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and is generally the CDR. Preferably, the surface-exposed position is determined using coordinates from a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface-exposed positions can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. Suitable software available for this purpose includes the SYBYL Biopolymer Module software (Tripos Associates). Generally, and preferably, when an algorithm requires user-input size parameters, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less. Furthermore, methods for determining surface exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1, 46-53).

[0178] Furthermore, in a non-limiting embodiment of the present invention, a naive library consisting of naive sequences, which are antibody sequences constructed from antibody genes derived from lymphocytes of healthy individuals and whose repertoire is unbiased, can also be particularly preferably used as a randomized variable region library (Gejima et al., Human Antibodies (2002) 11, 121-129, and Cardoso et al., Scand. J. Immunol. (2000) 51, 337-344). The amino acid sequence comprising a naive sequence described in the present invention refers to an amino acid sequence obtained from such a naive library.

[0179] In one embodiment of the present invention, antigen-binding domains of the present invention can be obtained from a library containing multiple antigen-binding molecules of the present invention with different sequences by combining a heavy chain variable region selected as a framework sequence that previously contains "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions" with a light chain variable region prepared as a randomized variable region sequence library. A non-limiting example of such a library, in which the ion concentration is calcium ion concentration, is a library that combines the heavy chain variable region sequence set forth in SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1) with a light chain variable region prepared as a randomized variable region sequence library. Alternatively, instead of the light chain variable region prepared as a randomized variable region sequence library, a library can be prepared by appropriately selecting from light chain variable regions with germline sequences. A suitable example is a library that combines the heavy chain variable region sequence set forth in SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1) with a light chain variable region having a germline sequence.

[0180] Furthermore, flexible residues can be included in the heavy chain variable region sequence selected as a framework sequence that already contains the aforementioned "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions." The number and location of the flexible residues are not limited to a particular embodiment, as long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on ion concentration conditions. That is, one or more flexible residues can be included in the heavy chain and / or light chain CDR sequences and / or FR sequences. For example, when the ion concentration is calcium ion concentration, non-limiting examples of flexible residues to be introduced into the heavy chain variable region sequence of SEQ ID NO: 9 (6RL#9-IgG1) include all amino acid residues in heavy chain CDR1 and CDR2, as well as amino acid residues in heavy chain CDR3 other than positions 95, 96, and / or 100a. Alternatively, non-limiting examples of flexible residues that may be introduced into the heavy chain variable region sequence set forth in SEQ ID NO: 10 (6KC4-1#85-IgG1) include all amino acid residues in heavy chain CDR1 and CDR2, as well as amino acid residues in heavy chain CDR3 other than positions 95 and / or 101.

[0181] Furthermore, a library containing multiple antigen-binding molecules with different sequences can also be prepared by combining a heavy chain variable region into which the aforementioned "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions" has been introduced with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence. A non-limiting example of such a library, when the ion concentration is calcium ion concentration, is a library that combines a heavy chain variable region sequence in which a specific residue in the heavy chain variable region has been substituted with at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentration conditions, with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence. Non-limiting examples of such amino acid residues include amino acid residues contained in the heavy chain CDR1. Other non-limiting examples of such amino acid residues include amino acid residues contained in the heavy chain CDR2. Another non-limiting example of such amino acid residues includes amino acid residues contained in the heavy chain CDR3. Non-limiting examples of such amino acid residues contained in the heavy chain CDR3 include amino acids at positions 95, 96, 100a, and / or 101 according to the Kabat numbering system in the CDR3 of the heavy chain variable region. Furthermore, these amino acid residues may be contained alone or in combination of two or more, as long as they form a calcium-binding motif and / or the antigen-binding activity of the antigen-binding molecule changes depending on calcium ion concentration.

[0182] Even when combining a heavy chain variable region into which at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions is introduced with a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence, as described above, it is possible to design the heavy chain variable region sequence to contain flexible residues. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on ion concentration conditions, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be contained in the heavy chain CDR sequence and / or FR sequence. Furthermore, a randomized variable region library can also be suitably used as the amino acid sequence of CDR1, CDR2, and / or CDR3 of the heavy chain variable region other than the amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on ion concentration conditions. When a germline sequence is used as the light chain variable region, non-limiting examples include germline sequences such as SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), and SEQ ID NO: 8 (Vk4).

[0183] As the amino acid that changes the antigen-binding activity of an antigen-binding molecule depending on calcium ion concentration conditions, any amino acid can be suitably used as long as it forms a calcium-binding motif, and specific examples of such amino acids include electron-donating amino acids, such as serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid.

[0184] Hydrogen ion concentration conditions In one embodiment of the present invention, the ion concentration condition refers to the hydrogen ion concentration condition or the pH condition. In the present invention, the concentration condition of protons, i.e., the atomic nuclei of hydrogen atoms, is also treated as the hydrogen exponent (pH) condition. When the activity of hydrogen ions in an aqueous solution is expressed as aH+, pH is defined as -log10aH+. When the ionic strength in an aqueous solution is (for example, 10 -3If the temperature is lower than 25°C, aH+ is approximately equal to the hydrogen ionic strength. For example, the ionic product of water at 25°C and 1 atmosphere is Kw = aH + aOH = 10 -14 Therefore, in pure water, aH+ = aOH = 10 -7 In this case, a pH of 7 is neutral, an aqueous solution with a pH of less than 7 is acidic, and an aqueous solution with a pH of more than 7 is alkaline.

[0185] In the present invention, when pH conditions are used as ion concentration conditions, pH conditions include high proton concentration or low pH, i.e., acidic pH conditions, and low proton concentration or high pH, ​​i.e., neutral pH conditions. "A change in binding activity depending on pH conditions" refers to a change in the antigen-binding activity of an antigen-binding molecule due to the difference between high proton concentration or low pH (acidic pH range) and low proton concentration or high pH (neutral pH range) conditions. For example, this may be the case when the antigen-binding activity of an antigen-binding molecule is higher in a neutral pH range than in an acidic pH range. Another example may be the case when the antigen-binding activity of an antigen-binding molecule is higher in an acidic pH range than in a neutral pH range.

[0186] As used herein, the neutral pH range is not limited to a specific numerical value, but may preferably be selected from the range of pH 6.7 to pH 10.0. In another embodiment, it may be selected from the range of pH 6.7 to pH 9.5. In a different embodiment, it may be selected from the range of pH 7.0 to pH 9.0, and in another embodiment, it may be selected from the range of pH 7.0 to pH 8.0. In particular, pH 7.4, which is close to the pH in plasma (blood) in vivo, is preferred.

[0187] As used herein, the acidic pH range is not limited to a specific numerical value, but may preferably be selected from the range of pH 4.0 to pH 6.5. In another embodiment, it may be selected from the range of pH 4.5 to pH 6.5. In a different embodiment, it may be selected from the range of pH 5.0 to pH 6.5, and in another embodiment, it may be selected from the range of pH 5.5 to pH 6.5. In particular, a pH of 5.8, which is close to the ionized calcium concentration in early endosomes in vivo, is preferred.

[0188] In the present invention, "the antigen-binding activity of an antigen-binding molecule at a high proton concentration or low pH (acidic pH range) is lower than that at a low proton concentration or high pH (neutral pH range)" means that the antigen-binding activity of the antigen-binding molecule at a pH selected from the range of pH 4.0 to pH 6.5 is weaker than that at a pH selected from the range of pH 6.7 to pH 10.0. Preferably, this means that the antigen-binding activity of the antigen-binding molecule at a pH selected from the range of pH 4.5 to pH 6.5 is weaker than that at a pH selected from the range of pH 6.7 to pH 9.5, and more preferably, that the antigen-binding activity of the antigen-binding molecule at a pH selected from the range of pH 5.0 to pH 6.5 is weaker than that at a pH selected from the range of pH 7.0 to pH 9.0. Preferably, this means that the antigen-binding activity of the antigen-binding molecule at a pH selected from the range of pH 5.5 to pH 6.5 is weaker than that at a pH selected from the range of pH 7.0 to pH 8.0. Particularly preferably, this means that the antigen-binding activity at the pH in early endosomes in vivo is weaker than the antigen-binding activity at the pH in plasma in vivo; specifically, it means that the antigen-binding activity of an antigen-binding molecule at pH 5.8 is weaker than the antigen-binding activity at pH 7.4.

[0189] Whether the antigen-binding activity of an antigen-binding molecule changes depending on pH conditions can be determined, for example, by using known measurement methods such as those described above in the section on binding activity. That is, the binding activity is measured under different pH conditions in the measurement method. For example, to confirm that the antigen-binding activity of an antigen-binding molecule changes more significantly under a neutral pH range than under an acidic pH range, the antigen-binding activities of the antigen-binding molecule under acidic and neutral pH ranges are compared.

[0190] Furthermore, in the present invention, the expression "the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range" can also be expressed as "the antigen-binding activity of an antigen-binding molecule at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than the antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range." In the present invention, "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" may also be expressed as "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is weaker than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range". Similarly, "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is reduced compared to that at a low proton concentration or high pH, ​​i.e., in a neutral pH range" may also be expressed as "antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is weakened than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range".

[0191] Conditions other than hydrogen ion concentration or pH when measuring antigen-binding activity can be appropriately selected by those skilled in the art and are not particularly limited. For example, measurement can be performed under conditions of HEPES buffer and 37°C. Measurement can be performed using, for example, Biacore (GE Healthcare). When the antigen is a soluble antigen, the binding activity to the soluble antigen can be evaluated by passing the antigen as an analyte through a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane-type antigen, the binding activity to the membrane-type antigen can be evaluated by passing the antigen-binding molecule as an analyte through a chip on which the antigen is immobilized.

[0192] In the antigen-binding molecules of the present invention, as long as the antigen-binding activity at a high proton concentration or low pH, i.e., an acidic pH range, is weaker than the antigen-binding activity at a low proton concentration or high pH, ​​i.e., a neutral pH range, the ratio of the antigen-binding activity at a high proton concentration or low pH, i.e., an acidic pH range, to the antigen-binding activity at a low proton concentration or high pH, ​​i.e., a neutral pH range, is not particularly limited. Preferably, the ratio of the KD (Dissociation constant) for the antigen at a high proton concentration or low pH, i.e., an acidic pH range, to the KD at a low proton concentration or high pH, ​​i.e., a neutral pH range, KD (pH5.8) / KD (pH7.4), is 2 or greater, more preferably 10 or greater, and even more preferably 40 or greater. The upper limit of the KD (pH 5.8) / KD (pH 7.4) value is not particularly limited, and may be any value, such as 400, 1000, or 10000, as long as it can be produced by those skilled in the art.

[0193] As a value of antigen-binding activity, KD (dissociation constant) can be used when the antigen is a soluble antigen, but apparent KD (apparent dissociation constant) can be used when the antigen is a membrane-type antigen. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc.

[0194] Alternatively, the dissociation rate constant kd (dissociation rate constant) can also be suitably used as an indicator of the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention at a high proton concentration or low pH, i.e., an acidic pH range, to that at a low proton concentration or high pH, ​​i.e., a neutral pH range. When kd (dissociation rate constant) is used instead of KD (dissociation constant) as an indicator of the binding activity ratio, the ratio of kd (dissociation rate constant) at a high proton concentration or low pH, i.e., an acidic pH range, to kd (dissociation rate constant) at a low proton concentration or high pH, ​​i.e., a neutral pH range, i.e., kd (at an acidic pH range) / kd (at a neutral pH range), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. The upper limit of the value of Kd (in the acidic pH range) / kd (in the neutral pH range) is not particularly limited, and may be any value, such as 50, 100, or 200, as long as it can be produced within the technical common sense of a person skilled in the art.

[0195] When the antigen is a soluble antigen, kd (dissociation rate constant) can be used as the value of antigen-binding activity, whereas when the antigen is a membrane-type antigen, apparent kd (apparent dissociation rate constant) can be used. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare) or a flow cytometer. In the present invention, when measuring the antigen-binding activity of an antigen-binding molecule at different hydrogen ion concentrations, i.e., pH, it is preferable to keep all conditions other than hydrogen ion concentration, i.e., pH, the same.

[0196] For example, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by screening for antigen-binding domains or antibodies, comprising the following steps (a) to (c): (a) obtaining the antigen-binding activity of an antigen-binding domain or antibody under an acidic pH range condition; (b) obtaining the antigen-binding activity of the antigen-binding domain or antibody under a neutral pH condition; (c) selecting an antigen-binding domain or antibody whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range.

[0197] Furthermore, an embodiment of the present invention, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, can be obtained by screening antigen-binding domains or antibodies or a library thereof, comprising the following steps (a) to (c): (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under a neutral pH range condition; (b) placing the antigen-binding domain or antibody bound to the antigen in step (a) under an acidic pH range; (c) isolating the antigen-binding domain or antibody dissociated in step (b).

[0198] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under an acidic pH range; (b) selecting antigen-binding domains or antibodies that do not bind to the antigen in step (a); (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to an antigen in a neutral pH range; (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0199] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (c): (a) contacting a library of antigen-binding domains or antibodies with a column onto which an antigen is immobilized at a neutral pH; (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under an acidic pH condition; (c) isolating the antigen-binding domain or antibody eluted in step (b).

[0200] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) passing a library of antigen-binding domains or antibodies through an antigen-immobilized column under an acidic pH condition; (b) recovering the antigen-binding domain or antibody that did not bind to the column and was eluted in step (a); (c) allowing the antigen-binding domain or antibody recovered in step (b) to bind to an antigen in a neutral pH range; (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0201] Furthermore, an antigen-binding domain or antibody whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than its antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, which is one embodiment of the present invention, can be obtained by a screening method comprising the following steps (a) to (d): (a) contacting a library of antigen-binding domains or antibodies with an antigen under a neutral pH range; (b) obtaining the antigen-binding domain or antibody that bound to the antigen in step (a); (c) placing the antigen-binding domain or antibody obtained in step (b) under an acidic pH condition; (d) isolating antigen-binding domains or antibodies whose antigen-binding activity in step (c) is weaker than the criterion selected in step (b).

[0202] The above steps may be repeated two or more times. Thus, the present invention provides antigen-binding domains or antibodies whose antigen-binding activity in an acidic pH range is lower than that in a neutral pH range, obtained by the above-mentioned screening method, which further comprises repeating steps (a) to (c) or (a) to (d) two or more times. The number of times steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is typically within 10 times.

[0203] In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody under high proton concentration conditions or low pH, i.e., in the acidic pH range, is not particularly limited as long as it is antigen-binding activity between pH 4.0 and 6.5, with a preferred pH being between pH 4.5 and 6.6. Another preferred pH is antigen-binding activity between pH 5.0 and 6.5, with a further preferred pH being between pH 5.5 and 6.5. A more preferred pH is the pH within early endosomes in vivo, specifically, antigen-binding activity at pH 5.8. Furthermore, the antigen-binding activity of an antigen-binding domain or antibody under low proton concentration conditions or high pH, ​​i.e., in the neutral pH range, is not particularly limited as long as it is antigen-binding activity between pH 6.7 and 10, with a preferred pH being between pH 6.7 and 9.5. Another preferred pH is antigen-binding activity between pH 7.0 and 9.5, with a further preferred pH being between pH 7.0 and 8.0. A more preferable pH is the pH in plasma in vivo, specifically, the antigen-binding activity at pH 7.4.

[0204] The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art, and conditions other than ionized calcium concentration can be appropriately determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), kd (Dissociation rate constant), or apparent kd (Apparent dissociation rate constant), etc. These can be measured by methods known to those skilled in the art, such as using Biacore (GE Healthcare), Scatchard plots, FACS, etc.

[0205] In the present invention, the step of selecting antigen-binding domains or antibodies whose antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than that at a high proton concentration or low pH, i.e., in an acidic pH range, means the same as the step of selecting antigen-binding domains or antibodies whose antigen-binding activity at a high proton concentration or low pH, i.e., in an acidic pH range, is lower than that at a low proton concentration or high pH, ​​i.e., in a neutral pH range.

[0206] As long as the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is higher than that at a high proton concentration or low pH, i.e., in an acidic pH range, the difference in antigen-binding activity between a low proton concentration or high pH, ​​i.e., in a neutral pH range, and a high proton concentration or low pH, i.e., in an acidic pH range, is not particularly limited; however, the antigen-binding activity at a low proton concentration or high pH, ​​i.e., in a neutral pH range, is preferably at least 2-fold, more preferably at least 10-fold, and even more preferably at least 40-fold, that at a high proton concentration or low pH, i.e., in an acidic pH range.

[0207] The antigen-binding domains or antibodies of the present invention to be screened by the above-mentioned screening methods may be any antigen-binding domains or antibodies, and it is possible to screen, for example, the antigen-binding domains or antibodies described above. For example, antigen-binding domains or antibodies having native sequences may be screened, or antigen-binding domains or antibodies with substituted amino acid sequences may be screened.

[0208] Antigen-binding domains or antibodies of the present invention to be screened by the above-mentioned screening methods may be prepared in any manner, and examples of such antibodies or libraries include pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, and antibodies or libraries obtained by introducing amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations into these antibodies or libraries (libraries with an increased content of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, and libraries in which amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acid mutations have been introduced at specific sites).

[0209] Antigen-binding domains or antibodies whose antigen-binding activity at low proton concentrations or high pH, ​​i.e., in the neutral pH range, is higher than that at high proton concentrations or low pH, i.e., in the acidic pH range, can be obtained from hybridomas obtained by immunizing animals or B cells from immunized animals by methods suitable for use in the production of such domains or antibodies. Examples of suitable methods include antigen-binding molecules or antibodies in which at least one amino acid in the antigen-binding domain or antibody is substituted with an amino acid with a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid mutation, as described in WO2009 / 125825, or in which an amino acid with a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid is inserted into the antigen-binding domain or antibody.

[0210] The position at which the mutation of an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid is introduced is not particularly limited, and may be any site as long as the antigen-binding activity in the acidic pH range is weaker than that in the neutral pH range compared to before the substitution or insertion (the value of KD(acidic pH range) / KD(neutral pH range) is increased, or the value of kd(acidic pH range) / kd(neutral pH range) is increased). For example, when the antigen-binding molecule is an antibody, suitable sites include the variable region and CDRs of the antibody. The number of amino acids to be substituted with amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0, or the number of amino acids to be inserted, can be determined appropriately by those skilled in the art. A single amino acid (e.g., histidine and glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0 may be substituted, a single amino acid (e.g., histidine and glutamic acid) or unnatural amino acid having a side chain pKa of 4.0-8.0 may be inserted, two or more amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0 may be substituted, or two or more amino acids (e.g., histidine and glutamic acid) or unnatural amino acids having a side chain pKa of 4.0-8.0 may be inserted. In addition to substitution with or insertion of an amino acid with a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, deletion, addition, insertion, and / or substitution of other amino acids may also be performed simultaneously.Substitution with or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids can be carried out randomly by methods known to those skilled in the art, such as histidine scanning, in which alanine in alanine scanning is replaced with histidine, and antigen-binding molecules with increased KD (acidic pH range) / KD (neutral pH range) or kd (acidic pH range) / kd (neutral pH range) values ​​compared to before mutation can be selected from antigen-binding domains or antibodies into which mutations such as substitution or insertion of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been randomly introduced.

[0211] Preferred examples of antigen-binding molecules that have been mutated to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids and have lower antigen-binding activity in the acidic pH range than in the neutral pH range include antigen-binding molecules whose antigen-binding activity in the neutral pH range after mutation to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids is equivalent to the antigen-binding activity in the neutral pH range before mutation to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids. In the present invention, an antigen-binding molecule after mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid has antigen-binding activity equivalent to that of an antigen-binding molecule before mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid means that, when the antigen-binding activity of the antigen-binding molecule before mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is taken as 100%, the antigen-binding activity of the antigen-binding molecule after mutation with an amino acid having a side chain pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or a non-natural amino acid is at least 10%, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more. The antigen-binding activity at pH 7.4 after mutation with an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid may be higher than the antigen-binding activity at pH 7.4 before mutation with an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid. If the antigen-binding activity of an antigen-binding molecule is reduced by substitution with or insertion of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, the antigen-binding activity can be made equivalent to the antigen-binding activity before substitution or insertion of an amino acid whose side chain has a pKa of 4.0-8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid by substituting, deleting, adding, and / or inserting one or more amino acids in the antigen-binding molecule.The present invention also encompasses antigen-binding molecules whose binding activity is equivalent to that of the antigen-binding molecules obtained by substituting or inserting an amino acid with a side chain pKa of 4.0 to 8.0 (e.g., histidine or glutamic acid) or an unnatural amino acid, followed by substituting, deleting, adding, and / or inserting one or more amino acids.

[0212] Furthermore, when the antigen-binding molecule is a substance comprising an antibody constant region, another preferred embodiment of the antigen-binding molecule has lower antigen-binding activity in an acidic pH range than in a neutral pH range, for example, by modifying the antibody constant region contained in the antigen-binding molecule. Specific examples of the modified antibody constant region include the constant regions set forth in SEQ ID NOs: 11, 12, 13, and 14.

[0213] Amino acids that change the binding activity of antigen-binding domains to antigens depending on hydrogen ion concentration conditions Antigen-binding domains or antibodies of the present invention to be screened by the above-described screening methods may be prepared in any manner. For example, when the ion concentration conditions are hydrogen ion concentration conditions or pH conditions, pre-existing antibodies, pre-existing libraries (such as phage libraries), antibodies or libraries prepared from hybridomas obtained by immunizing animals or B cells from immunized animals, antibodies or libraries obtained by introducing mutations of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids into these antibodies or libraries (libraries with an increased content of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, libraries in which mutations of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids have been introduced at specific sites, and the like can be used.

[0214] In one embodiment of the present invention, a library comprising a plurality of antigen-binding molecules of the present invention having different sequences can be prepared by combining a light chain variable region into which "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions" has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library.

[0215] Non-limiting examples of such amino acid residues include those contained in light chain CDR1. Other non-limiting examples of such amino acid residues include those contained in light chain CDR2. Further non-limiting examples of such amino acid residues include those contained in light chain CDR3.

[0216] As described above, non-limiting examples of amino acid residues contained in the light chain CDR1 include those at positions 24, 27, 28, 31, 32, and / or 34 according to the Kabat numbering system in the CDR1 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR2 include those at positions 50, 51, 52, 53, 54, 55, and / or 56 according to the Kabat numbering system in the CDR2 of the light chain variable region. Non-limiting examples of amino acid residues contained in the light chain CDR3 include those at positions 89, 90, 91, 92, 93, 94, and / or 95A according to the Kabat numbering system in the CDR3 of the light chain variable region. Furthermore, these amino acid residues may be contained alone or in combination of two or more, as long as the binding activity of the antigen-binding molecule to an antigen changes depending on the hydrogen ion concentration.

[0217] Even when combining a light chain variable region into which the above-described "at least one amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on hydrogen ion concentration conditions" has been introduced with a heavy chain variable region prepared as a randomized variable region sequence library, the light chain variable region sequence can be designed to contain flexible residues, as described above. As long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on hydrogen ion concentration conditions, the number and position of the flexible residues are not limited to a particular embodiment. That is, one or more flexible residues can be contained in the CDR sequence and / or FR sequence of the heavy chain and / or light chain. For example, non-limiting examples of flexible residues to be introduced into the light chain variable region sequence include the amino acid residues listed in Table 3 or Table 4. Furthermore, germline sequences such as Vk1 (SEQ ID NO: 5), Vk2 (SEQ ID NO: 6), Vk3 (SEQ ID NO: 7), and Vk4 (SEQ ID NO: 8) can be suitably used as the amino acid sequence of the light chain variable region excluding the amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on hydrogen ion concentration conditions and the flexible residues.

[0218] [Table 3] (The positions represent the Kabat numbering)

[0219] [Table 4] (The positions represent the Kabat numbering)

[0220] Any amino acid residue can be suitably used as the amino acid residue that changes the antigen-binding activity of an antigen-binding molecule depending on hydrogen ion concentration conditions. Specific examples of such amino acid residues include amino acids with a side chain pKa of 4.0 to 8.0. Suitable examples of such electron-donating amino acids include natural amino acids such as histidine and glutamic acid, as well as histidine analogs (US2009 / 0035836) and unnatural amino acids such as m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768). Particularly suitable examples of such amino acid residues include amino acids whose side chain pKa is 6.0 to 7.0. Suitable examples of such electron-donating amino acids include histidine.

[0221] To modify amino acids in the antigen-binding domain, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. Furthermore, several known methods can also be used to modify amino acids by substituting amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), a type of stop codon, can also be used.

[0222] A suitable example of a heavy chain variable region to be combined is a randomized variable region library. A randomized variable region library can be prepared by appropriately combining known methods. In a non-limiting aspect of the present invention, an immune library constructed from antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infectious diseases or humans with increased blood antibody titers after vaccination, cancer patients, or patients with autoimmune diseases can be suitably used as a randomized variable region library.

[0223] In a non-limiting embodiment of the present invention, a synthetic library in which the CDR sequences of V genes in genomic DNA or reconstructed functional V genes are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of appropriate length can also be used as a randomized variable region library, as described above. In this case, since diversity in the gene sequences of heavy chain CDR3 is observed, it is also possible to replace only the CDR3 sequence. The criterion for generating amino acid diversity in the variable regions of antigen-binding molecules is to provide diversity to amino acid residues at surface-exposed positions of the antigen-binding molecule. A surface-exposed position refers to a position that is determined to be surface-exposed and / or capable of contacting an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule, and is generally the CDR. Preferably, the surface-exposed position is determined using coordinates from a three-dimensional model of the antigen-binding molecule using a computer program such as the InsightII program (Accelrys). Surface-exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface-exposed positions can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. Suitable software available for this purpose includes the SYBYL Biopolymer Module software (Tripos Associates). Generally, and preferably, when an algorithm requires a user-input size parameter, the "size" of the probe used in the calculation is set to a radius of about 1.4 angstroms or less. Furthermore, methods for determining surface exposed regions and areas using software for personal computers are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1, 46-53).

[0224] Furthermore, in a non-limiting embodiment of the present invention, a naive library consisting of naive sequences, which are antibody sequences constructed from antibody genes derived from lymphocytes of healthy individuals and whose repertoire is unbiased, can also be particularly preferably used as a randomized variable region library (Gejima et al., Human Antibodies (2002) 11, 121-129, and Cardoso et al., Scand. J. Immunol. (2000) 51, 337-344).

[0225] FcRn Unlike Fcγ receptors, which belong to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides, sharing 22–29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18 (12), 592–598). FcRn is expressed as a heterodimer consisting of a transmembrane α or heavy chain complexed with a soluble β or light chain (β2-microglobulin). Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, and α3), and a short cytoplasmic domain anchors the protein to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain in the Fc region of antibodies (Raghavan et al., Immunity (1994) 1, 303–315).

[0226] FcRn is expressed in the maternal placenta or yolk sac of mammals and is involved in the transfer of IgG from mother to fetus. Additionally, in the small intestine of neonatal rodents, where FcRn is expressed, FcRn is involved in the transfer of maternal IgG from ingested colostrum or milk across the brush border epithelium. FcRn is expressed in numerous other tissues across multiple species, as well as in various endothelial cell lines. It is also expressed in human adult vascular endothelium, muscle vasculature, and liver sinusoids. FcRn is thought to play a role in maintaining plasma IgG concentrations by binding IgG and recycling it to serum. The binding of FcRn to IgG molecules is usually strictly pH-dependent, with optimal binding occurring in the acidic pH range below 7.0.

[0227] Human FcRn, whose precursor is a polypeptide containing the signal sequence represented by SEQ ID NO: 15, forms a complex with human β2-microglobulin in vivo (the polypeptide containing the signal sequence is shown in SEQ ID NO: 16). As shown later in the Reference Examples, soluble human FcRn complexed with β2-microglobulin can be produced using standard recombinant expression techniques. The binding activity of the Fc regions of the present invention to such soluble human FcRn complexed with β2-microglobulin can be evaluated. Unless otherwise specified, human FcRn herein refers to a form capable of binding to the Fc regions of the present invention, including, for example, a complex between human FcRn and human β2-microglobulin.

[0228] Fc area The Fc region contains an amino acid sequence derived from the constant region of an antibody heavy chain, which is the portion of the antibody heavy chain constant region, spanning from the N-terminus of the hinge region to the papain cleavage site, at approximately 216 amino acids (EU numbering), including the hinge, CH2, and CH3 domains.

[0229] The binding activity of the Fc regions provided by the present invention to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described above in the section on binding activity. Conditions other than pH can be appropriately determined by those skilled in the art. The antigen-binding activity and human FcRn-binding activity of an antigen-binding molecule can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Apparent dissociation rate), or apparent kd (Apparent dissociation rate). These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc. may be used.

[0230] Conditions other than pH when measuring the binding activity of the Fc region of the present invention to human FcRn are not particularly limited and can be appropriately selected by those skilled in the art. For example, as described in WO2009125825, measurements can be performed in MES buffer at 37°C. Furthermore, the binding activity of the Fc region of the present invention to human FcRn can be measured by methods known to those skilled in the art, such as using Biacore (GE Healthcare). The binding activity of the Fc region of the present invention to human FcRn can be assessed by passing human FcRn, Fc region, or antigen-binding molecule of the present invention containing an Fc region, as an analyte, over a chip onto which an Fc region, an antigen-binding molecule of the present invention containing an Fc region, or human FcRn has been immobilized.

[0231] The neutral pH range, as a condition under which the Fc region contained in the antigen-binding molecule of the present invention has binding activity to FcRn, typically refers to pH 6.7 to pH 10.0. The neutral pH range is preferably a range indicated by any pH value between pH 7.0 and pH 8.0, and is preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, with pH 7.4 being particularly preferred, which is close to the pH of plasma (blood) in vivo. When the binding affinity between a human FcRn-binding domain and human FcRn is low at pH 7.4, making it difficult to evaluate the binding affinity, pH 7.0 can be used instead of pH 7.4. In the present invention, the acidic pH range, as a condition under which the Fc region contained in the antigen-binding molecule of the present invention has binding activity to FcRn, typically refers to pH 4.0 to pH 6.5. The term "pH" preferably refers to a pH of 5.5 to 6.5, and particularly preferably refers to a pH of 5.8 to 6.0, which is close to the pH in early endosomes in vivo. Regarding the temperature used as a measurement condition, the binding affinity between a human FcRn-binding domain and human FcRn may be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature of 15°C to 40°C is used to determine the binding affinity between a human FcRn-binding domain and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, may also be used to determine the binding affinity between a human FcRn-binding domain and human FcRn. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0232] According to The Journal of Immunology (2009) 182: 7663-7671, the human FcRn-binding activity of native human IgG1 is 1.7 μM (KD) in the acidic pH range (pH 6.0), but barely detectable activity in the neutral pH range. Thus, in a preferred embodiment, antigen-binding molecules of the present invention having human FcRn-binding activity in the acidic and neutral pH ranges can be screened, including antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is 20 μM or stronger and whose human FcRn-binding activity in the neutral pH range is equivalent to or stronger than that of native human IgG. In a more preferred embodiment, antigen-binding molecules of the present invention can be screened, including antigen-binding molecules whose human FcRn-binding activity in the acidic pH range is 2.0 μM or stronger and whose human FcRn-binding activity in the neutral pH range is 40 μM or stronger. In an even more preferred embodiment, antigen-binding molecules of the present invention may be screened for that have a human FcRn-binding activity (KD) of 0.5 μM or stronger in the acidic pH range and a human FcRn-binding activity (KD) of 15 μM or stronger in the neutral pH range. The KD values ​​are determined by the method described in The Journal of Immunology (2009) 182: 7663-7671 (in which the antigen-binding molecule is immobilized on a chip and human FcRn is injected as an analyte).

[0233] In the present invention, an Fc region having human FcRn-binding activity in the acidic and neutral pH ranges is preferred. The domain can be used as is if it already has human FcRn-binding activity in the acidic and neutral pH ranges. When the domain has no or weak human FcRn-binding activity in the acidic and / or neutral pH ranges, an Fc region having the desired human FcRn-binding activity can be obtained by modifying amino acids in the antigen-binding molecule. Alternatively, an Fc region having the desired human FcRn-binding activity in the acidic and / or neutral pH ranges can also be preferably obtained by modifying amino acids in the human Fc region. Furthermore, an Fc region having the desired human FcRn-binding activity can also be obtained by modifying amino acids in an Fc region that already has human FcRn-binding activity in the acidic and / or neutral pH ranges. Amino acid modifications in the human Fc region that confer such desired binding activity can be identified by comparing the human FcRn-binding activity in the acidic and / or neutral pH ranges before and after the amino acid modifications. Those skilled in the art can appropriately modify amino acids using known techniques.

[0234] In the present invention, "amino acid modification" or "amino acid modification" of an Fc region includes modifying the amino acid sequence to a different amino acid sequence from that of the starting Fc region. Any Fc region can be used as the starting domain, as long as the modified variant of the starting Fc region can bind to human FcRn in the acidic pH range (thus, the starting Fc region does not necessarily need to have binding activity to human FcRn under neutral pH conditions). Preferred examples of starting Fc regions include the Fc region of an IgG antibody, i.e., a native Fc region. Furthermore, modified Fc regions obtained by further modifying an already modified Fc region can also be preferably used as the modified Fc region of the present invention. The starting Fc region may refer to the polypeptide itself, a composition comprising the starting Fc region, or the amino acid sequence encoding the starting Fc region. Examples of starting Fc regions include the Fc regions of known IgG antibodies produced by recombinant methods, as outlined in the antibody section. The source of the starting Fc region is not limited, and it may be obtained from any non-human animal or human. Preferably, the organism is selected from the group consisting of mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels, and non-human primates. In another embodiment, the starting Fc region can also be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. Preferably, the starting Fc region is obtained from human IgG1, but is not limited to a specific IgG subclass. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the starting Fc region. Similarly, as used herein, it is meant that the Fc region of any class or subclass of IgG from any of the aforementioned organisms can preferably be used as the starting Fc region.Examples of naturally occurring IgG variants or engineered forms are described in known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338), but are not limited thereto.

[0235] Examples of modifications include one or more mutations, such as substitution of amino acid residues different from those of the starting Fc region, or insertion of one or more amino acid residues into or deletion of one or more amino acids from the starting Fc region. Preferably, the amino acid sequence of the modified Fc region comprises at least a portion of a non-naturally occurring Fc region. Such variants necessarily have less than 100% sequence identity or similarity with the starting Fc region. In a preferred embodiment, the variant has an amino acid sequence identity or similarity of about 75% to less than 100% with the amino acid sequence of the starting Fc region, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100%, and most preferably about 95% to less than 100%. In one non-limiting embodiment of the present invention, there is at least one amino acid difference between the starting Fc region and the modified Fc region of the present invention. The amino acid differences between the starting Fc region and the modified Fc region can also be preferably identified by the amino acid differences at the specified amino acid residue positions represented by the EU numbering system described above.

[0236] To modify amino acids in the Fc region, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. Furthermore, several known methods can be used to modify amino acids by substituting amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), a type of stop codon, can also be suitably used.

[0237] An Fc region having human FcRn-binding activity in the neutral pH range contained in an antigen-binding molecule of the present invention can be obtained by any method. Specifically, an Fc region having human FcRn-binding activity in the neutral pH range can be obtained by modifying amino acids in a human IgG immunoglobulin used as the starting Fc region. Preferred examples of IgG immunoglobulin Fc regions for modification include the Fc region of human IgG (IgG1, IgG2, IgG3, or IgG4, and variants thereof). Modifications to other amino acids can be made at any amino acid position, as long as the Fc region retains human FcRn-binding activity in the neutral pH range or enhances human FcRn-binding activity in the neutral pH range. When an antigen-binding molecule contains a human IgG1 Fc region as the human Fc region, it is preferable that the Fc region contains a modification that enhances human FcRn binding in the neutral pH range compared to the binding activity of the starting human IgG1 Fc region. Amino acids that can be modified in this way include, for example, amino acids at positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, 291 to 312, 315 to 320, 321 to 322, and 323 to 324 (EU numbering). Examples of amino acid modifications include those at positions 4, 325, 327 to 339, 341, 343, 345, 360, 362, 370, 375 to 378, 380, 382, ​​385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442. More specifically, examples of amino acid modifications include those listed in Table 5. These amino acid modifications enhance the binding of the Fc region of IgG immunoglobulins to human FcRn in the neutral pH range.

[0238] Among these modifications, modifications that enhance binding to human FcRn even in the neutral pH range are appropriately selected for use in the present invention. Particularly preferred amino acids in Fc region variants include, for example, amino acids at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering). By substituting at least one amino acid selected from these amino acids with another amino acid, the binding activity of the Fc region contained in the antigen-binding molecule towards human FcRn in the neutral pH range can be enhanced.

[0239] Particularly preferred modifications include, for example, the following modifications, as represented by EU numbering in the Fc region: The amino acid at position 237 is Met; The amino acid at position 248 is Ile; The amino acid at position 250 is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; The amino acid at position 252 is either Phe, Trp, or Tyr; The amino acid at position 254 is Thr; The amino acid at position 255 is Glu; the amino acid at position 256 is Asp, Asn, Glu, or Gln; the amino acid at position 257 is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; The amino acid at position 258 is His; The amino acid at position 265 is Ala, The amino acid at position 286 is either Ala or Glu, The amino acid at position 289 is His; The amino acid at position 297 is Ala, The amino acid at position 303 is Ala, The amino acid at position 305 is Ala, the amino acid at position 307 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; the amino acid at position 308 is Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; The amino acid at position 309 is Ala, Asp, Glu, Pro, or Arg; the amino acid at position 311 is Ala, His, or Ile; The amino acid at position 312 is either Ala or His; The amino acid at position 314 is either Lys or Arg; the amino acid at position 315 is Ala, Asp, or His; The amino acid at position 317 is Ala, The amino acid at position 332 is Val; The amino acid at position 334 is Leu, The amino acid at position 360 is His, The amino acid at position 376 is Ala, The amino acid at position 380 is Ala, The amino acid at position 382 is Ala, The amino acid at position 384 is Ala, The amino acid at position 385 is either Asp or His; The amino acid at position 386 is Pro, The amino acid at position 387 is Glu; The amino acid at position 389 is either Ala or Ser, The amino acid at position 424 is Ala, the amino acid at position 428 is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; The amino acid at position 433 is Lys. The amino acid at position 434 is Ala, Phe, His, Ser, Trp, or Tyr, and The amino acid at position 436 is His, Ile, Leu, Phe, Thr, or Val; The number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, or two or more amino acids may be modified. Examples of combinations of amino acid modifications at two or more amino acid positions include those shown in Table 6.

[0240] antigen binding molecule In the present invention, the term "antigen-binding molecule" is used in the broadest sense to refer to a molecule comprising an antigen-binding domain and an Fc region. Specifically, various molecular types are included as long as they exhibit antigen-binding activity. For example, antibodies are an example of a molecule in which an antigen-binding domain is linked to an Fc region. Examples of antibodies include single monoclonal antibodies (including agonist and antagonist antibodies), human antibodies, humanized antibodies, chimeric antibodies, and the like. Antibody fragments are preferably antigen-binding domains and antigen-binding fragments (e.g., Fab, F(ab')2, scFv, and Fv). Scaffold molecules, in which existing stable α / β barrel protein structures or other three-dimensional structures are used as scaffolds, and only partial structures of these structures are compiled into libraries for constructing antigen-binding domains, are also included in the antigen-binding molecules of the present invention.

[0241] Antigen-binding molecules of the present invention can comprise at least a portion of an Fc region that mediates binding to FcRn and Fcγ receptors. For example, in one non-limiting embodiment, the antigen-binding molecule can be an antibody or an Fc fusion protein. A fusion protein is a chimeric polypeptide comprising a polypeptide comprising a first amino acid sequence linked to a polypeptide having a second amino acid sequence to which it is not naturally linked. For example, a fusion protein can comprise an amino acid sequence encoding at least a portion of an Fc region (e.g., a portion of the Fc region that confers binding to FcRn or a portion of the Fc region that confers binding to Fcγ receptors) and a non-immunoglobulin polypeptide comprising an amino acid sequence encoding, for example, a ligand-binding domain of a receptor or a receptor-binding domain of a ligand. The amino acid sequences can reside in separate proteins that are carried together in the fusion protein, or they can normally reside in the same protein but are rearranged in a new configuration in the fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis or by recombinant techniques in which polynucleotides encoding the peptide regions in the desired relationship are constructed and expressed.

[0242] The domains of the present invention can be linked directly by a polypeptide bond or via a linker. The linker can be any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (e.g., a linker disclosed in Protein Engineering (1996) 9 (3), 299-305), but peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited and can be selected appropriately by those skilled in the art depending on the purpose. The preferred length is 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and 15 amino acids is particularly preferred.

[0243] For example, for a peptide linker: Ser Gly·Ser Gly Gly Ser Ser Gly Gly Gly·Gly·Gly·Ser (SEQ ID NO: 17) Ser·Gly·Gly·Gly (SEQ ID NO: 18) Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 19) Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 20) Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 21) Ser·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 22) Gly·Gly·Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 23) Ser·Gly·Gly·Gly·Gly·Gly·Gly·Gly (SEQ ID NO: 24) (Gly·Gly·Gly·Gly·Ser (SEQ ID NO: 19))n (Ser·Gly·Gly·Gly·Gly (SEQ ID NO: 20))n [n is an integer of 1 or more], etc. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0244] Synthetic chemical linkers (chemical crosslinkers) are crosslinkers commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available.

[0245] When multiple linkers are used to connect the domains, all of the linkers may be of the same type, or different types of linkers may be used.

[0246] In addition to the linkers exemplified above, linkers having peptide tags such as His tags, HA tags, myc tags, and FLAG tags can also be used as appropriate. Furthermore, the property of binding to each other through hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions, or combinations of these bonds can also be suitably utilized. For example, the affinity between antibody CH1 and CL can be utilized, or Fc regions derived from the aforementioned bispecific antibodies can be used to associate heterologous Fc regions. Furthermore, disulfide bonds formed between domains can also be suitably utilized.

[0247] To link each domain via a peptide bond, polynucleotides encoding the domains are linked in-frame. Methods for linking polynucleotides in-frame include restriction fragment ligation, fusion PCR, and overlap PCR, and these methods can be used alone or in combination as appropriate to produce the antigen-binding molecules of the present invention. In the present invention, the terms "linked," "fused," "linked," and "fused" are used interchangeably. These terms refer to the linking of two or more elements or components, such as polypeptides, to form a single structure by any means, including the above-mentioned chemical conjugation or recombinant techniques. "In-frame fusion," when two or more elements or components are polypeptides, refers to the linking of two or more open reading frames to form a continuous, longer open reading frame while maintaining the correct reading frame of the polypeptide. When two Fab molecules are used as the antigen-binding domain, an antibody, which is an antigen-binding molecule of the present invention, in which the antigen-binding domain and Fc region are linked in-frame by a peptide bond without a linker can be used as a preferred antigen-binding molecule of the present application.

[0248] Fcγ receptor An Fcγ receptor (also referred to as FcγR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies, and refers to any member of a family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγRs or FcγR isoforms or allotypes. FcγRs may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), as well as any unidentified mouse FcγRs or FcγR isoforms or allotypes. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).The polynucleotide sequence and amino acid sequence of human FcγRI are shown in SEQ ID NOs: 25 (NM_000566.3) and 26 (NP_000557.1), respectively. The polynucleotide sequence and amino acid sequence of human FcγRIIa (allotype H131) are shown in SEQ ID NOs: 27 (BC020823.1) and 28 (AAH20823.1), respectively (allotype R131 is a sequence in which the 166th amino acid of SEQ ID NO: 28 is substituted with Arg). The polynucleotide sequence and amino acid sequence of FcγRIIb are shown in SEQ ID NOs: 28 (BC020823.1) and 28 (AAH20823.1), respectively. The polynucleotide and amino acid sequences of FcγRIIIa are set forth in SEQ ID NOs: 29 (BC146678.1) and 30 (AAI46679.1), respectively; the polynucleotide and amino acid sequences of FcγRIIIb are set forth in SEQ ID NOs: 31 (BC033678.1) and 32 (AAH33678.1), respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are set forth in SEQ ID NOs: 33 (BC128562.1) and 34 (AAI28563.1), respectively (RefSeq accession numbers are indicated in parentheses). For example, in Reference Example 27, etc., allotype V158 is used, but this is not to be construed as particularly limiting the allotype of the FcγRIIIa isoform described herein. Whether an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the FACS or ELISA formats described above, as well as by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method utilizing the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0249] "Fc ligand" or "effector ligand" refers to any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. Binding of the Fc ligand to Fc preferably induces one or more effector functions. Fc ligands include, but are not limited to, Fc receptors, FcγR, FcαR, FcεR, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, staphylococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRHs), a family of Fc receptors homologous to FcγR (Davis et al., (2002) Immunological Reviews 190, 123-136). Fc ligands may also include undiscovered molecules that bind to Fc.

[0250] FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), are composed of an α chain that binds to the Fc portion of IgG and a common γ chain with ITAMs that transduce activation signals intracellularly. On the other hand, FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc, contains ITAMs in its cytoplasmic domain. These receptors are expressed on many immune cells, including macrophages, mast cells, and antigen-presenting cells. Binding of these receptors to the Fc portion of IgG transmits activation signals that promote the phagocytic activity of macrophages, the production of inflammatory cytokines, mast cell degranulation, and enhanced function of antigen-presenting cells. Fcγ receptors capable of transmitting activation signals as described above are also referred to as activating Fcγ receptors in the present invention.

[0251] On the other hand, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM, which transmits inhibitory signals. In B cells, cross-linking of FcγRIIb with the B cell receptor (BCR) suppresses activation signals from the BCR, resulting in the suppression of antibody production by the BCR. In macrophages, cross-linking of FcγRIII with FcγRIIb suppresses phagocytic ability and the ability to produce inflammatory cytokines. Fcγ receptors that have the ability to transmit inhibitory signals as described above are also referred to as inhibitory Fcγ receptors in the present invention.

[0252] The ALPHA screen is performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead and the two beads are in close proximity. A photosensitizer inside the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. If the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescent reaction occurs.

[0253] For example, an antigen-binding molecule containing a biotin-labeled Fc region is bound to donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to acceptor beads. In the absence of a competing antigen-binding molecule containing an Fc region variant, a polypeptide complex having a wild-type Fc region interacts with the Fcγ receptor, generating a signal at 520-620 nm. An antigen-binding molecule containing an untagged Fc region variant competes with the interaction between an antigen-binding molecule having a native Fc region and the Fcγ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. Biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin or similar is known. Methods for tagging Fcγ receptors with GST include expressing a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in frame in a vector operably linked to the fusion gene in cells, etc., and purifying the gene using a glutathione column. The resulting signal is suitably analyzed by fitting it to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0254] One of the substances (ligand) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). When the other substance (analyte) whose interaction is to be observed is passed over the surface of the sensor chip, binding occurs between the ligand and the analyte, increasing the mass of the immobilized ligand molecule and changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation returns the signal position). The Biacore system plots the amount of shift (i.e., the change in mass on the sensor chip surface) on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics (association rate constant (ka) and dissociation rate constant (kd)) can be calculated from the sensorgram curve, and affinity (KD) can be calculated from the ratio of these constants. Inhibition assays are also suitable for use with the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0255] Heterocomplex containing two FcRn molecules and one activating Fcγ receptor molecule Crystallographic studies of FcRn and IgG antibodies have shown that the FcRn-IgG complex is composed of two molecules of FcRn and one molecule of IgG, and that the binding of the two molecules occurs near the contact surface of the CH2 and CH3 domains located on either side of the IgG Fc region (Burmeister et al. (Nature (1994) 372, 336-343)). Meanwhile, as confirmed in Example 3 described below, it has been revealed that the Fc region of an antibody can form a four-component complex containing two molecules of FcRn and one molecule of an activating Fcγ receptor (Figure 48). The formation of this heterocomplex is a phenomenon that became clear as a result of further analysis of the properties of antigen-binding molecules containing an Fc region that has FcRn-binding activity under neutral pH conditions.

[0256] Although the present invention is not limited to a particular theory, it is believed that the formation of a four-component heterocomplex containing the Fc region of an antigen-binding molecule, two molecules of FcRn, and one molecule of an activating Fcγ receptor may have the following effects on the pharmacokinetics (plasma retention) of the antigen-binding molecule when administered to a living body, and on the immune response (immunogenicity) to the administered antigen-binding molecule. As mentioned above, in addition to various activating Fcγ receptors, FcRn is expressed on immune cells. It is suggested that the formation of such a four-component complex by an antigen-binding molecule on immune cells improves its affinity for immune cells and further enhances the internalization signal by associating the intracellular domains, thereby promoting uptake into immune cells. The same is true for antigen-presenting cells, suggesting that the formation of a four-component complex on the cell membrane of antigen-presenting cells may facilitate uptake of the antigen-binding molecule into antigen-presenting cells. Generally, antigen-binding molecules taken up by antigen-presenting cells are degraded in lysosomes within the antigen-presenting cells and presented to T cells. As a result, the formation of the above-mentioned quaternary complex on the cell membrane of antigen-presenting cells may promote uptake of antigen-binding molecules into antigen-presenting cells, thereby potentially worsening the plasma retention of antigen-binding molecules and potentially inducing (exacerbating) immune responses.

[0257] Therefore, when an antigen-binding molecule with a reduced ability to form such a quaternary complex is administered to a living body, it is thought that the plasma retention of the antigen-binding molecule will be improved and the induction of an immune response by the living body will be suppressed. Preferred embodiments of antigen-binding molecules that inhibit the formation of such complexes on immune cells, including antigen-presenting cells, include the following three types.

[0258] (Mode 1) An antigen-binding molecule comprising an Fc region that has FcRn-binding activity under neutral pH conditions and whose binding activity to activating FcγR is lower than that of a native Fc region.

[0259] The antigen-binding molecule of embodiment 1 forms a ternary complex by binding to two FcRn molecules, but does not form a complex including activating FcγR ( Figure 49 ). An Fc region whose binding activity to activating FcγR is lower than that of a native Fc region can be prepared by modifying the amino acids of the native Fc region as described above. Whether the binding activity of an altered Fc region to activating FcγR is lower than that of a native Fc region can be determined appropriately using the methods described above in the section on binding activity.

[0260] Preferred examples of activating Fcγ receptors include FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including allotypes R131 and H131); and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2).

[0261] The pH conditions for measuring the binding activity of the Fc region contained in the antigen-binding molecules of the present invention to Fcγ receptors can be appropriately set to an acidic pH range or a neutral pH range. The neutral pH range, as used to measure the binding activity of the Fc region contained in the antigen-binding molecules of the present invention to Fcγ receptors, typically refers to a pH range of 6.7 to 10.0. The neutral pH range is preferably a range indicated by any pH value between 7.0 and 8.0, and is preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, with pH 7.4, which is close to the pH of plasma (blood) in vivo, being particularly preferred. In the present invention, the acidic pH range, as used to measure the binding activity of the Fc region contained in the antigen-binding molecules of the present invention to Fcγ receptors, typically refers to a pH range of 4.0 to 6.5. Preferably, this refers to a pH of 5.5 to 6.5, and particularly preferably to a pH of 5.8 to 6.0, which is close to the pH in early endosomes in vivo. Regarding the temperature used as a measurement condition, the binding affinity between an Fc region and a human Fcγ receptor can be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature of 15°C to 40°C is used to determine the binding affinity between a human Fc region and an Fcγ receptor. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, can also be used to determine the binding affinity between an Fc region and an Fcγ receptor. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0262] As used herein, "the binding activity of an Fc region variant toward an activating Fcγ receptor is lower than the binding activity of a native Fc region toward an activating Fcγ receptor" means that the binding activity of the Fc region variant toward any one of the human Fcγ receptors, FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb, is lower than the binding activity of the native Fc region toward these human Fcγ receptors. For example, based on the above-mentioned analytical methods, this refers to the binding activity of an antigen-binding molecule comprising an Fc region variant being 95% or less, preferably 90% or less, 85% or less, 80% or less, or 75% or less, and particularly preferably 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the binding activity of an antigen-binding molecule comprising a control native Fc region. As the native Fc region, a starting Fc region or an Fc region of a different isotype of a wild-type antibody can be used.

[0263] Furthermore, the native binding activity to activating FcγR is preferably the binding activity to human IgG1 Fcγ receptor, and in addition to the above-mentioned alterations, the Fcγ receptor-binding activity can also be reduced by changing the isotype to human IgG2, human IgG3, or human IgG4. In addition to the above-mentioned alterations, the Fcγ receptor-binding activity can also be reduced by expressing an antigen-binding molecule comprising an Fc region that has Fcγ receptor-binding activity in a non-glycosylated host such as Escherichia coli.

[0264] As a control antigen-binding molecule containing an Fc region, an antigen-binding molecule containing the Fc region of an IgG monoclonal antibody can be used as appropriate. The structures of the Fc regions are set forth in SEQ ID NOS: 1 (RefSeq Accession No. AAC82527.1 with an A added to the N-terminus), 2 (RefSeq Accession No. AAB59393.1 with an A added to the N-terminus), 3 (RefSeq Accession No. CAA27268.1), and 4 (RefSeq Accession No. AAB59394.1 with an A added to the N-terminus). Furthermore, when an antigen-binding molecule containing the Fc region of an antibody of a certain isotype is used as a test substance, the effect of the Fcγ receptor-binding activity of the antigen-binding molecule containing the Fc region is verified by using an antigen-binding molecule containing the Fc region of an IgG monoclonal antibody of that specific isotype as a control. As described above, an antigen-binding molecule containing an Fc region verified to have high Fcγ receptor-binding activity is appropriately selected.

[0265] In one non-limiting embodiment of the present invention, examples of Fc regions whose binding activity to activating FcγR is lower than the binding activity of a native Fc region to activating FcγR include: Preferred examples of the Fc region include those in which one or more of the amino acids 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, 328, and 329 (EU numbering) have been modified to amino acids different from those in the native Fc region. However, modifications of the Fc region are not limited to the above modifications. For example, deglycosylation (N297A, N297Q), IgG1-L234A / L235A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, IgG4-L236E, and other modifications. The modifications may include modifications such as G236R / L328R, L235G / G236R, N325A / L328R, and N325LL328R described in WO 2008 / 092117, insertion of amino acids at positions 233, 234, 235, and 237 (EU numbering), and modifications at positions described in WO 2000 / 042072.

[0266] In a non-limiting embodiment of the present invention, the amino acids of the Fc region are represented by EU numbering: the amino acid at position 234 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, or Trp; the amino acid at position 235 is Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg; the amino acid at position 236 is selected from Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, and Tyr; the amino acid at position 237 is Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg; the amino acid at position 238 is Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg; the amino acid at position 239 is either Gln, His, Lys, Phe, Pro, Trp, Tyr or Arg; the amino acid at position 265 is Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; The amino acid at position 266 is either Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr; The amino acid at position 267 is selected from Arg, His, Lys, Phe, Pro, Trp, and Tyr; the amino acid at position 269 is Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; the amino acid at position 270 is Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; The amino acid at position 271 is either Arg, His, Phe, Ser, Thr, Trp, or Tyr; The amino acid at position 295 is selected from Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, and Tyr; The amino acid at position 296 is selected from Arg, Gly, Lys, and Pro; The amino acid at position 297 is Ala, The amino acid at position 298 is selected from Arg, Gly, Lys, Pro, Trp, and Tyr; The amino acid at position 300 is either Arg, Lys, or Pro. The amino acid at position 324 is either Lys or Pro. The amino acid at position 325 is Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, TrpTyr, or Val; the amino acid at position 327 is selected from Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; The amino acid at position 328 is selected from Arg, Asn, Gly, His, Lys, and Pro; the amino acid at position 329 is selected from Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, and Arg; The amino acid at position 330 is either Pro or Ser, The amino acid at position 331 is either Arg, Gly, or Lys, or The amino acid at position 332 is either Arg, Lys, or Pro; Preferred examples of such Fc regions include those in which one or more of the following modifications have been made:

[0267] (Aspect 2) An antigen-binding molecule comprising an Fc region that has FcRn-binding activity under neutral pH conditions and whose binding activity to inhibitory FcγR is higher than that to activating Fcγ receptors.

[0268] The antigen-binding molecule of Mode 2 can form a complex containing these four molecules by binding to two molecules of FcRn and one molecule of inhibitory FcγR. However, because one molecule of an antigen-binding molecule can only bind to one molecule of FcγR, it cannot bind to another activating FcγR while bound to an inhibitory FcγR (Figure 50). Furthermore, it has been reported that antigen-binding molecules that are internalized into cells while bound to an inhibitory FcγR are recycled to the cell membrane and avoid intracellular degradation (Immunity (2005) 23, 503-514). In other words, it is thought that an antigen-binding molecule with selective binding activity to an inhibitory FcγR cannot form a heterocomplex containing an activating FcγR and two molecules of FcRn, which are responsible for immune responses.

[0269] Suitable examples of activating Fcγ receptors include FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including allotypes R131 and H131); and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). Suitable examples of inhibitory Fcγ receptors include FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

[0270] As used herein, "higher binding activity to inhibitory FcγRs than to activating Fcγ receptors" means that the binding activity of an Fc region variant to FcγRIIb is higher than the binding activity to any of the human Fcγ receptors FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb. For example, based on the above-mentioned analytical methods, the binding activity of an antigen-binding molecule comprising an Fc region variant to FcγRIIb is 105% or more, preferably 110% or more, 120% or more, 130% or more, 140% or more, particularly preferably 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 450% or more, 500% or more, 750% or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, or 50 times or more that of the binding activity to any one of the human Fcγ receptors FcγRI, FcγRIIa, FcγRIIIa, and / or FcγRIIIb.

[0271] It is most preferable that the binding activity to FcγRIIb is higher than that of all of FcγRIa, FcγRIIa (including allotypes R131 and H131), and FcγRIIIa (including allotypes V158 and F158). Since FcγRIa has extremely high affinity for native IgG1, its binding is thought to be saturated by large amounts of endogenous IgG1 in vivo. Therefore, its binding activity to FcγRIIb is higher than that of FcγRIIa and FcγRIIIa, and even if it is lower than that of FcγRIa, it is thought that it is possible to inhibit the formation of the complex.

[0272] As a control antigen-binding molecule containing an Fc region, an antigen-binding molecule containing an Fc region of an IgG monoclonal antibody can be used as appropriate. The structures of the Fc regions are set forth in SEQ ID NOs: 11 (RefSeq Accession No. AAC82527.1 with an A added to the N-terminus), 12 (RefSeq Accession No. AAB59393.1 with an A added to the N-terminus), 13 (RefSeq Accession No. CAA27268.1), and 14 (RefSeq Accession No. AAB59394.1 with an A added to the N-terminus). Furthermore, when an antigen-binding molecule containing an Fc region of an antibody of a certain isotype is used as a test substance, the effect of the Fcγ receptor-binding activity of the antigen-binding molecule containing the Fc region is verified by using an antigen-binding molecule containing an Fc region of an IgG monoclonal antibody of the specific isotype as a control. As described above, an antigen-binding molecule containing an Fc region verified to have high Fcγ receptor-binding activity is appropriately selected.

[0273] In a non-limiting embodiment of the present invention, preferred examples of Fc regions that have selective binding activity to inhibitory FcγRs include Fc regions in which the amino acid at position 238 or 328 (EU numbering) has been altered to an amino acid different from that of a native Fc region. Furthermore, Fc regions or alterations described in US 2009 / 0136485 can also be appropriately selected as Fc regions that have selective binding activity to inhibitory Fcγ receptors.

[0274] In a non-limiting embodiment of the present invention, preferred examples of the Fc region include an Fc region in which the amino acids at position 238 (EU numbering) have been altered to Asp or the amino acid at position 328 (EU numbering) have been altered to Glu.

[0275] In a further non-limiting embodiment of the present invention, the amino acid at position 238 (EU numbering) is substituted with Asp, and the amino acid at position 237 (EU numbering) is Trp, the amino acid at position 237 (EU numbering) is Phe, the amino acid at position 267 (EU numbering) is Val, the amino acid at position 267 (EU numbering) is Gln, the amino acid at position 268 (EU numbering) is Asn, the amino acid at position 271 (EU numbering) is Gly, the amino acid at position 326 (EU numbering) is Leu, and the amino acid at position 328 (EU numbering) is Val. The amino acid at position 326 represented by EU numbering is Gln, the amino acid at position 326 represented by EU numbering is Glu, the amino acid at position 326 represented by EU numbering is Met, the amino acid at position 239 represented by EU numbering is Asp, the amino acid at position 267 represented by EU numbering is Ala, the amino acid at position 234 represented by EU numbering is Trp, the amino acid at position 234 represented by EU numbering is Tyr, the amino acid at position 237 represented by EU numbering is Ala, the amino acid at position 237 represented by EU numbering is Asp, the amino acid at position 23 The amino acid at position 7 is Glu, the amino acid at position 237 in EU numbering is Leu, the amino acid at position 237 in EU numbering is Met, the amino acid at position 237 in EU numbering is Tyr, the amino acid at position 330 in EU numbering is Lys, the amino acid at position 330 in EU numbering is Arg, the amino acid at position 233 in EU numbering is Asp, the amino acid at position 268 in EU numbering is Asp, the amino acid at position 268 in EU numbering is Glu, the amino acid at position 326 in EU numbering The amino acid at position 326 in EU numbering is Asp, the amino acid at position 326 in EU numbering is Ser, the amino acid at position 326 in EU numbering is Thr, the amino acid at position 323 in EU numbering is Ile, the amino acid at position 323 in EU numbering is Leu, the amino acid at position 323 in EU numbering is Met, the amino acid at position 296 in EU numbering is Asp, the amino acid at position 326 in EU numbering is Ala, the amino acid at position 326 in EU numbering is Asn, the amino acid at position 330 in EU numbering is Met,Preferred examples of such Fc regions include those in which one or more of the following modifications have been made:

[0276] (Mode 3) An antigen-binding molecule comprising an Fc domain, one of two polypeptides constituting the Fc domain having FcRn-binding activity under neutral pH conditions, and the other having no FcRn-binding activity under neutral pH conditions.

[0277] The antigen-binding molecule of embodiment 3 can form a ternary complex by binding to one molecule of FcRn and one molecule of FcγR, but does not form a quaternary heterocomplex containing two molecules of FcRn and one molecule of FcγR (Figure 51). As an Fc region contained in the antigen-binding molecule of embodiment 3, in which one of the two polypeptides constituting the Fc region has FcRn-binding activity in a neutral pH range and the other polypeptide does not have FcRn-binding activity in a neutral pH range, an Fc region derived from a bispecific antibody can also be used as appropriate. Bispecific antibodies are two types of antibodies that have specificity for different antigens. IgG-type bispecific antibodies can be secreted by hybrid hybridomas (quadromas) generated by fusing two types of IgG antibody-producing hybridomas (Milstein et al. (Nature (1983) 305, 537-540)).

[0278] When the antigen-binding molecule of the above embodiment 3 is produced using a recombinant technique such as that described in the antibody section, a method can be used in which genes encoding the polypeptides constituting the two desired Fc regions are introduced into cells and co-expressed. However, the produced Fc regions are mixtures of Fc regions in which one of the two polypeptides constituting the Fc region has FcRn-binding activity at neutral pH and the other polypeptide does not have FcRn-binding activity at neutral pH, Fc regions in which both of the polypeptides constituting the Fc region have FcRn-binding activity at neutral pH, and Fc regions in which neither of the polypeptides constituting the Fc region has FcRn-binding activity at neutral pH, in a molecular ratio of 2:1:1. It is difficult to purify antigen-binding molecules containing the desired combination of Fc regions from three types of IgG.

[0279] When producing antigen-binding molecules of embodiment 3 using such recombinant techniques, antigen-binding molecules comprising heterologous combinations of Fc regions can be preferentially secreted by modifying the CH3 domains that constitute the Fc region with appropriate amino acid substitutions. Specifically, the amino acid side chains in the CH3 domain of one heavy chain are substituted with larger side chains (knobs) and the amino acid side chains in the CH3 domain of the other heavy chain are substituted with smaller side chains (holes) so that the knobs can be positioned in the holes, promoting heterologous H chain formation and inhibiting homologous H chain formation (WO1996027011, Ridgway et al. (Protein Engineering (1996) 9, 617-621), Merchant et al. (Nat. Biotech. (1998) 16, 677-681)).

[0280] Furthermore, techniques for producing bispecific antibodies are also known that utilize methods for controlling the association of polypeptides or heteromultimers composed of polypeptides to control the association of two polypeptides that constitute Fc domains. Specifically, a method can be used to produce bispecific antibodies by modifying amino acid residues that form the interface between the two polypeptides that constitute Fc domains, thereby inhibiting the association of polypeptides that constitute Fc domains with identical sequences and controlling the formation of polypeptide complexes composed of two Fc domains with different sequences (WO 2006 / 106905). Such methods can also be used to produce the antigen-binding molecules of embodiment 3 of the present invention.

[0281] In one non-limiting embodiment of the present invention, two polypeptides constituting an Fc domain derived from the bispecific antibody described above can be used as the Fc domain. More specifically, two polypeptides constituting an Fc domain are preferably used, wherein the amino acid at position 349 (EU numbering) in the amino acid sequence of one polypeptide is Cys and the amino acid at position 366 is Trp, and the amino acid at position 356 (EU numbering) in the amino acid sequence of the other polypeptide is Cys, Ser, Ala, and Val at positions 368 and 407, all of which are represented by EU numbering.

[0282] In another non-limiting embodiment of the present invention, an Fc region preferably comprises two polypeptides that constitute an Fc region, wherein the amino acid at position 409 (EU numbering) in the amino acid sequence of one polypeptide is Asp, and the amino acid at position 399 (EU numbering) in the amino acid sequence of the other polypeptide is Lys. In this embodiment, the amino acid at position 409 may be Glu instead of Asp, and the amino acid at position 399 may be Arg instead of Lys. Furthermore, in addition to the Lys at amino acid 399, Asp may also be suitably added as amino acid 360 or Asp as amino acid 392.

[0283] In another non-limiting embodiment of the present invention, the Fc region preferably includes two polypeptides that constitute the Fc region, wherein the amino acid at position 370 (EU numbering) in the amino acid sequence of one of the polypeptides is Glu, and the amino acid at position 357 (EU numbering) in the amino acid sequence of the other polypeptide is Lys.

[0284] In yet another non-limiting embodiment of the present invention, the Fc region preferably comprises two polypeptides that constitute the Fc region, wherein the amino acid at position 439 (EU numbering) in the amino acid sequence of one of the polypeptides is Glu, and the amino acid at position 356 (EU numbering) in the amino acid sequence of the other polypeptide is Lys.

[0285] In another non-limiting embodiment of the present invention, the Fc region may be any of the following embodiments in combination: two polypeptides forming an Fc domain, wherein in the amino acid sequence of one of the polypeptides, the amino acid at position 409 is Asp and the amino acid at position 370 is Glu according to EU numbering, and in the amino acid sequence of the other polypeptide, the amino acid at position 399 is Lys and the amino acid at position 357 is Lys according to EU numbering (in this embodiment, the Glu at amino acid position 370 according to EU numbering may be replaced by Asp, and the Glu at amino acid position 370 according to EU numbering may be replaced by Asp at amino acid position 392); two polypeptides forming an Fc domain, wherein in the amino acid sequence of one of the polypeptides, the amino acid at position 409 is Asp and the amino acid at position 439 is Glu according to EU numbering, and in the amino acid sequence of the other polypeptide, the amino acid at position 399 is Lys and the amino acid at position 356 is Lys according to EU numbering (in this embodiment, the Glu at position 439 according to EU numbering may be replaced by Asp at position 360, Asp at position 392, or Asp at position 439); Two polypeptides constituting an Fc region, wherein the amino acid at position 370 and the amino acid at position 439 are Glu and Glu, respectively, according to EU numbering in the amino acid sequence of one polypeptide, and the amino acid at position 357 and the amino acid at position 356 are Lys and Lys, respectively, according to EU numbering in the amino acid sequence of the other polypeptide; or two polypeptides forming an Fc domain, wherein in the amino acid sequence of one of the polypeptides, the amino acid at position 409 is Asp, the amino acid at position 370 is Glu, and the amino acid at position 439 is Glu, as indicated by EU numbering, and in the amino acid sequence of the other polypeptide, the amino acid at position 399 is Lys, the amino acid at position 357 is Lys, and the amino acid at position 356 is Lys, as indicated by EU numbering (in this embodiment, the amino acid at position 370 (EU numbering) does not need to be substituted with Glu, and further, in addition to not substituting the amino acid at position 370 with Glu, the Glu at position 439 may be replaced with Asp, or the Glu at position 439 may be replaced with Asp, or the Glu at position 392 may be replaced with Asp); is preferably used.

[0286] Furthermore, in another non-limiting embodiment of the present invention, two polypeptides that constitute an Fc domain, wherein the amino acid at position 356 (EU numbering) in the amino acid sequence of one of the polypeptides is Lys, and the amino acid at position 435 (EU numbering) is Arg and the amino acid at position 439 (EU numbering) in the amino acid sequence of the other polypeptide are also preferably used.

[0287] Furthermore, in another non-limiting embodiment of the present invention, two polypeptides that constitute an Fc domain, wherein the amino acids at positions 356 and 357 in the amino acid sequence of one polypeptide are Lys and Lys, respectively, according to EU numbering, and the amino acids at positions 370, 435, and 439 in the amino acid sequence of the other polypeptide are Glu, Arg, and Glu, respectively, according to EU numbering, are also suitable for use.

[0288] These antigen-binding molecules of modes 1 to 3 are all expected to have reduced immunogenicity and improved plasma retention compared to antigen-binding molecules that can form quaternary complexes.

[0289] Reduced immune response (improved immunogenicity) Whether or not the immune response to the antigen-binding molecule of the present invention has been altered can be evaluated by measuring the response of a living organism to which a pharmaceutical composition containing the antigen-binding molecule as an active ingredient has been administered. The response of a living organism mainly includes two immune responses: cellular immunity (induction of cytotoxic T cells that recognize peptide fragments of the antigen-binding molecule bound to MHC class I) and humoral immunity (induction of antibody production that binds to the antigen-binding molecule). In particular, in the case of protein pharmaceuticals, antibody production against the administered antigen-binding molecule is called immunogenicity. There are two methods for evaluating immunogenicity: evaluating antibody production in vivo and evaluating immune cell responses in vitro.

[0290] The in vivo immune response (immunogenicity) can be evaluated by measuring the antibody titer when an antigen-binding molecule is administered to a living body. For example, when antigen-binding molecules A and B are administered to mice and the antibody titer is measured, if the antibody titer is higher for antigen-binding molecule A than for B, or if the incidence of individuals with elevated antibody titers is higher when antigen-binding molecule A is administered to multiple mice, then antigen-binding molecule A is determined to be more immunogenic than B. Antibody titers can be measured using methods known to those skilled in the art, such as ELISA, ECL, or SPR, which measure molecules that specifically bind to the administered molecule (J. Pharm. Biomed. Anal. (2011) 55 (5), 878-888).

[0291] One method for evaluating the living body's immune response (immunogenicity) to an antigen-binding molecule in vitro is to react human peripheral blood mononuclear cells (or fractionated cells) isolated from a donor with the antigen-binding molecule in vitro and measure the number or percentage of cells such as helper T cells that react or proliferate, or the amount of cytokines produced (Clin. Immunol. (2010) 137 (1), 5-14, Drugs R D. (2008) 9 (6), 385-396). For example, when antigen-binding molecules A and B are evaluated in such an in vitro immunogenicity test, if antigen-binding molecule A shows a higher response than antigen-binding molecule B, or if antigen-binding molecule A shows a higher positive response rate when evaluated from multiple donors, then antigen-binding molecule A is judged to be more immunogenic than antigen-binding molecule B.

[0292] Although the present invention is not limited to a particular theory, it is believed that antigen-binding molecules with FcRn-binding activity in the neutral pH range can form a four-component heterocomplex containing two FcRn molecules and one FcγR molecule on the cell membrane of antigen-presenting cells, thereby promoting their uptake into antigen-presenting cells and facilitating the induction of immune responses. Phosphorylation sites exist in the intracellular domains of FcγR and FcRn. Phosphorylation of the intracellular domain of receptors expressed on the cell surface generally occurs upon receptor association, leading to receptor internalization. When native IgG1 forms a binary FcγR / IgG1 complex on antigen-presenting cells, the intracellular domains of FcγR do not assemble. However, if an IgG molecule with binding activity to FcRn under neutral pH conditions were to form a tetrameric complex (FcγR / two FcRn / IgG), the three intracellular domains of FcγR and FcRn would assemble, potentially inducing internalization of the FcγR / two FcRn / IgG heterocomplex. The formation of a tetrameric FcγR / two FcRn / IgG heterocomplex is thought to occur on antigen-presenting cells that express both FcγR and FcRn, potentially increasing the amount of antibody molecules taken up by antigen-presenting cells and potentially worsening immunogenicity. By using any of the methods of aspects 1, 2, and 3 discovered in the present invention, it is possible that the formation of the above-mentioned complex on antigen-presenting cells can be inhibited, thereby reducing uptake into antigen-presenting cells and resulting in improved immunogenicity.

[0293] Improved pharmacokinetics Although the present invention is not bound by any particular theory, for example, when an antigen-binding molecule containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions (e.g., its antigen-binding activity in an acidic pH range is lower than that in a neutral pH range) and an Fc region that has human FcRn-binding activity in a neutral pH range is administered to a living body, its uptake into cells in the body is promoted, thereby increasing the number of antigens that can be bound by a single antigen-binding molecule and promoting the elimination of plasma antigen concentration. This can be explained, for example, as follows.

[0294] For example, when an antibody whose antigen-binding molecule binds to a membrane antigen is administered to the body, the antibody binds to the antigen and is internalized together with the antigen into intracellular endosomes. The antibody then migrates to lysosomes while still bound to the antigen and is degraded by the lysosome along with the antigen. This elimination from plasma via internalization is called antigen-dependent elimination, and has been reported for many antibody molecules (Drug Discov Today (2006) 11(1-2), 81-88). When a single IgG antibody molecule binds to an antigen in a bivalent manner, the single antibody molecule is internalized while bound to two antigen molecules and is degraded in the lysosome. Therefore, in the case of a conventional antibody, a single IgG antibody molecule cannot bind to three or more antigen molecules. For example, a single IgG antibody molecule with neutralizing activity cannot neutralize three or more antigen molecules.

[0295] The relatively long plasma retention of IgG molecules (slow elimination) is due to the function of human FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to human FcRn expressed in endosomes under acidic conditions within the endosome. IgG molecules that fail to bind to human FcRn subsequently migrate to lysosomes where they are degraded. On the other hand, IgG molecules bound to human FcRn migrate to the cell surface. Under the neutral conditions of plasma, IgG molecules dissociate from human FcRn and are recycled back into the plasma.

[0296] Furthermore, when the antigen-binding molecule is an antibody that binds to a soluble antigen, the antibody binds to the antigen after administration and is then taken up into the cell while still bound to the antigen. Most of the antibodies taken up into the cell bind to FcRn in the endosome and then migrate to the cell surface. Under the neutral conditions of plasma, the antibody dissociates from human FcRn and is released extracellularly. However, antibodies containing a normal antigen-binding domain whose antigen-binding activity does not change depending on ionic concentration conditions such as pH are released extracellularly while still bound to the antigen and cannot re-bind to the antigen. Therefore, like antibodies that bind to membrane antigens, a normal single-molecule IgG antibody whose antigen-binding activity does not change depending on ionic concentration conditions such as pH cannot bind to three or more antigens.

[0297] Antibodies that bind to antigens in a pH-dependent manner (antibodies that bind to antigens in a neutral pH range in plasma and dissociate from antigens in the acidic pH range in endosomes) can dissociate from antigens in endosomes. Antibodies that bind to antigens in a calcium ion concentration-dependent manner (antibodies that bind to antigens in a high calcium ion concentration range in plasma and dissociate from antigens in the low calcium ion concentration range in endosomes) can dissociate from antigens in endosomes. Antibodies that bind to antigens in a pH-dependent manner or calcium ion concentration-dependent manner can dissociate from antigens after dissociation and are recycled into plasma by FcRn, allowing them to re-bind to antigens. This allows a single antibody molecule to repeatedly bind to multiple antigen molecules. Furthermore, antigens bound to antigen-binding molecules dissociate from antibodies in endosomes, resulting in degradation in lysosomes rather than being recycled into plasma. By administering such antigen-binding molecules to living organisms, the uptake of antigens into cells is promoted, making it possible to reduce the antigen concentration in plasma.

[0298] By imparting human FcRn-binding ability under neutral pH conditions (pH 7.4) to antibodies that bind to antigens in a pH-dependent manner, i.e., they bind strongly to antigens under neutral pH conditions in plasma and dissociate from antigens under acidic pH conditions in endosomes (antibodies that bind to antigens under neutral pH conditions and dissociate under acidic pH conditions), or antibodies that bind to antigens in a calcium ion concen...

Claims

1. Methods for producing antibodies, including the following: (A) A library of polypeptides in which each polypeptide contains an antigen-binding domain comprising a heavy chain variable domain (VH) sequence and a light chain variable domain (VL) sequence, and a selection of antigen-binding domains that bind to an antigen with the antigen-binding activity described in either (i) or (ii) below: (i) The antigen-binding activity is lower at an acidic pH than at a neutral pH; where the acidic pH is in the range of 4.0 to 6.5 and the neutral pH is in the range of 7.0 to 9.5; or (ii) The antigen-binding activity is lower at low calcium ion concentrations than at high calcium ion concentrations; where the low calcium ion concentration is a calcium ion concentration in the range of 0.1 μM to 30 μM, and the high calcium ion concentration is a calcium ion concentration in the range of 100 μM to 10 mM; and (B) A step to produce an antibody comprising (1) an antigen-binding domain comprising VH and VL of the selected antigen-binding domain, and (2) an Fc region in which EU numbering position 238 is Asp and EU numbering position 328 is Glu, wherein the Fc region (a) binds to human FcRn at pH 7.0 more strongly than the natural human IgG1 Fc region binds to the human FcRn region, and (b) binds to human FcγRIIb more strongly than to an active Fcγ receptor selected from human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), and human FcγRIIIa(F).

2. The method according to claim 1, wherein at least one of the following EU numbering positions in the Fc region contains the amino acid residue shown below: Asp ranked 233rd; Trp or Tyr ranked 234th; Ranked 237th are Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr; Asp ranked 239th; Ala, Gln, or Val are ranked 267th; Asn, Asp, or Glu are ranked 268th; Gly ranked 271st; Ranked 326th are Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr; Arg, Lys, or Met ranked 330th; Ile, Leu, or Met are ranked 323rd; Asp ranked 296th.

3. The method according to claim 1, wherein the antigen-binding activity of the selected antigen-binding domain changes at the pH described in (i) above, and the selected antigen-binding domain contains a histidine residue at one or more of the following Kabat numbering positions: VH ranked 27th, 31st, 32nd, 33rd, 35th, 50th, 58th, 59th, 61st, 62nd, 99th, 100bth, and 102nd, as well as VL ranked 24th, 27th, 28th, 31st, 32nd, 50th, 52nd, 53rd, 54th, 55th, 56th, 89th, 90th, 91st, 92nd, 93rd, and 94th.

4. The method according to claim 1, wherein the antigen-binding activity of the selected antigen-binding domain changes at the calcium ion concentration described in (ii) above, and at least one of the following Kabat numbering positions of the selected antigen-binding domain contains an amino acid residue having metal-chelating activity: VH ranked 95th, 96th, 100a, and 101st, and VL ranked 30th, 31st, 32nd, 50th, and 92nd.

5. The method according to claim 1, wherein the Fc region of the antibody is different from the natural human IgG1 Fc region by amino acid substitution at one or more positions, including at least one of EU numbering positions 248, 289, 314, 315, 360, 384, 386, 387, 389, and 424.

6. The method according to claim 1, wherein the Fc region of the antibody is different from the natural human IgG1 Fc region by amino acid substitution at one or more positions, including at least one of EU numbering positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436.

7. The method according to claim 1, wherein at least one of the following EU numbering positions in the Fc region of the antibody contains the amino acid residue shown below: Met ranked 237th; Ile ranked 248th; In 250th place are Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; Phe, Trp, or Tyr are ranked 252nd; Thr ranked 254th; Glu ranked 255th; Ranked 256th are Asn, Asp, Glu, or Gln; Ranked 257th are Ala, Gly, Ile, Met, Asn, Ser, Thr, or Val; His ranked 258th; Ala is ranked 265th; Ala or Glu ranked 286th; His ranked 289th; Ala is ranked 297th; Gly ranked 298th; Ala is ranked 303rd; Ala is ranked 305th; Ranked 307th are Ala, Asp, Phe, Gly, his, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; Ranked 308th are Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; Ala, Asp, Glu, Pro, or Arg are ranked 309th; Ala, His, or Ile are ranked 311th; Ala or His is ranked 312th; Lys or Arg are ranked 314th; Ala, Asp, or His are ranked 315th; Ala is ranked 317th; Val is ranked 332nd; Leu is ranked 334th; His ranked 360th; Ala is ranked 376th; Ala is ranked 380th; Ala is ranked 382nd; Ala is ranked 384th; Asp or His is ranked 385th; Pro ranked 386th; Glu ranked 387th; Ala or Ser is ranked 389th; Ala is ranked 424th; Ranked 428th are Ala, Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; Lys is ranked 433rd; 434th place: Ala, Phe, His, Ser, Trp, or Tyr; Ranked 436th are His, Ile, Leu, Phe, Thr, or Val.

8. The method according to claim 1, wherein the antigen-binding activity is lower at low calcium ion concentrations than at high calcium ion concentrations.

9. The method according to claim 8, wherein the low calcium ion concentration is a calcium ion concentration in the range of 0.5 μM to 10 μM, and the high calcium ion concentration is a calcium ion concentration in the range of 200 μM to 5 mM.

10. The method according to claim 8, wherein the low calcium ion concentration is a calcium ion concentration in the range of 1 μM to 5 μM, and the high calcium ion concentration is a calcium ion concentration in the range of 500 μM to 2.5 mM.

11. The method according to claim 1, wherein the antigen-binding activity is lower at an acidic pH than at a neutral pH.

12. The method according to claim 11, wherein the acidic pH is in the range of pH 4.5 to 6.5, and the neutral pH is in the range of pH 6.7 to 9.

5.

13. The method according to claim 11, wherein the acidic pH is in the range of pH 5.0 to 6.5, and the neutral pH is in the range of pH 7.0 to 9.

0.

14. The method according to claim 11, wherein the acidic pH is in the range of pH 5.5 to 6.5, and the neutral pH is in the range of pH 7.0 to 8.

0.

15. The method according to claim 11, wherein the acidic pH is pH 5.8 and the neutral pH is pH 7.

4.

16. An antibody produced by the method described in any one of claims 1 to 15.

Images