Il-8 binding antibody and use thereof

Abstract

To provide a human IL-8 binding antibody having improved pharmacokinetic characteristics pertaining to an antibody such as antibody half-life and / or ion concentration dependant antigen binding properties that improve antigen clearance from blood plasma.SOLUTION: The antibody includes an antigen binding domain the avidity of which varies with respect to an antigen depending on ion concentration conditions and is an IL-8 antibody including an amino acid replacement of a specific amino acid sequence. The increase of the isoelectric point (pI) by altering at least one amino acid residue exposed on the surface of the antibody enhances the clearance capacity of the antigen from plasma.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims priority from Japanese Priority Patent Application No. 2015-185254, filed in Japan on September 18, 2015, the contents of which are incorporated by reference in their entirety.

[0002] Technical Field In one non-exclusive aspect, the present disclosure provides anti-IL-8 antibodies, pharmaceutical compositions comprising the antibodies, nucleic acids encoding the antibodies, and host cells comprising the nucleic acids. It also provides methods for producing and using the IL-8 antibodies and pharmaceutical compositions, for example, in the treatment of IL-8-associated diseases. [Background technology]

[0003] Antibodies have attracted attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Numerous IgG-type therapeutic antibodies are commercially available, and numerous therapeutic antibodies are currently being developed (Reichert et al., Nat. Biotechnol. 23:1073-1078 (2005) (Non-Patent Document 1); Pavlou et al., Eur. J. Pharm. Biopharm. 59(3):389-396 (2005) (Non-Patent Document 2)). Meanwhile, various technologies have been developed for second-generation therapeutic antibodies, including those aimed at improving effector function, antigen binding ability, pharmacokinetics, or stability, and reducing the risk of immunogenicity (Kim et al., Mol. Cells. 20(1):17-29 (2005) (Non-Patent Document 3)). Because therapeutic antibodies generally require very high dosages, the development of therapeutic antibodies faces challenges such as the difficulty of preparing subcutaneously administered formulations and high production costs. Methods for improving the pharmacokinetics, pharmacodynamics, and antigen binding of therapeutic antibodies provide a means for reducing the dosage and manufacturing costs associated with therapeutic antibodies.

[0004] Substitution of amino acid residues in the constant region provides one method for improving the pharmacokinetics of antibodies (Hinton et al., J. Immunol. 176 (1):346-356 (2006) (Non-Patent Document 4); Ghetie et al., Nat. Biotechnol. 15(7):637-640 (1997) (Non-Patent Document 5)). Affinity maturation technology provides a method for increasing the antigen-neutralizing ability of antibodies (Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471 (2005) (Non-Patent Document 6); Wu et al., J. Mol. Biol. 368:652 (2007) (Non-Patent Document 7)), and introducing mutations into amino acid residues in the CDRs and / or framework regions of the antibody variable domain can increase the binding activity to an antigen. Improving the antigen-binding ability of an antibody can improve the in vitro biological activity of the antibody or reduce the dosage, and can also improve the drug efficacy in vivo (in the body) (Wu et al., J. Mol. Biol. 368:652-665 (2007) (Non-Patent Document 8)).

[0005] The amount of antigen that can be neutralized per antibody molecule depends on the affinity of the antibody for that antigen, so increasing the affinity allows for antigen neutralization with a smaller amount of antibody. It is conventionally possible to increase the affinity of an antibody for an antigen using various known methods (see, for example, Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471 (2005) (Non-Patent Document 6)). Furthermore, if an antibody can be covalently bound to an antigen and its affinity increased to infinity, it is theoretically possible for one antibody molecule to neutralize one antigen molecule (or two antigens if the antibody is bivalent). However, one of the limitations of therapeutic antibody development to date is that a single antibody molecule typically binds to and neutralizes only one antigen molecule (or two antigens if the antibody is bivalent). In recent years, it has been reported that the use of an antibody that binds to an antigen in a pH-dependent manner (hereinafter also referred to as a "pH-dependent antibody" or "pH-dependent binding antibody") enables a single antibody molecule to bind to and neutralize multiple antigen molecules (see, for example, WO2009 / 125825 (Patent Document 1); Igawa et al., Nat. Biotechnol. 28:1203-1207 (2010) (Non-Patent Document 9)). pH-dependent antibodies bind strongly to antigens under the neutral pH conditions of plasma and dissociate from the antigen under the acidic pH conditions in cellular endosomes. After dissociating from the antigen, the antibody is recycled into plasma by FcRn and is free to bind to and neutralize other antigen molecules, making it possible for a single pH-dependent antibody to repeatedly bind to and neutralize multiple antigen molecules.

[0006] It has been reported recently that antibody recycling properties can be achieved by using antibodies with calcium-dependent antigen-antibody interactions (hereinafter also referred to as "calcium ion concentration-dependent antibodies"), taking advantage of the difference in calcium (Ca) ion concentrations between plasma and endosomes (WO2012 / 073992 (Patent Document 2)). (Hereinafter, pH-dependent antibodies and calcium ion concentration-dependent antibodies will be collectively referred to as "pH / Ca concentration-dependent antibodies.")

[0007] IgG antibodies have a long plasma retention period due to their binding to FcRn. The binding between IgG antibodies and FcRn is strong under acidic pH conditions (e.g., pH 5.8), but hardly binds under neutral pH conditions (e.g., pH 7.4). IgG antibodies are nonspecifically taken up by cells, but return to the cell surface by binding to FcRn in endosomes under the acidic pH conditions of the endosome. IgG then dissociates from FcRn under the neutral pH conditions of plasma.

[0008] It has been reported that pH-dependent antibodies modified to enhance FcRn binding under neutral pH conditions have the ability to repeatedly bind to antigen molecules and eliminate them from plasma, and that administration of such antibodies can eliminate antigens from plasma (WO 2011 / 122011 (Patent Document 3)). According to this report, pH-dependent antibodies modified to enhance FcRn binding under neutral pH conditions (e.g., pH 7.4) can further accelerate antigen elimination compared to pH-dependent antibodies comprising the Fc region of a native IgG antibody (WO 2011 / 122011 (Patent Document 3)).

[0009] On the other hand, if mutations are introduced into the Fc region of an IgG antibody to abolish FcRn binding under acidic pH conditions, the antibody cannot be recycled from endosomes into plasma, significantly impairing plasma retention. Therefore, a method for improving plasma retention of IgG antibodies has been reported that enhances FcRn binding under acidic pH conditions. Introducing amino acid modifications into the Fc region of an IgG antibody to improve FcRn binding under acidic pH conditions increases the recycling efficiency from endosomes into plasma, thereby improving plasma retention. For example, the modifications M252Y / S254T / T256E (YTE; Dall'Acqua et al., J. Biol. Chem. 281:23514-235249 (2006) (Non-Patent Document 10)), M428L / N434S (LS; Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)) (Non-Patent Document 11), and N434H (Zheng et al., Clin. Pharm. & Ther. 89(2):283-290 (2011) (Non-Patent Document 12)) have been reported to result in increased antibody half-life compared to native IgG1.

[0010] However, antibodies containing such Fc region variants that have increased FcRn-binding activity under neutral or acidic pH conditions may be concerned about worsening immunogenicity or the incidence of aggregate formation. Furthermore, it has been reported that they also increase binding activity to anti-drug antibodies (hereinafter also referred to as "existing ADAs") (e.g., rheumatoid factor) that are present in patients prior to administration of a therapeutic antibody (WO 2013 / 046722 (Patent Document 4) and WO 2013 / 046704 (Patent Document 5)). WO 2013 / 046704 (Patent Document 5) reports that Fc region variants containing specific mutations (typically, two residue modifications, Q438R / S440E, as expressed in EU numbering) increase FcRn-binding activity under acidic pH conditions, and also demonstrate significantly reduced binding to rheumatoid factor compared to unmodified native Fc. However, WO2013 / 046704 (Patent Document 5) does not specifically demonstrate that this Fc region variant has superior plasma retention compared to antibodies having a native Fc region.

[0011] Therefore, there is a demand for safer and more advantageous Fc region variants that do not bind to existing ADAs and have improved plasma retention.

[0012] Reported effector functions of IgG antibodies include antibody-dependent cellular cytotoxicity (hereinafter referred to as "ADCC"), complement-dependent cytotoxicity (hereinafter referred to as "CDC"), and antibody-dependent cell-mediated phagocytosis (ADCP), which is the phagocytosis of target cells mediated by IgG antibodies. For an IgG antibody to mediate ADCC or ADCP activity, binding of the Fc region of the IgG antibody to an antibody receptor (also referred to as "Fcγ receptor," "FcgR," "Fc gamma receptor," or "FcγR" within the scope of Disclosure A of this specification) present on the surface of effector cells such as killer cells, natural killer cells, or activated macrophages is required. In humans, the FcγR family of proteins has been reported to include FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms, and each allotype has also been reported (Jefferis et al., Immunol. Lett. 82:57-65 (2002) (Non-Patent Document 13)). The balance of an antibody's affinity for activating receptors, including FcγRIa, FcγRIIa, FcγRIIIa, or FcγRIIIb, and inhibitory receptors, including FcγRIIb, is an important factor in optimizing the effector function of an antibody.

[0013] Various techniques have been reported to enhance or improve the activity of therapeutic antibodies against antigens. For example, because the binding activity of an antibody to activating FcγR plays an important role in the cytotoxic activity of the antibody, antibodies have been developed that target membrane antigens and have enhanced cytotoxic activity due to their enhanced binding activity to activating FcγR. See, for example, WO2000 / 042072 (Patent Document 6); WO2006 / 019447 (Patent Document 7); Lazar et al., Proc. Nat. Acad. Sci. USA. 103:4005-4010 (2006) (Non-Patent Document 14); Shinkawa et al., J. Biol. Chem. 278, 3466-3473 (2003) (Non-Patent Document 15); Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998) (Non-Patent Document 16); Clynes et al., Nat. Med. 6:443-446 (2000) (Non-Patent Document 17). Similarly, because binding activity to inhibitory FcγR (FcγRIIb in humans) plays an important role in immunosuppressive and agonistic activity, research has been conducted on antibodies targeting membrane antigens with enhanced binding activity to inhibitory FcγR (Li et al., Proc. Nat. Acad. Sci. USA. 109 (27):10966-10971 (2012) (Non-Patent Document 18)). Furthermore, the effects of FcγR binding of antibodies that bind to soluble antigens have been investigated primarily from the perspective of side effects (Scappaticci et al., J. Natl. Cancer Inst. 99 (16):1232-1239 (2007) (Non-Patent Document 19)). For example, when an antibody with enhanced binding to FcγRIIb is used as a pharmaceutical, it is expected to reduce the risk of producing anti-drug antibodies (Desai et al., J. Immunol. 178(10):6217-6226 (2007) (Non-Patent Document 20)).

[0014] Recently, it has been reported that introducing amino acid modifications into the Fc region of an IgG antibody to increase the binding activity of an antibody targeting a soluble antigen to activating and / or inhibitory FcγRs can further accelerate the elimination of the antigen from serum (WO2012 / 115241 (Patent Document 8), WO2013 / 047752 (Patent Document 9), WO2013 / 125667 (Patent Document 10), WO2014 / 030728 (Patent Document 11)). Furthermore, Fc region variants have been discovered that have binding activity to FcγRIIb that is not significantly different from that of the Fc region of a native IgG antibody, but have reduced activity against other activating FcγRs (WO2014 / 163101 (Patent Document 12)).

[0015] Because the plasma retention of soluble antigens is significantly shorter than that of antibodies with an FcRn-mediated recycling mechanism, soluble antigens can exhibit increased plasma retention and plasma concentration by binding to antibodies with such recycling mechanisms (e.g., antibodies that do not have the properties of pH / Ca concentration-dependent antibodies). Therefore, for example, if a soluble antigen in plasma has multiple physiological functions, even if one physiological function is blocked by antibody binding, the increased plasma retention and / or plasma concentration of the antigen due to antibody binding may result in the plasma concentration of the antigen exacerbating pathogenic symptoms caused by other physiological functions. In such cases, in addition to the methods of applying the above-mentioned modifications to antibodies to accelerate antigen elimination, methods have been reported that utilize the formation of multivalent immune complexes between multiple pH / Ca concentration-dependent antibodies and multiple antigens to enhance binding to FcRn, FcγR, and complement receptors (WO 2013 / 081143 (Patent Document 13)).

[0016] It has been reported that even when the Fc region is not modified, the blood half-life of an antibody can be controlled regardless of the target antigen or type of antibody by modifying amino acid residues that may be exposed on the surface of the antibody variable region so as to change the charge of those residues, thereby increasing or decreasing the isoelectric point (pI) of the antibody (WO 2007 / 114319 (Patent Document 14): Amino acid substitution technology mainly in FR; WO 2009 / 041643 (Patent Document 15): Amino acid substitution technology mainly in CDR). These documents show that lowering the pI of an antibody can extend the plasma half-life of the antibody, and conversely, increasing the pI of the antibody can shorten the plasma half-life of the antibody.

[0017] Regarding modification of the charge of amino acid residues in the constant region of an antibody, it has been reported that modifying the charge of specific amino acid residues, particularly in the CH3 domain, to increase the pI of the antibody can promote antigen uptake into cells, and it has also been described that such modifications preferably do not interfere with FcRn binding (WO 2014 / 145159 (Patent Document 16)). It has also been reported that modifying the charge of amino acid residues in the constant region of an antibody (mainly the CH1 domain) to lower the pI can extend the plasma half-life of the antibody, and that combining this with mutations in amino acid residues that increase FcRn binding can extend the plasma half-life of the antibody while increasing FcRn binding (WO 2012 / 016227 (Patent Document 17)).

[0018] On the other hand, when such modification techniques intended to increase or decrease the pI of an antibody are combined with other modification techniques that increase or decrease the binding to FcRn or FcγR, the effect on the plasma retention of the antibody or the promotion of antigen elimination from the plasma has not been clarified.

[0019] The extracellular matrix (ECM) is a structure that surrounds cells in vivo and is composed primarily of glycoproteins such as collagen, proteoglycans, fibronectin, and laminin. The role of the ECM in vivo is to create a microenvironment for cell survival, and the ECM is important for various cell functions such as cell proliferation and cell adhesion.

[0020] ECM has been reported to be involved in the in vivo pharmacokinetics of proteins administered to the body. The blood concentration of the VEGF-Trap molecule, a fusion protein of VEGF receptor and Fc, following subcutaneous administration was examined (Holash et al., Proc. Natl. Acad. Sci., 99(17):11393-11398 (2002) (Non-Patent Document 21)). VEGF-Trap molecules with a high pI have low plasma concentrations following subcutaneous administration, resulting in low bioavailability. Modified VEGF-Trap molecules with a lower pI due to amino acid substitutions have higher plasma concentrations and improved bioavailability. Furthermore, the correlation between the change in bioavailability and the binding strength to the ECM has revealed that the bioavailability of VEGF-Trap molecules following subcutaneous administration depends on the binding strength to the ECM at the subcutaneous site.

[0021] WO2012 / 093704 (Patent Document 18) reports that there is an inverse correlation between the binding ability of an antibody to ECM and its plasma retention, and therefore antibody molecules that do not bind to ECM have better plasma retention than antibodies that bind to ECM.

[0022] While techniques for reducing extracellular matrix binding have been reported to improve the in vivo bioavailability and plasma retention of proteins, the benefits of increasing antibody ECM binding have not been previously recognized.

[0023] Human IL-8 (Interleukin 8) is a member of the chemokine family, consisting of 72 or 77 amino acid residues in length. The term "chemokine" is a general term for a family of proteins with a molecular weight of 8-12 kDa and containing four cysteine residues that form intermolecular disulfide bonds. Chemokines are classified into CC chemokines, CXC chemokines, C chemokines, and CA3C chemokines based on the arrangement of their cysteines. IL-8 is classified as a CXC chemokine and is also called CXCL8.

[0024] IL-8 exists in solution in both monomeric and homodimeric forms. The IL-8 monomer contains an antiparallel β-sheet with a C-terminal α-helix that spans the β-sheet. The IL-8 monomer contains two disulfide bridges, between cysteines 7 and 34 and between cysteines 9 and 50 in the 72-amino acid form of IL-8. The IL-8 homodimer is stabilized by non-covalent interactions between the β-sheets of the two monomers, as there are no covalent bonds between the homodimer molecules.

[0025] IL-8 expression is induced in various cells, including peripheral blood monocytes, tissue macrophages, NK cells, fibroblasts, and vascular endothelial cells, in response to stimulation by inflammatory cytokines (Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014)).

[0026] Chemokines are generally undetectable or only weakly detectable in normal tissues, but are strongly detected at sites of inflammation, promoting leukocyte infiltration into inflamed tissues and contributing to the initiation of inflammation. IL-8 is a proinflammatory cytokine known to activate neutrophils, increase the expression of cell adhesion molecules, and enhance neutrophil adhesion to vascular endothelial cells. IL-8 also possesses neutrophil chemotactic activity, and IL-8 produced in injured tissues promotes the chemotaxis of neutrophils adhering to vascular endothelial cells into the tissue, inducing inflammation associated with neutrophil infiltration. IL-8 is also known to be a potent angiogenic factor for endothelial cells and to be involved in promoting tumor angiogenesis.

[0027] Inflammatory diseases associated with elevated (e.g., excessive) IL-8 levels include inflammatory skin diseases such as inflammatory keratosis (e.g., psoriasis), atopic dermatitis, and contact dermatitis; chronic inflammatory diseases that are autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and Behcet's disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, and drug-induced allergic hepatitis; inflammatory kidney diseases such as glomerulonephritis; inflammatory respiratory diseases such as bronchitis and asthma; inflammatory chronic vascular diseases such as atherosclerosis; multiple sclerosis, stomatitis, vocal cord inflammation, and inflammation associated with the use of artificial organs and / or blood vessels. Elevated (e.g., excessive) IL-8 levels are also associated with malignancies such as ovarian cancer, lung cancer, prostate cancer, gastric cancer, breast cancer, melanoma, head and neck cancer, and kidney cancer; sepsis due to infection; cystic fibrosis; and pulmonary fibrosis (see, e.g., Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014) (Non-Patent Document 22), which is incorporated herein by reference in its entirety).

[0028] High-affinity human anti-IL-8 antibodies have been developed as pharmaceutical compositions for some of these diseases (Desai et al., J. Immunol. 178(10):6217-6226 (2007) (Non-Patent Document 23)), but they have not yet been released on the market. To date, only one pharmaceutical composition containing an anti-IL-8 antibody has been commercially available, and this is a murine anti-IL-8 antibody for psoriasis as a topical medication. Novel anti-IL-8 antibodies for disease treatment are needed. [Prior art documents] [Patent documents]

[0029] [Patent Document 1] WO2009 / 125825 [Patent Document 2] WO2012 / 073992 [Patent Document 3] WO2011 / 122011 [Patent Document 4] WO2013 / 046722 [Patent Document 5] WO2013 / 046704 [Patent Document 6] WO2000 / 042072 [Patent Document 7] WO2006 / 019447 [Patent Document 8] WO2012 / 115241 [Patent Document 9] WO2013 / 047752 [Patent Document 10] WO2013 / 125667 [Patent Document 11] WO2014 / 030728 [Patent Document 12] WO2014 / 163101 [Patent Document 13] WO2013 / 081143 [Patent Document 14] WO2007 / 114319

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Non-licensed literature

[0030] [Non-licensed document 1] Reichert et al., Nat. Biotechnol. 23:1073-1078 (2005) [Non-licensed document 2] Pavlou et al., Eur. J. Pharm. Biopharm. 59(3):389-396 (2005) [Non-licensed document 3] Kim et al., Mol. Cells. 20 (1):17-29 (2005)

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[0031] In one non-exclusive aspect, a non-limiting object of an embodiment of Disclosure A is to provide molecules with improved pharmacokinetic properties relative to antibodies, such as ion concentration-dependent antigen binding, which improves antibody half-life and / or antigen clearance from plasma.

[0032] In one non-exclusive aspect, a non-limiting object of an embodiment of Disclosure B is to provide safer and more advantageous Fc region variants with increased half-life and reduced binding to pre-existing anti-drug antibodies (ADA).

[0033] In one non-exclusive aspect, a non-limiting object of an embodiment of Disclosure C is to provide an anti-IL-8 antibody having pH-dependent binding affinity for IL-8. An additional embodiment relates to an anti-IL-8 antibody that, when administered to an individual, has the effect of rapidly eliminating IL-8 compared to a reference antibody. In another embodiment, Disclosure C relates to an anti-IL-8 antibody that, when administered to an individual, can stably maintain IL-8 neutralizing activity. In some embodiments, the anti-IL-8 antibody exhibits reduced immunogenicity. In additional embodiments, Disclosure C relates to methods of producing and using the anti-IL-8 antibody. Another alternative, non-limiting object of Disclosure C is to provide a novel IL-8 antibody that can be included in a pharmaceutical composition.

[0034] In one non-exclusive aspect within the scope of Disclosure A provided herein, the present inventors surprisingly found that the ability of an ion concentration-dependent antibody (an antibody comprising an ion concentration-dependent antigen-binding domain (an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions)) to eliminate an antigen from plasma can be enhanced by increasing the isoelectric point (pI) by modifying at least one surface-exposed amino acid residue of the antibody. In another non-exclusive aspect, the present inventors found that an ion concentration-dependent antibody with an increased pI can further increase the binding ability of the antibody to the extracellular matrix. Without being bound by any particular theory, the present inventors have found that increasing the binding ability of the antibody to the extracellular matrix can enhance the elimination of antigens from plasma.

[0035] In one non-exclusive aspect within the scope of Disclosure B of the present specification, the present inventors conducted extensive research into safer and more advantageous Fc region variants that do not bind to anti-drug antibodies (existing ADAs) and can further improve plasma retention. As a result, the present inventors surprisingly found that, to achieve plasma retention of an antibody while maintaining significantly reduced binding to rheumatoid factor, an Fc region variant in which the amino acid at position 434 (EU numbering) is substituted with Ala (A) and which contains a specific two-residue mutation (typically Q438R / S440E (EU numbering)) as a combination of amino acid residue mutations is preferred.

[0036] In one non-exclusive embodiment within the scope of Disclosure C of the present specification, the inventors have produced a number of pH-dependent anti-IL-8 antibodies (anti-IL-8 antibodies that bind to IL-8 in a pH-dependent manner). After various tests, the inventors have found pH-dependent anti-IL-8 antibodies that, when administered to an individual, have the effect of rapidly eliminating IL-8 compared to a reference antibody. In some embodiments, Disclosure C relates to pH-dependent anti-IL-8 antibodies that can stably maintain IL-8 neutralizing activity. In a further non-limiting embodiment, the pH-dependent anti-IL-8 antibodies have low immunogenicity and excellent expression levels.

[0037] Furthermore, within the scope of Disclosure C, the present inventors have succeeded in obtaining an anti-IL-8 antibody comprising an Fc region whose binding affinity to FcRn at acidic pH is increased compared to the binding affinity of a native Fc region. In an alternative embodiment, the present inventors have succeeded in obtaining an anti-IL-8 antibody comprising an Fc region whose binding affinity to a pre-existing ADA is decreased compared to the binding affinity of a native Fc region. In an alternative embodiment, the present inventors have succeeded in obtaining an anti-IL-8 antibody comprising an Fc region whose plasma half-life is increased compared to the plasma half-life of a native Fc region. In an alternative embodiment, the present inventors have succeeded in obtaining a pH-dependent anti-IL-8 antibody comprising an Fc region whose binding affinity to an effector receptor is decreased compared to the binding affinity of a native Fc region. In another embodiment, the present inventors have discovered a nucleic acid encoding the above-mentioned anti-IL-8 antibody. In another embodiment, the present inventors have also discovered a host comprising the above-mentioned nucleic acid. In another aspect, the present inventors have discovered a method for producing the anti-IL-8 antibody, which comprises culturing the host described above. In another aspect, the present inventors have discovered a method for enhancing elimination of IL-8 from an individual relative to a reference antibody, which comprises administering the anti-IL-8 antibody to the individual.

[0038] In one embodiment, Disclosure A relates to, but is not limited to: [1] An antibody comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, wherein at least one amino acid residue that can be exposed on the antibody surface has been modified to increase the isoelectric point (pI). [2] The antibody according to [1], wherein the antigen is a soluble antigen. [3] the antibody of [1] or [2], wherein the antigen-binding domain has higher antigen-binding activity under a high ion concentration condition than under a low ion concentration condition; [4] The antibody according to any one of [1] to [3], wherein the ion concentration is hydrogen ion concentration (pH) or calcium ion concentration. [5] The antibody according to [4], wherein the ratio of KD (acidic pH range) / KD (neutral pH range) for the antigen in the acidic pH range to KD in the neutral pH range is 2 or more. [6] The antibody according to any one of [1] to [5], wherein at least one amino acid residue in the antigen-binding domain is substituted with histidine, or at least one histidine is inserted. [7] The antibody according to any one of [1] to [6], which can promote elimination of the antigen from plasma compared to the antibody before modification. [8] The antibody according to any one of [1] to [7], which has increased binding activity to an extracellular matrix compared to the antibody before modification. [9] The antibody according to any one of [1] to [8], wherein the amino acid residue modification is an amino acid residue substitution.

[10] The modification of the amino acid residue is (a) Substitution of a negatively charged amino acid residue with an uncharged amino acid residue; (b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue; and (c) Substitution of an uncharged amino acid residue with a positively charged amino acid residue The antibody according to any one of [1] to [9], selected from the group consisting of:

[11] The antibody according to any one of [1] to

[10] , wherein the antibody comprises a variable region and / or a constant region, and the amino acid residue modification is a modification of an amino acid residue in the variable region and / or the constant region.

[12] The antibody according to

[11] , wherein the variable region comprises a complementarity-determining region (CDR) and / or a framework region (FR).

[13] The variable region comprises a heavy chain variable region and / or a light chain variable region, and at least one amino acid residue is represented by the Kabat numbering system. (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44 in the heavy chain variable region FR; 46th, 68th, 71st, 72nd, 73rd, 75th, 76th, 77th, 81st, 82nd, 82a, 82b, 83rd, 84th, 85th, 86th, 105th, 108th, 110th, and 112th; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49 in the FR of the light chain variable region, and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region The antibody of

[12] , wherein the antibody is modified at a position in the CDR or FR selected from the group consisting of:

[14] At least one amino acid residue is (a) positions 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region The antibody of

[13] , wherein the antibody is modified at a position in the CDR or FR selected from the group consisting of:

[15] At least one amino acid residue is represented by EU numbering. 196th, 253rd, 254th, 256th, 258th, 278th, 280th, 281st, 282nd, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th, 342nd, 343rd, 345th, 356th, 358th, 359th, 361st, 362nd, 373th, 382nd, 384th, 385th, 386th, 387th, 389th, 399th, 400th, 401st, 402nd, 413th, 415th, 418th, 419th, 421st, 424th, 430th, 433th, 434th, and 443rd The antibody of any one of

[11] to

[14] , wherein the antibody is modified at a position in the constant region selected from the group consisting of:

[16] At least one amino acid residue is 254th, 258th, 281st, 282nd, 285th, 309th, 311th, 315th, 327th, 330th, 342nd, 343rd, 345th, 356th, 358th, 359th, 361st, 362nd, 384th, 385th, 386th, 387th, 389th, 399th, 400th, 401st, 402nd, 413th, 418th, 419th, 421st, 433rd, 434th, and 443rd The antibody of

[15] , wherein the antibody is modified at a position in the constant region selected from the group consisting of:

[17] At least one amino acid residue is represented by EU numbering. 282nd, 309th, 311th, 315th, 342nd, 343rd, 384th, 399th, 401st, 402nd, and 413th The antibody of

[16] , wherein the antibody is modified at a position in the constant region selected from the group consisting of:

[18] The antibody according to any one of [1] to

[17] , wherein the constant region has binding activity to Fc gamma receptor (FcγR), and the binding activity to FcγR is enhanced under neutral pH conditions, as compared to a reference antibody comprising the constant region of a native IgG.

[19] The antibody of

[18] , wherein the FcγR is FcγRIIb.

[20] The antibody of any one of [1] to

[17] , wherein the constant region has binding activity to one or more activating FcγRs selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa, as well as binding activity to FcγRIIb, and compared to a reference antibody that differs only in having a constant region that is the constant region of a native IgG, the binding activity to the FcγRIIb is maintained or enhanced, while the binding activity to the activating FcγR is decreased.

[21] The antibody according to any one of [1] to

[20] , wherein the constant region has FcRn-binding activity and has increased FcRn-binding activity under neutral pH conditions (e.g., pH 7.4) compared to a reference antibody that differs only in having a constant region that is the constant region of native IgG.

[22] The antibody according to any one of [1] to

[21] , which is a multispecific antibody that binds to at least two types of antigens.

[23] The antibody according to any one of [1] to

[22] , wherein the antibody is an IgG antibody.

[24] A pharmaceutical composition comprising the antibody according to any one of [1] to

[23] .

[25] The pharmaceutical composition according to

[24] , for promoting elimination of an antigen from plasma.

[26] The pharmaceutical composition according to

[24] or

[25] , for increasing antibody binding to an extracellular matrix.

[27] A nucleic acid encoding the antibody according to any one of [1] to

[23] .

[28] A vector comprising the nucleic acid according to

[27] .

[29] A host cell comprising the vector according to

[28] .

[30] A method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, the method comprising the steps of culturing the host cell of

[29] and recovering the antibody from the cell culture. [30A] A method for producing an antibody containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, the method comprising the step of modifying at least one amino acid residue that can be exposed on the antibody surface so as to increase the isoelectric point (pI). [30B] At least one amino acid residue is (I) Represented by Kabat numbering, (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44 in the heavy chain variable region FR; 46th, 68th, 71st, 72nd, 73rd, 75th, 76th, 77th, 81st, 82nd, 82a, 82b, 83rd, 84th, 85th, 86th, 105th, 108th, 110th, and 112th; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49 in the FR of the light chain variable region, and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region or a position in the CDR or FR selected from the group consisting of: (II) represented by EU numbering, 196th, 253rd, 254th, 256th, 258th, 278th, 280th, 281st, 282nd, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th, 342nd, 343rd, 345th, 356th, 358th, 359th, 361st, 362nd, 373th, 382nd, 384th, 385th, 386th, 387th, 389th, 399th, 400th, 401st, 402nd, 413th, 415th, 418th, 419th, 421st, 424th, 430th, 433th, 434th, and 443rd a position in the constant region selected from the group consisting of: The method according to [30A], wherein the method is modified by

[31] The modification of the amino acid residue is (a) Substitution of a negatively charged amino acid residue with an uncharged amino acid residue; (b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue; (c) substitution of an uncharged amino acid residue with a positively charged amino acid residue; and (d) Substitution of or insertion of histidine in CDR or FR The method of [30A] or [30B], comprising a modification selected from the group consisting of:

[32] Compared to a reference antibody, (a) selecting an antibody capable of promoting elimination of an antigen from plasma; (b) selecting an antibody having increased binding activity to the extracellular matrix; (c) selecting antibodies with increased FcγR-binding activity under neutral pH conditions (e.g., pH 7.4); (d) selecting an antibody having increased binding activity to FcγRIIb under a neutral pH condition (e.g., pH 7.4); (e) selecting antibodies that have reduced binding activity to one or more activating FcγRs preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa, while maintaining or increasing binding activity to FcγRIIb; (f) selecting antibodies with increased FcRn-binding activity under neutral pH conditions (e.g., pH 7.4); (g) selecting antibodies with increased isoelectric point (pI); (h) checking the isoelectric point (pI) of the recovered antibodies and then selecting antibodies with an increased isoelectric point (pI); and (i) selecting antibodies whose antigen-binding activity is altered or increased depending on ion concentration conditions; The method according to any one of

[30] or [30A] to [30C], optionally further comprising any one or more of:

[0039] Disclosure A relates, in alternative embodiments, to, but is not limited to, the following: [A1] An antibody having a constant region, wherein at least one amino acid residue selected from the same group of modification sites as the modification sites in the group defined in

[15] or

[16] has been modified in the constant region. [A2] The antibody of [A1], further comprising a heavy chain variable region and / or a light chain variable region, wherein the variable region comprises CDRs and / or FRs, and wherein at least one amino acid residue in the CDRs and / or FRs has been modified, the amino acid residue being selected from the same group of modification sites as the modification sites in the group defined in

[13] or

[14] . [A3] An antibody having a constant region, wherein at least one amino acid residue in the constant region selected from the group of modification sites identical to the modification sites in the group defined in

[15] or

[16] has been modified to increase the pI. [A4] The antibody according to [A3], further comprising a heavy chain variable region and / or a light chain variable region, wherein the variable region comprises CDRs and / or FRs, and wherein at least one amino acid residue selected from the same group of modification sites as the modification sites in the group defined in

[13] or

[14] has been modified in the CDRs and / or FRs. [A5] An antibody comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, wherein the antibody has a constant region in which at least one amino acid residue has been modified, the amino acid residue being selected from the same group of modification sites as the group of modification sites in

[15] or

[16] . [A6] The antibody of [A5], further comprising a heavy chain variable region and / or a light chain variable region, wherein the variable region comprises CDRs and / or FRs, and wherein at least one amino acid residue selected from the same group of modification sites as the modification sites in the group defined in

[13] or

[14] has been modified in the CDRs and / or FRs. [A7] Use of the antibody according to any one of [1] to

[23] and [A1] to [A6] in the manufacture of a pharmaceutical for promoting elimination of an antigen from plasma. [A8] Use of the antibody according to any one of [1] to

[23] and [A1] to [A6] in the manufacture of a pharmaceutical for increasing binding to an extracellular matrix. [A9] Use of the antibody according to any one of [1] to

[23] and [A1] to [A6] for eliminating an antigen from plasma. [A10] Use of the antibody according to any one of [1] to

[23] and [A1] to [A6] to enhance binding to an extracellular matrix. [A11] An antibody obtained by the method described in any one of

[30] , [30A], [30B],

[31] , and

[32] .

[0040] According to various embodiments, a combination of part or all of one or more elements described in any of [1] to

[30] , [30A], [30B],

[31] ,

[32] , and [A1] to [A11] above is included in Disclosure A, provided that such combination is not technically inconsistent based on the common general knowledge in the art. For example, in some embodiments, Disclosure A includes a method for producing a modified antibody comprising an antigen-binding domain that accelerates elimination of the antigen from plasma compared to before the antibody modification, comprising: (a) (I) Positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in the FR of the heavy-chain variable region, as indicated by Kabat numbering; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDR of the heavy-chain variable region; and (c) positions 31, 61, 62, 63, 64, 65, and 97 in the FR of the light-chain variable region. and (d) at positions in the CDRs or FRs selected from the group consisting of positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in the light chain variable region CDRs; or (II) 196th, 253rd, 254th, 256th, 258th, 278th, 280th, 281st, 282nd, expressed in EU numbering; , 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th, 342nd, 343rd, 345th, 356th, 3 58th, 359th, 361st, 362nd, 373rd, 382nd, 384th, 385th, 386th, 387th, 389th, 399th, 400th 401st, 402nd, 413th, 415th, 418th, 419th, 421st, 424th, 430th, 433rd, 434th, and 443rd at a position in the constant region selected from the group consisting of: modifying at least one amino acid residue that may be exposed on the surface of the antibody; (b) modifying the antigen-binding domain in such a manner that the resulting antigen-binding activity varies depending on ion concentration conditions, wherein (a) and (b) can be performed simultaneously or sequentially; (c) culturing the host cell to express nucleic acid encoding the modified antibody; and (d) recovering the modified antibody from the host cell culture. In further embodiments, the method optionally further comprises any one or more of the following: Compared to the antibody before modification, (e) selecting an antibody capable of promoting elimination of the antigen from plasma; (f) selecting an antibody having increased binding activity to the extracellular matrix; (g) selecting antibodies with increased FcγR-binding activity under neutral pH conditions (e.g., pH 7.4); (h) selecting an antibody having increased binding activity to FcγRIIb under a neutral pH condition (e.g., pH 7.4); (i) selecting an antibody that has reduced binding activity to one or more activating FcγRs preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa, while maintaining or increasing binding activity to FcγRIIb; (j) selecting an antibody having increased binding activity to FcRn under a neutral pH condition (e.g., pH 7.4); (k) selecting antibodies with increased isoelectric point (pI); (l) checking the isoelectric point (pI) of the recovered antibodies and then selecting antibodies with an increased isoelectric point (pI); and (m) A step of selecting antibodies whose antigen-binding activity is changed or increased depending on ion concentration conditions.

[0041] Other embodiments of Disclosure A relate to, for example but not limited to, the following inventions: [D1] (a) represented by EU numbering, 196th, 253rd, 254th, 256th, 258th, 278th, 280th, 281st, 282nd, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th, 342nd, 343rd, 345th, 356th, 358th, 359th, 361st, 362nd, 373th, 382nd, 384th, 385th, 386th, 387th, 389th, 399th, 400th, 401st, 402nd, 413th, 415th, 418th, 419th, 421st, 424th, 430th, 433th, 434th, and 443rd modifying a nucleic acid encoding the antibody before modification so as to alter the charge of at least one amino acid residue at a position selected from the group consisting of: (b) culturing the host cell so that the nucleic acid is expressed; and (c) recovering the antibody from the host cell culture a method for producing a modified antibody having a plasma half-life extended or shortened compared to the antibody before modification, comprising: [D2] Represented by EU numbering, 196th, 253rd, 254th, 256th, 258th, 278th, 280th, 281st, 282nd, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th, 342nd, 343rd, 345th, 356th, 358th, 359th, 361st, 362nd, 373th, 382nd, 384th, 385th, 386th, 387th, 389th, 399th, 400th, 401st, 402nd, 413th, 415th, 418th, 419th, 421st, 424th, 430th, 433th, 434th, and 443rd modifying at least one amino acid residue at a position selected from the group consisting of: 10. A method for extending or shortening the plasma half-life of an antibody, comprising:

[0042] Disclosure B, in one embodiment, relates to, for example, but not limited to, the following:

[33] A variant Fc region comprising an FcRn-binding domain, wherein the FcRn-binding domain comprises, as represented by EU numbering, Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440.

[34] the Fc region variant of

[33] , wherein the FcRn-binding domain comprises, as represented by EU numbering, Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440;

[35] the Fc region variant of

[33] or

[34] , wherein the FcRn-binding domain further comprises Ile or Leu at position 428 and / or Ile, Leu, Val, Thr, or Phe at position 436, as represented by EU numbering;

[36] the Fc region variant of

[35] , wherein the FcRn-binding domain comprises Leu at position 428 and / or Val or Thr at position 436, as represented by EU numbering;

[37] The FcRn-binding domain is represented by EU numbering. N434A / Q438R / S440E;N434A / Q438R / S440D; N434A / Q438K / S440E;N434A / Q438K / S440D; N434A / Y436T / Q438R / S440E;N434A / Y436T / Q438R / S440D;N434A / Y436T / Q438K / S440E;N434A / Y436T / Q438K / S440D;N434A / Y436V / Q438R / S440E;N434A / Y436V / Q438R / S440D;N434A / Y436V / Q438K / S440E;N434A / Y436V / Q438K / S440D;N434A / R435H / F436T / Q438R / S440E;N434A / R435H / F436T / Q438R / S440D;N434A / R435H / F436T / Q438K / S440E;N434A / R435H / F436T / Q438K / S440D;N434A / R435H / F436V / Q438R / S440E;N434A / R435H / F436V / Q438R / S440D;N434A / R435H / F436V / Q438K / S440 E;N434A / R435H / F436V / Q438K / S440D;M428L / N434A / Q438R / S440E;M428L / N434A / Q438R / S440D;M428L / N434A / Q438 K / S440E;M428L / N434A / Q438K / S440D;M428L / N434A / Y436T / Q438R / S440E;M428L / N434A / Y436T / Q438R / S440D;M42 8L / N434A / Y436T / Q438K / S440E;M428L / N434A / Y436T / Q438K / S440D;M428L / N434A / Y436V / Q438R / S440E;M428L / N43 4A / Y436V / Q438R / S440D;M428L / N434A / Y436V / Q438K / S440E;M428L / N434A / Y436V / Q438K / S440D;L235R / G236R / S2 39K / M428L / N434A / Y436T / Q438R / S440E; and L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E The Fc region variant of any of

[33] to

[36] , comprising a combination of substituted amino acids selected from the group consisting of:

[38] The FcRn-binding domain comprises any one of the following, represented by EU numbering: N434A / Q438R / S440E;N434A / Y436T / Q438R / S440E;N434A / Y436V / Q438R / S440E ;M428L / N434A / Q438R / S440E;M428L / N434A / Y436T / Q438R / S440E;M428L / N434 A / Y436V / Q438R / S440E;L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S44 0E; and L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E The Fc region variant described in

[37] , comprising a combination of substituted amino acids selected from the group consisting of:

[39] The Fc region variant of any of

[33] to

[38] , which has increased FcRn-binding activity under acidic pH conditions (e.g., pH 5.8) compared to the Fc region of native IgG.

[40] The Fc region variant according to any one of

[33] to

[39] , which does not have significantly increased binding activity to an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of a native IgG.

[41] The Fc region variant according to

[40] , wherein the anti-drug antibody (ADA) is rheumatoid factor (RF).

[42] The Fc region variant of any of

[33] to

[41] , which has a reduced plasma clearance (CL), an increased plasma residence time, or an increased plasma half-life (t1 / 2), compared to the Fc region of a native IgG.

[43] The Fc region variant of any of

[33] to

[42] , which has increased plasma retention compared to a reference Fc region variant comprising the following amino acid substitution combination: N434Y / Y436V / Q438R / S440E, as represented by EU numbering.

[44] An antibody comprising the Fc region variant of any one of

[33] to

[43] .

[45] The antibody according to

[44] , wherein the antibody is an IgG antibody.

[46] A pharmaceutical composition comprising the antibody of

[44] or

[45] .

[47] The pharmaceutical composition according to

[46] , for increasing the plasma retention of an antibody.

[48] A nucleic acid encoding the Fc region variant of any one of

[33] to

[43] , or the antibody of

[44] or

[45] .

[49] A vector comprising the nucleic acid according to

[48] .

[50] A host cell comprising the vector according to

[49] .

[51] A method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the same, comprising the steps of culturing the host cell of

[50] and recovering the Fc region variant or an antibody comprising the same from the cell culture.

[52] (a) selecting an Fc region variant that has increased FcRn-binding activity under acidic pH conditions compared to the Fc region of a native IgG; (b) selecting Fc region variants whose binding activity to anti-drug antibodies (ADA) under neutral pH conditions is not significantly increased compared to the Fc region of native IgG; (c) selecting an Fc region variant that exhibits increased plasma retention compared to the Fc region of native IgG; and (d) selecting an antibody comprising an Fc region variant that can promote elimination of the antigen from plasma, compared to a reference antibody comprising the Fc region of native IgG; The method of

[51] , optionally further comprising any one or more steps selected from the group consisting of:

[53] A method for producing an Fc region variant containing an FcRn-binding domain or an antibody comprising said variant, comprising the steps of substituting amino acids such that the resulting Fc region variant or an antibody comprising said variant contains Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, as indicated by EU numbering.

[0043] Disclosure B, in one embodiment, relates to, for example, but not limited to, the following: [B1] Use of an Fc region variant described in any one of

[33] to

[43] or an antibody described in

[44] or

[45] in the manufacture of a pharmaceutical for increasing plasma retention. [B2] Use of an Fc region variant according to any one of

[33] to

[43] or an antibody according to

[44] or

[45] in the manufacture of a pharmaceutical product that does not significantly increase the binding activity to an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of a native IgG. [B3] Use of an Fc region variant described in any one of

[33] to

[43] or an antibody described in

[44] or

[45] to increase the plasma retention time. [B4] Use of the Fc region variant described in any one of

[33] to

[43] or the antibody described in

[44] or

[45] to prevent a significant increase in binding activity to an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of native IgG. [B5] An Fc region variant or an antibody comprising said variant, obtained by the method described in any one of

[51] ,

[52] , and

[53] .

[0044] According to various embodiments, a combination of some or all of one or more elements described in any of

[33] to

[53] and [B1] to [B5] above is included in Disclosure B, provided that such a combination is not technically inconsistent based on common general knowledge in the art. For example, in some embodiments, Disclosure B includes Fc region variants comprising an FcRn-binding domain that may include the following: (a) Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, as expressed in EU numbering; (b) Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, as expressed in EU numbering; (c) Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr, or Phe at position 436; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, as indicated by EU numbering. (d) Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr, or Phe at position 436; Arg or Lys at position 438; and Glu or Asp at position 440, as indicated by EU numbering. (e) Leu at position 428; Ala at position 434; Val or Thr at position 436; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, as indicated by EU numbering; or (f) Leu at position 428; Ala at position 434; Val or Thr at position 436; Arg or Lys at position 438; and Glu or Asp at position 440, as indicated by EU numbering.

[0045] Disclosure C also relates, in one embodiment, to, for example, but not limited to, the following:

[54] An isolated anti-IL-8 antibody that binds to human IL-8, wherein the anti-IL-8 antibody contains at least one amino acid substitution in at least one of the following (a) to (f): (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

[55] The anti-IL-8 antibody of

[54] , which comprises amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO: 68, arginine at position 11 of the amino acid sequence of SEQ ID NO: 68, and tyrosine at position 3 of the amino acid sequence of SEQ ID NO: 69.

[56] the anti-IL-8 antibody of

[54] or

[55] , further comprising an amino acid substitution of alanine at position 6 of the amino acid sequence of SEQ ID NO: 68 and glycine at position 8 of the amino acid sequence of SEQ ID NO: 68;

[57] The anti-IL-8 antibody of any one of

[54] to

[56] , which comprises an amino acid substitution of asparagine at position 1 of the amino acid sequence of SEQ ID NO: 71, leucine at position 5 of the amino acid sequence of SEQ ID NO: 71, and glutamine at position 1 of the amino acid sequence of SEQ ID NO: 72.

[58] An anti-IL-8 antibody described in any one of

[54] to

[57] , comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 73; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 74.

[59] An anti-IL-8 antibody described in any one of

[54] to

[58] , comprising: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76.

[60] The anti-IL-8 antibody of any one of

[54] to

[59] , comprising a heavy chain variable region of SEQ ID NO: 78 and a light chain variable region of SEQ ID NO: 79.

[61] The anti-IL-8 antibody of any one of

[54] to

[60] , comprising an Fc region having at least one property selected from the following properties (a) to (f): (a) the binding affinity of the Fc region to FcRn at acidic pH is increased compared to the binding affinity of a native Fc region to FcRn; (b) the binding affinity of the Fc region to a pre-existing ADA is reduced compared to the binding affinity of a native Fc region to a pre-existing ADA; (c) the plasma half-life of the Fc region is increased compared to the plasma half-life of a native Fc region; (d) the plasma clearance of the Fc region is reduced compared to the plasma clearance of a native Fc region; and (e) the binding affinity of the Fc region to an effector receptor is reduced compared to the binding affinity of a native Fc region to the effector receptor; and (f) Increased binding to the extracellular matrix.

[62] the anti-IL-8 antibody of

[61] , wherein the Fc region comprises an amino acid substitution at one or more positions selected from the group consisting of positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438, and 440 (EU numbering);

[63] the anti-IL-8 antibody of

[62] , which comprises an Fc region containing one or more amino acid substitutions selected from the group consisting of L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E;

[64] the anti-IL-8 antibody of

[63] , wherein the Fc region comprises the following amino acid substitutions: L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E;

[65] the anti-IL-8 antibody of

[63] , wherein the Fc region comprises the following amino acid substitutions: L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E;

[66] An anti-IL-8 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence of SEQ ID NO: 82.

[67] An anti-IL-8 antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 80 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 82.

[68] An isolated nucleic acid encoding the anti-IL-8 antibody of any one of

[54] to

[67] .

[69] A vector containing the nucleic acid of

[68] .

[70] A host cell containing the vector of

[69] .

[71] A method for producing an anti-IL-8 antibody, comprising culturing the host according to

[70] .

[72] A method for producing the anti-IL-8 antibody of

[71] , comprising the step of isolating the antibody from the culture supernatant.

[73] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of

[54] to

[67] and a pharmaceutically acceptable carrier.

[74] The anti-IL-8 antibody of any one of

[54] to

[67] , for use in a pharmaceutical composition.

[75] The anti-IL-8 antibody of any one of

[54] to

[67] , for use in the treatment of a disease in which IL-8 is present in excess.

[76] Use of the anti-IL-8 antibody of any one of

[54] to

[67] in the manufacture of a pharmaceutical composition for a disease associated with the excessive presence of IL-8.

[77] A method for treating a patient suffering from a disease associated with excessive IL-8, comprising administering to the patient an anti-IL-8 antibody according to any one of

[54] to

[67] .

[78] A method for promoting elimination of IL-8 from an individual, comprising administering to the individual the anti-IL-8 antibody of any one of

[54] to

[67] .

[79] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of

[54] to

[67] , wherein the antibody binds to IL-8 and binds to the extracellular matrix.

[80] A method for producing an anti-IL-8 antibody comprising a variable region whose IL-8-binding activity is pH-dependent, comprising: (a) evaluating the binding of an anti-IL-8 antibody to an extracellular matrix; (b) selecting an anti-IL-8 antibody that has high binding to the extracellular matrix; (c) culturing a host containing a vector comprising nucleic acid encoding the antibody; and (d) isolating the antibody from the culture medium; A method for producing an anti-IL-8 antibody, comprising:

[0046] Disclosure C, in an alternative embodiment, relates to the following: [C1] Use of the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for inhibiting the accumulation of biologically active IL-8. [C2] Use of the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in suppressing the accumulation of biologically active IL-8. [C3] Use of an anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for inhibiting angiogenesis. [C4] Use of the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in inhibiting angiogenesis. [C5] Use of an anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for inhibiting the promotion of neutrophil migration. [C6] Use of the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in inhibiting the promotion of neutrophil migration. [C7] An anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31], for use in suppressing the accumulation of biologically active IL-8. [C8] A method for suppressing the accumulation of biologically active IL-8, comprising the step of administering to an individual the anti-IL-8 antibody described in any one of

[54] to

[67] and [C26] to [C31]. [C9] A pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, comprising the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C10] An anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] for use in inhibiting angiogenesis. [C11] A method for inhibiting angiogenesis in an individual, comprising the step of administering to the individual the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C12] A pharmaceutical composition for inhibiting angiogenesis, comprising the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C13] An anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] for use in inhibiting the promotion of neutrophil migration. [C14] A method for inhibiting the promotion of neutrophil migration in an individual, comprising the step of administering to the individual the anti-IL-8 antibody described in any one of

[54] to

[67] and [C26] to [C31]. [C15] A pharmaceutical composition for inhibiting the promotion of neutrophil migration, comprising the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C16] An anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31], for use in the treatment of a disease in which IL-8 is present in excess. [C17] Use of an anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for treating a disease in which IL-8 is present in excess. [C18] Use of the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in the treatment of a disease in which IL-8 is present in excess. [C19] A method for treating a disease in an individual where IL-8 is present in excess, comprising the step of administering to the individual the anti-IL-8 antibody described in any one of

[54] to

[67] and [C26] to [C31]. [C20] A pharmaceutical composition for treating a disease associated with excessive IL-8, comprising the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C21] An anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] for use in promoting elimination of IL-8. [C22] Use of an anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for promoting elimination of IL-8. [C23] Use of an anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31] in promoting elimination of IL-8. [C24] A method for promoting elimination of IL-8 in an individual, comprising the step of administering to the individual an anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C25] A pharmaceutical composition for promoting elimination of IL-8, comprising the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C26] An anti-IL-8 antibody comprising an Fc region containing an amino acid substitution at one or more positions selected from the group consisting of positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438, and 440 (EU numbering). [C27] The anti-IL-8 antibody of [C26], comprising an Fc region having at least one property selected from the following properties (a) to (f): (a) the binding affinity of the Fc region to FcRn at acidic pH is increased compared to the binding affinity of a native Fc region to FcRn; (b) the binding affinity of the Fc region to a pre-existing ADA is reduced compared to the binding affinity of a native Fc region to a pre-existing ADA; (c) the plasma half-life of the Fc region is increased compared to the plasma half-life of a native Fc region; (d) the plasma clearance of the Fc region is reduced compared to the plasma clearance of a native Fc region; (e) the binding affinity of the Fc region to an effector receptor is reduced compared to the binding affinity of a native Fc region to the effector receptor; and (f) Increased binding to the extracellular matrix. [C28] The anti-IL-8 antibody of [C26] or [C27], which comprises an Fc region containing one or more amino acid substitutions selected from the group consisting of L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E as defined by EU numbering. [C29] The anti-IL-8 antibody of [C28], comprising an Fc region containing one or more amino acid substitutions selected from the group consisting of: (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E; or (b) L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E, as represented by EU numbering. [C30] The anti-IL-8 antibody of [C26], which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence of SEQ ID NO: 82. [C31] The anti-IL-8 antibody of [C26], which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 82. [C32] An isolated nucleic acid encoding the anti-IL-8 antibody according to any one of [C26] to [C31]. [C33] A vector comprising the nucleic acid according to [C32]. [C34] A host cell containing the vector according to [C33]. [C35] A method for producing an anti-IL-8 antibody, comprising the step of culturing the host cell according to [C34]. [C36] A method for producing the anti-IL-8 antibody according to any one of [C26] to [C31], further comprising the step of isolating the antibody from a host cell culture. [C37] A pharmaceutical composition comprising the anti-IL-8 antibody according to any one of [C26] to [C31] and a pharmaceutically acceptable carrier. [C38] A method for treating a patient suffering from a disease associated with excessive IL-8, comprising administering to the patient the anti-IL-8 antibody described in any one of [C26] to [C31]. [C39] A method for promoting the elimination of IL-8 from an individual, comprising the step of administering to the individual an anti-IL-8 antibody according to any one of [C26] to [C31]. [C40] A method for inhibiting IL-8, comprising the step of contacting IL-8 with the anti-IL-8 antibody according to any one of

[54] to

[67] and [C26] to [C31]. [C41] The method according to [C40], wherein the biological activity of IL-8 is inhibited.

[0047] According to various embodiments, any combination of part or all of one or more elements described in any of

[54] to

[80] and [C1] to [C41] above is included in Disclosure C, unless such combination is technically inconsistent based on common general knowledge in the art. [Brief explanation of the drawings]

[0048] [Figure 1] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice administered with a human IL-6 receptor-binding antibody (Low_pI-IgG1) that binds to human IL-6 receptor in a pH-dependent manner and whose constant region is composed of native IgG1, and an antibody (High_pI-IgG1) in which the pI of the variable region of the same antibody has been increased. [Figure 2] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice administered with a human IL-6 receptor-binding antibody (Low_pI-F939) that binds to human IL-6 receptor in a pH-dependent manner and has been conferred FcRn-binding ability under neutral pH conditions, and with antibodies (Middle_pI-F939 and High_pI-F939) in which the pI of the variable region of said antibody has been increased. [Figure 3] FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice administered with a human IL-6 receptor-binding antibody (Low_pI-F1180) that binds to human IL-6 receptor in a pH-dependent manner and has enhanced FcγR-binding activity under neutral pH conditions, and antibodies (Middle_pI-F1180, High_pI-F1180) in which the pI of the variable region of said antibody has been increased. [Figure 4]FIG. 1 shows the time course of human IL-6 receptor concentration in the plasma of human FcRn transgenic mice, which maintain a steady-state plasma concentration of soluble human IL-6 receptor, when the following antibodies were administered to them: a human IL-6 receptor-binding antibody (Low_pI-IgG1) that binds to human IL-6 receptor in a pH-dependent manner and whose constant region is composed of native IgG1; an antibody (Low_pI-F11) that contains an Fc region modified to enhance FcRn binding under neutral pH conditions; and antibodies with increased pI of the variable regions of these antibodies (High_pI-IgG1, High_pI-F11). [Figure 5] The graph shows the degree of binding to the extracellular matrix of three antibodies with different pIs (Low_pI-IgG1, Middle_pI-IgG1, and High_pI-IgG1) that exhibit pH-dependent binding to the human IL-6 receptor, and two antibodies with different pIs (Low_pI(NPH)-IgG1 and High_pI(NPH)-IgG1) that do not exhibit pH-dependent binding to the human IL-6 receptor. Within the scope of Disclosure A of this specification, "NPH" means pH-independent. [Figure 6] The degree of binding to soluble human FcγRIIb of antibodies containing Fc region variants in which the pI of each antibody was increased by altering a single amino acid residue in the constant region of Ab1H-P600, an antibody that binds to IgE in a pH-dependent manner, was measured using BIACORE (registered trademark) and is shown as a relative value, with the value for Ab1H-P600 set to 1.00. [Figure 7] The intracellular uptake rate of antibodies containing Fc region variants in which the pI of each antibody has been increased by modifying a single amino acid residue in the constant region of Ab1H-P600 into hFcγRIIb-expressing cell lines was evaluated as a relative value, with the value for Ab1H-P600 set to 1.00. [Figure 8] 1 shows the degree of binding of Fv4-IgG1, which has the Fc region of native human IgG1, to rheumatoid factor in individual sera of RA patients. [Figure 9]1 shows the degree of binding of Fv4-YTE, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual sera of RA patients. [Figure 10] 1 shows the degree of binding of Fv4-LS, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 11] 1 shows the degree of binding of Fv4-N434H, an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 12] 1 shows the degree of binding of Fv4-F1847m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 13] 1 shows the degree of binding of Fv4-F1848m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 14] 1 shows the degree of binding of Fv4-F1886m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 15] 1 shows the degree of binding of Fv4-F1889m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 16] 1 shows the degree of binding of Fv4-F1927m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 17] 1 shows the degree of binding of Fv4-F1168m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients. [Figure 18] The graph shows the average binding affinity to rheumatoid factor in the serum of RA patients for Fv4-IgG1, which has the Fc region of native human IgG1, and antibodies containing novel Fc region variants that have Fc region variants with enhanced binding to various FcRns. [Figure 19]FIG. 1 shows the time courses of concentrations of various anti-human IgE antibodies in the plasma of cynomolgus monkeys when the anti-human IgE antibodies OHB-IgG1, which has the Fc region of native human IgG1, and antibodies containing novel Fc region variants with Fc region variants that have enhanced binding to FcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m) were administered to the cynomolgus monkeys. [Figure 20] FIG. 1 shows the time course of plasma concentration of anti-human IL-6 receptor antibody in human FcRn transgenic mice when Fv4-IgG1, an anti-human IL-6 receptor antibody having the Fc region of native human IgG1, or Fv4-F1718, an anti-human IL-6 receptor antibody with enhanced FcRn-binding ability under acidic pH conditions, was administered. [Figure 21] 1 shows sensorgrams obtained when the binding of H998 / L63 and Hr9 to IL-8 was measured by Biacore at pH 7.4 and pH 5.8. [Figure 22] This shows the time course of human IL-8 concentration in the plasma of mice administered with 2 mg / kg of H998 / L63 or H89 / L118 mixed with human IL-8. [Figure 23] This shows the time course of human IL-8 concentration in the plasma of mice administered with 2 mg / kg or 8 mg / kg of H89 / L118 mixed with human IL-8. [Figure 24] This shows the time course of human IL-8 concentration in the plasma of mice when H89 / L118 or H553 / L118 mixed with human IL-8 was administered to the mice at 2 mg / kg or 8 mg / kg, respectively. [Figure 25A] The graph shows the change in the relative chemiluminescence intensity of Hr9, H89 / L118, or H553 / L118 antibodies before storage in plasma over time as a function of antibody concentration. [Figure 25B] This shows the change in the relative chemiluminescence intensity of Hr9, H89 / L118, or H553 / L118 antibodies over time as a function of antibody concentration after storage in plasma for one week. [Figure 25C]This shows the change in the relative chemiluminescence intensity of Hr9, H89 / L118, or H553 / L118 antibodies over time as a function of antibody concentration after storage in plasma for 2 weeks. [Figure 26] The predicted ADA incidence rates for each anti-IL-8 antibody (hWS4, Hr9, H89 / L118, H496 / L118, or H553 / L118) as predicted by EpiMatrix and other existing therapeutic antibodies are shown. [Figure 27] The predicted ADA incidence rates for each anti-IL-8 antibody (H496 / L118, H496v1 / L118, H496v2 / L118, H496v3 / L118, H1004 / L118, or H1004 / L395) as predicted by EpiMatrix are shown, along with the predicted ADA incidence rates for other existing therapeutic antibodies. [Figure 28A] The graph shows the change in the relative chemiluminescence intensity of Hr9, H89 / L118, or H1009 / L395-F1886s antibodies before storage in plasma over time as a function of antibody concentration. [Figure 28B] The graph shows the change in the relative chemiluminescence intensity of Hr9, H89 / L118, or H1009 / L395-F1886s antibodies over time as a function of antibody concentration after storage in plasma for one week. [Figure 28C] This shows the change in the relative chemiluminescence intensity of Hr9, H89 / L118, or H1009 / L395-F1886s antibodies over time as a function of antibody concentration after storage in plasma for 2 weeks. [Figure 29] 1 shows the time course of human IL-8 concentration in the plasma of mice when H1009 / L395, H553 / L118, and H998 / L63 mixed with human IL-8 were each administered to the mice. [Figure 30] The graph shows the amount of Hr9, H89 / L118, or H1009 / L395 bound to the extracellular matrix when added alone to the extracellular matrix, and when added in combination with human IL-8. [Figure 31]This figure shows the time course of antibody concentration in the plasma of human FcRn transgenic mice when an antibody having the variable region of H1009 / L395 and an Fc region (F1942m) that does not bind to FcRn was administered to the mice alone or in mixture with human IL-8. [Figure 32] The predicted ADA incidence rates for H1009 / L395 and H1004 / L395, as well as the predicted ADA incidence rates for other existing therapeutic antibodies, are shown in Figure 1. [Figure 33] FIG. 1 shows the time courses of concentrations of various anti-human IL-8 antibodies in the plasma of cynomolgus monkeys when H89 / L118-IgG1, which has the variable region of H89 / L118 and the Fc region of native human IgG1, and antibodies with altered Fc regions that have enhanced FcRn binding (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m, and H89 / L118-F1927m) were administered to the cynomolgus monkeys. [Figure 34] FIG. 1 shows the binding ability to various FcγRs of antibodies having the variable region of H1009 / L395 and whose Fc region is a variant (F1886m, F1886s, or F1974m). [Figure 35] This graph shows the time course of human IL-8 concentration in the plasma of human FcRn transgenic mice administered with anti-IL-8 antibodies mixed with human IL-8. The anti-IL-8 antibodies used were H1009 / L395-IgG1 (2 mg / kg), which contains the variable region of H1009 / L395 and the Fc region of native human IgG1, and H1009 / L395-F1886s (2, 5, or 10 mg / kg), which contains the variable region of H1009 / L395 and an altered Fc region. [Figure 36] FIG. 1 shows the time course of antibody concentrations in the plasma of cynomolgus monkeys when Hr9-IgG1 or H89 / L118-IgG1, both of which contain the Fc region of native human IgG1, or H1009 / L395-F1886s or H1009 / L395-F1974m, both of which contain modified Fc regions, were administered to the cynomolgus monkeys. [Figure 37] 1 shows the time profiles of plasma IgE concentrations of several anti-IgE antibodies in C57BL6J mice from the perspective of antibody variable region modifications. [Figure 38A] Octet sensorgrams of 25 selected pH-dependent and / or calcium-dependent antigen-binding clones are shown. [Figure 38B] This is a continuation of Figure 38A. [Figure 38C] This is a continuation of Figure 38B. [Figure 38D] This is a continuation of Figure 38C. [Figure 39] 1 shows the time profiles of plasma C5 concentrations of several anti-C5 bispecific antibodies in C57BL6J mice in terms of antibody variable region modifications. [Figure 40] 1 shows the time profiles of plasma IgE concentrations of several anti-IgE antibodies in C57BL6J mice in terms of antibody constant region modifications. DETAILED DESCRIPTION OF THE INVENTION

[0049] Detailed Description The following describes non-limiting embodiments of Disclosures A, B, and C. All embodiments described in the following examples are intended to be considered as being described in the "Detailed Description" section without being bound by any patent practice, custom, law, or other restrictions that may restrict the content of the examples in any country in which the patent application is intended to be granted.

[0050] Disclosure A or Disclosure B In some embodiments, Disclosure A relates to an antibody comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, wherein at least one amino acid residue that can be exposed on the antibody surface has been modified to increase its isoelectric point (pI) (within the scope of Disclosure A, this antibody is also referred to as an "ion concentration-dependent antibody with increased pI," and the antigen-binding domain of this antibody is also referred to as an "ion concentration-dependent antigen-binding domain with increased pI"). This invention is based in part on the present inventors' surprising discovery that an ion concentration-dependent antibody in which at least one amino acid residue that can be exposed on the antibody surface has been modified to increase its isoelectric point (pI), can promote antigen elimination from plasma (e.g., when the antibody is administered in vivo); further, an ion concentration-dependent antibody with increased pI can increase the antibody's binding ability to the extracellular matrix. This invention is also based in part on the present inventors' surprising discovery that this advantageous effect is achieved by combining two entirely different concepts: the concept of an ion concentration-dependent antigen-binding domain or an ion concentration-dependent antibody, and the concept of an antibody whose pI has been increased by modifying at least one amino acid residue that can be exposed to the surface (also referred to as an "antibody with increased pI" within the scope of Disclosure A; conversely, within the scope of Disclosure A, an antibody whose pI has been decreased by modifying at least one amino acid residue that can be exposed to the surface is referred to as an "antibody with reduced pI"). Therefore, this invention is classified as a type of pioneer invention that can bring about significant technical innovation in the field to which Disclosure A belongs (e.g., the medical field).

[0051] For example, antibodies comprising an antigen-binding domain in which at least one amino acid residue that can be exposed on the antibody surface has been modified to increase the pI, and in which the antigen-binding domain has been further modified so that its antigen-binding activity changes depending on ion concentration conditions, are naturally included within the scope of Disclosure A of this specification (such antibodies are also referred to as "ion concentration-dependent antibodies with increased pI" within the scope of Disclosure A of this specification).

[0052] For example, antibodies comprising an ion concentration-dependent antigen-binding domain in which at least one amino acid residue that can be exposed on the antibody surface has a charge that differs from the charge of at least one amino acid residue at a corresponding position in the antibody before modification (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody or parent antibody (e.g., an antibody before modification, or an antibody before library creation or in the process of library creation)), and in which the pI of the antibody as a whole is increased, are naturally included in Disclosure A described herein (such antibodies are also referred to as "ion concentration-dependent antibodies with increased pI" within the scope of the description of Disclosure A in the present specification).

[0053] For example, antibodies that have an ion concentration-dependent antigen-binding domain and whose pI has been increased by modifying at least one amino acid residue that can be exposed on the surface of an unmodified antibody (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody or parent antibody (e.g., an antibody before modification, or an antibody before or in the process of being made into a library)) are naturally also included in Disclosure A described in the present specification (such antibodies are also referred to as "ion concentration-dependent antibodies with increased pI" within the scope of the description of Disclosure A in the present specification).

[0054] For example, antibodies comprising an ion concentration-dependent antigen-binding domain in which at least one amino acid residue that can be exposed on the antibody surface has been modified for the purpose of increasing the pI of the antibody are naturally included in Disclosure A described herein (such antibodies are also referred to as "ion concentration-dependent antibodies with increased pI" within the scope of Disclosure A described in the present specification).

[0055] Within the scope of Disclosures A and B herein, the term "amino acid" includes not only natural amino acids but also unnatural amino acids. Within the scope of Disclosures A and B herein, amino acids or amino acid residues may be designated by a one-letter code (e.g., A) or a three-letter code (e.g., Ala), or a combination thereof (e.g., Ala (A)).

[0056] In the context of Disclosures A and B, when the terms "amino acid modification," "amino acid residue modification," or equivalents are used, they may be understood to refer to, but are not limited to, chemical molecular modifications to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) specific amino acids (residues) in the amino acid sequence of an antibody, or the addition, deletion, substitution, or insertion of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) amino acids. Amino acid addition, deletion, substitution, or insertion can be performed on a nucleic acid encoding the amino acid sequence by, for example, site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82, 488-492 (1985)), overlap extension PCR, or by affinity maturation of antibodies, chain shuffling of antibody heavy or light chains, or by panning against an antigen using a phage display library (Smith et al., Methods Enzymol. 217, 228-257 (1993)). These methods may be performed alone or in combination, as appropriate. Such amino acid modifications are not limited to, but preferably involve substituting one or more amino acid residues in the antibody amino acid sequence with other amino acids (respectively). Amino acid addition, deletion, substitution, or insertion, as well as modification of the amino acid sequence by humanization or chimerization, can be performed by methods known in the art. Similarly, the variable regions or constant regions of the antibodies used to produce the antibodies of Disclosure A or B as recombinant antibodies may also be subjected to changes or modifications of amino acids (residues), such as addition, deletion, substitution, or insertion of amino acids.

[0057] In one embodiment within the scope of Disclosures A and B in the present specification, the substitution of an amino acid (residue) refers to a substitution with another amino acid (residue), and may be designed to modify, for example, the following points (a) to (c): (a) The backbone structure of the polypeptide in the sheet or helical regions; (b) the charge or hydrophobicity at the target site, or (c) Side chain size.

[0058] Amino acid residues are classified based on the properties of the side chains contained in their structure, for example into the following groups: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) Aromaticity: Trp, Tyr, Phe.

[0059] Substitutions of amino acid residues within each of these groups are called conservative substitutions, while substitutions of amino acid residues between other groups are called non-conservative substitutions. Amino acid substitutions may be conservative, non-conservative, or a combination thereof. Substitutions with amino acids other than natural amino acids can be achieved by a variety of known methods (Wang et al., Annu. Rev. Biophys. Biomol. Struct. 35, 225-249 (2006); Forster et al., Proc. Natl. Acad. Sci. USA 100 (11), 6353-6357 (2003)). 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, may be used.

[0060] Within the scope of Disclosures A and B herein, it is understood that the structure of an "antigen" is not limited to a specific structure, as long as the antigen contains an epitope that binds to an antibody. Antigens may be inorganic or organic. Antigens may be any ligand, including various cytokines such as interleukins, chemokines, and growth factors. Alternatively, receptors present in soluble form in biological fluids such as plasma or modified to be soluble may also be used as antigens. A non-limiting example of such a soluble receptor is the soluble IL-6 receptor described in Mullberg et al., J. Immunol. 152 (10), 4958-4968 (1994). Furthermore, antigens may be monovalent (e.g., soluble IL-6 receptor) or multivalent (e.g., IgE).

[0061] In one embodiment, the antigen that can be bound by the antibodies of Disclosures A and B is preferably a soluble antigen present in a biological fluid (e.g., a biological fluid listed in WO2013 / 125667, preferably plasma, interstitial fluid, lymph, ascites, or pleural effusion) of a subject (a subject to which the administration (application) of the antibody is intended within the scope of the description of Disclosures A and B in this specification, and which may be virtually any animal, for example, a human, a mouse, etc.), but may also be a membrane antigen.

[0062] Within the scope of Disclosures A and B of the present specification, the "prolongation of plasma half-life" or "shortening of plasma half-life" or equivalent expressions of a molecule of interest (which may be an antigen or an antibody) can be more specifically expressed by any other parameters, such as mean plasma residence time, plasma clearance (CL), and area under the concentration curve (AUC), in addition to the parameter of plasma half-life (t1 / 2) ("Pharmacokinetics: Understanding through Exercises" (Nanzando)). For example, these parameters can be specifically evaluated by performing noncompartmental analysis according to the instructions provided with the in vivo kinetic analysis software WinNonlin (Pharsight). It is known to those skilled in the art that these parameters are generally correlated with each other.

[0063] Within the scope of Disclosures A and B herein, the term "epitope" refers to an antigenic determinant present in an antigen, and means the site on the antigen to which an antigen-binding domain of an antibody binds. Thus, for example, an epitope can be defined based on its structure. Alternatively, an epitope can be defined by the binding activity of an antibody 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 glycan, the epitope can also be identified based on a specific glycan structure. The antigen-binding domains in Disclosures A and B may bind to the same or different epitopes in the antigen.

[0064] A linear epitope may be a primary sequence of amino acids, typically comprising at least three, and usually at least five, e.g., 8-10, or 6-20, unique amino acids.

[0065] In a conformational epitope, the amino acids that make up the epitope typically do not exist consecutively in the primary sequence.Antibodies can recognize conformational epitopes in the three-dimensional structure of a peptide or protein.Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-directed spin labeling, and electron paramagnetic resonance spectroscopy (Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.)).

[0066] Within the scope of disclosures A and B of this specification, the term "antibody" is not particularly limited and is used in the broadest sense as long as it can bind to a target antigen. Non-limiting examples of such antibodies include commonly known antibodies (e.g., natural immunoglobulins (abbreviated as "Ig")) or molecules or variants derived therefrom, such as Fab, Fab', F(ab')2, diabodies, and ScFv (Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; WO 93 / 11161; Peer et al., Nature Nanotechnology 2: 751-760 (2007)), minibodies (minibodies) (Orita et al., Blood 105: 562-566). (2005)), scaffold proteins, single-armed antibodies (including all embodiments of the single-armed antibodies described in WO2005 / 063816), and multispecific antibodies (e.g., bispecific antibodies: antibodies with specificity for two different epitopes, including antibodies that recognize different antigens and antibodies that recognize different epitopes on the same antigen). Within the scope of Disclosures A and B herein, "bispecific antibodies" may be prepared, but are not limited to, as antibody molecules having a common L chain, as described in WO2005 / 035756, or by a method described in WO2008 / 119353 in which two conventional antibodies having IgG4-like constant regions are mixed together and an exchange reaction occurs between these two such antibodies (known to those skilled in the art as the "Fab arm exchange" method). Alternatively, the antibody may be an antibody having a structure in which the heavy chain variable region and the light chain variable region are linked as a single chain (e.g., sc(Fv)2). Alternatively, it may be an antibody-like molecule (e.g., scFv-Fc) in which an scFv (or sc(Fv)2) consisting of a heavy chain variable region (VH) and a light chain variable region (VL) is linked to an Fc region (a constant region lacking the CH1 domain). Multispecific antibodies consisting of scFv-Fc have an (scFv)2-Fc structure in which the first polypeptide is VH1-linker-VL1-Fc and the second polypeptide is VH2-linker-VL2-Fc.Alternatively, the present invention may include antibody-like molecules in which a single-domain antibody is linked to an Fc region (Marvin et al., Curr. Opin. Drug Discov. Devel., 9(2): 184-193 (2006)), Fc fusion proteins (e.g., immunoadhesins) (US2013 / 0171138), functional fragments thereof, functional equivalents thereof, and glycosylated variants thereof. As used herein, native IgG (e.g., native IgG1) refers to a polypeptide that comprises the same amino acid sequence as an IgG found in nature (e.g., native IgG1) and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. Native IgG may also include naturally occurring mutants thereof.

[0067] Typically, when an antibody has a structure substantially identical to or similar to that of native IgG, the basic structure may be a Y-shaped four-chain structure (two heavy chain polypeptides and two light chain polypeptides). Typically, the heavy chain and the light chain can be linked together via disulfide bonds (SS bonds) to form a heterodimer. Such heterodimers may be linked together via disulfide bonds to form a Y-shaped heterotetramer. Note that the two heavy chains or the two light chains may be the same or different.

[0068] For example, papain digestion of an IgG antibody can cleave the hinge region (also referred to as "hinge" within the scope of Disclosures A and B of this specification) linking the Fab region of the heavy chain to the Fc region, yielding two Fab regions and one Fc region. Typically, the Fab region contains an antigen-binding domain. Phagocytes such as leukocytes and macrophages possess receptors (Fc receptors) that can bind to the Fc region, and through these Fc receptors, they can recognize and phagocytose antigen-bound antibodies (opsonization). The Fc region also mediates immune responses such as ADCC and CDC, and possesses effector functions that trigger responses after antibody antigen binding. It is known that antibody effector functions vary depending on the type of immunoglobulin (isotype). The Fc region of the IgG class can refer to, for example, but is not limited to, the region from cysteine at position 226 (in EU numbering) or proline at position 230 to the C-terminus. Alternatively, the Fc region can be suitably obtained by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody or the like with a protease such as pepsin, and then eluting the fraction adsorbed to a protein A or protein G column.

[0069] In the disclosures A and B of this specification, the positions of amino acid residues in the variable regions (CDRs and / or FRs) of an antibody are indicated according to Kabat, and the positions of amino acid residues in the constant region or Fc region are indicated according to EU numbering based on Kabat amino acid positions (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)).

[0070] Within the scope of disclosures A and B in this specification, the term "library" may refer to a molecule (population) of a plurality of antibodies having sequence diversity, each of whose individual sequences may be identical or different, a plurality of fusion polypeptides containing antibodies, or nucleic acids or polynucleotides encoding these amino acid sequences, as described in detail in WO2013 / 125667 (e.g., paragraphs 0121 to 0125), etc. For example, the library may contain antibodies in a population of at least 10 4 molecules, more preferably at least 10 5 molecules, more preferably at least 10 6 molecules, particularly preferably at least 10 7The library may contain more than one molecule. The library may also be a phage library. The term "mainly consisting of" means that a certain percentage of the numerous independent clones having different sequences in the library are antibodies that may differ in their antigen-binding activity. In one embodiment, an immune library constructed based on antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infectious diseases, humans whose blood antibody titers have been increased after vaccination, or patients with cancer or autoimmune diseases may be suitably used as a randomized variable region library. In an alternative embodiment, a naive library constructed from antibody genes derived from lymphocytes of healthy individuals and containing naive sequences, which are antibody sequences with no bias in its repertoire, may also be suitably used as a randomized variable region library (Gejima et al., Human Antibodies 11: 121-129 (2002); Cardoso et al., Scand. J. Immunol. 51: 337-344 (2000)). An amino acid sequence containing a naive sequence can refer to an amino acid sequence obtained from such a naive library. In an alternative embodiment, 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 sequences encoding codon sets of appropriate lengths can also be suitably used as a randomized variable region library. In this case, since sequence diversity of CDR3 genes is observed, it is also possible to replace only the heavy chain CDR3 sequence. A standard method for generating amino acid diversity in antibody variable regions may be to increase the variation of amino acid residues at positions that can be exposed on the antibody surface.

[0071] In one embodiment, when an antibody of Disclosure A or B has a structure substantially identical to or similar to that of, for example, a native Ig antibody, the antibody typically has a variable region ("V region") [a heavy chain variable region ("VH region") and a light chain variable region ("VL region")] and a constant region ("C region") [a heavy chain constant region ("CH region") and a light chain constant region ("CL region")]. The CH region is further divided into three regions, CH1 to CH3. Typically, the Fab region of the heavy chain contains a VH region and CH1, and the Fc region of the heavy chain typically contains CH2 and CH3. Typically, a hinge region is located between CH1 and CH2. Furthermore, the variable region typically has complementarity-determining regions ("CDRs") and framework regions ("FRs"). Typically, the VH region and VL region each contain three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR4). Typically, a total of six CDRs present in the variable regions of the heavy and light chains interact to form the antigen-binding domain of an antibody. In contrast, an antibody having only one CDR is known to have a lower binding affinity to an antigen than an antibody having six CDRs, but still has the ability to recognize and bind to the antigen.

[0072] Ig antibodies are divided into several classes (isotypes) based on differences in the structure of their constant regions. In most mammals, immunoglobulins are classified into five classes: IgG, IgA, IgM, IgD, and IgE, based on differences in the structure of their constant regions. In humans, IgG has four subclasses: IgG1, IgG2, IgG3, and IgG4, and IgA has two subclasses: IgA1 and IgA2. Heavy chains are divided into gamma, mu, alpha, delta, and epsilon chains based on differences in their constant regions, and based on these differences, there are five classes (isotypes) of immunoglobulins: IgG, IgM, IgA, IgD, and IgE. On the other hand, there are two types of light chains: lambda and kappa, and all immunoglobulins contain one of these.

[0073] In one embodiment, when an antibody of Disclosure A or B has a heavy chain, the heavy chain may be, for example, any one of a gamma chain, a mu chain, an alpha chain, a delta chain, and an epsilon chain, or may be derived from any one of them, and when an antibody of Disclosure A or B has a light chain, the light chain may be, for example, any one of a kappa chain and a lambda chain, or may be derived from either. Furthermore, within the scope of Disclosures A and B described herein, the antibody may be, but is not limited to, any isotype (e.g., IgG, IgM, IgA, IgD, or IgE) and any subclass (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, mouse IgG1, IgG2a, IgG2b, and IgG3), or may be derived from any one of them.

[0074] Within the scope of Disclosures A and B herein, the "antigen-binding domain" may have any structure as long as it binds to the target antigen. Examples of such domains include the variable regions of the heavy and light chains of an antibody (e.g., 1 to 6 CDRs); a module of about 35 amino acids called an A domain contained in Avimer, a cell membrane protein present in the body (WO2004 / 044011 and WO2005 / 040229); Adnectin, which contains a 10Fn3 domain that binds to a protein in fibronectin, a glycoprotein expressed on the cell membrane (WO2002 / 032925); Affibody, which has as its scaffold an IgG-binding domain that constitutes a bundle of three helices consisting of 58 amino acids of protein A (WO1995 / 001937); and DARPin (Designed Ankyrin Repeat: AR), which is a region exposed on the molecular surface of ankyrin repeats (AR) that have a structure in which subunits each consisting of one turn, two antiparallel helices, and one loop containing 33 amino acid residues are repeatedly stacked. Examples of such proteins include anticalins, which are highly conserved eight antiparallel chains supporting one side of a barrel structure twisted toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462); and variable lymphocyte receptors (VLRs), which do not have an immunoglobulin structure and are used as the adaptive immune system of jawless fish such as lampreys and hagfish, have a concave region formed by a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules are repeatedly stacked (WO2008 / 016854). Suitable antigen-binding domains in Disclosure A or B may include antigen-binding domains comprising the heavy and light chain variable regions of an IgG antibody, and more specifically, may include ScFv, single-chain antibody, Fv, scFv2 (single-chain Fv2), Fab, and F(ab')2.

[0075] In one embodiment of Disclosure A, the term "ion concentration" refers to, but is not limited to, hydrogen ion concentration (pH) or metal ion concentration. Here, "metal ions" can be any one of ions of Group I elements, such as alkali metals and copper group elements excluding hydrogen, Group II elements, such as alkaline earth metals and zinc group elements, Group III elements excluding boron, Group IV elements excluding carbon and silicon, Group VIII elements, such as iron group and platinum group elements, elements belonging to subgroups A of groups V, VI, and VII, and 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.

[0076] In one embodiment of Disclosure A, a suitable metal ion may be calcium ion, as described in detail in WO2012 / 073992 and WO2013 / 125667.

[0077] In one embodiment of Disclosure A, the "ionic concentration conditions" may be conditions that focus on the difference in biological behavior of an ion concentration-dependent antibody between low and high ionic concentrations. Furthermore, "the antigen-binding activity changes depending on the ionic concentration conditions" may mean that the antigen-binding activity of an ion concentration-dependent antigen-binding domain or ion concentration-dependent antibody in Disclosure A or B changes between low and high ionic concentrations. Examples of this include, but are not limited to, cases where the antigen-binding activity is higher (stronger) or lower (weaker) under high ionic concentrations than under low ionic concentrations.

[0078] In one embodiment of Disclosure A, the ion concentration can be hydrogen ion concentration (pH) or calcium ion concentration. When the ion concentration is hydrogen ion concentration (pH), the ion concentration-dependent antigen-binding domain can also be referred to as a "pH-dependent antigen-binding domain," and when the ion concentration is calcium ion concentration, it can also be referred to as a "calcium ion concentration-dependent antigen-binding domain."

[0079] In one embodiment within the context of Disclosure A, ion concentration-dependent antigen-binding domains, ion concentration-dependent antibodies, ion concentration-dependent antigen-binding domains with increased pIs, and ion concentration-dependent antibodies with increased pIs may be obtained from a library primarily consisting of antibodies with different sequences (having diversity), in which the antigen-binding domain contains at least one amino acid residue that changes the antigen-binding activity of the antigen-binding domain or antibody depending on ion concentration conditions. The antigen-binding domain may preferably be present in a light chain variable region (which may be modified) and / or a heavy chain variable region (which may be modified). Alternatively, a library may be prepared by combining such a light chain variable region or heavy chain variable region with a heavy chain variable region or light chain variable region prepared as a randomized variable region sequence library. Non-limiting examples of libraries include those in which the ion concentration is hydrogen ion concentration or calcium ion concentration, combining heavy chain variable regions prepared as a randomized variable region sequence library with light chain variable region sequences in which amino acid residues in germline sequences such as SEQ ID NO: 1 (Vk1), SEQ ID NO: 2 (Vk2), SEQ ID NO: 3 (Vk3), or SEQ ID NO: 4 (Vk4) are substituted with at least one amino acid residue that can alter antigen-binding activity in an ion concentration-dependent manner. Furthermore, when the ion concentration is calcium ion concentration, examples of libraries include those in which the heavy chain variable region sequence of SEQ ID NO: 5 (6RL#9-IgG1) or SEQ ID NO: 6 (6KC4-1#85-IgG1) is combined with light chain variable regions prepared as a randomized variable region sequence library or light chain variable regions with germline sequences.

[0080] In one embodiment, when the ion concentration is calcium ion concentration, the high calcium ion concentration is not particularly limited to a specific value, but may be a concentration selected from 100 μM to 10 mM, 200 μM to 5 mM, 400 μM to 3 mM, 200 μM to 2 mM, or 400 μM to 1 mM. A concentration selected from 500 μM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood) in vivo, may also be preferred. A low calcium ion concentration is not particularly limited to a specific value, but may also be a concentration selected from 0.1 μM to 30 μM, 0.2 μM to 20 μM, 0.5 μM to 10 μM, 1 μM to 5 μM, or 2 μM to 4 μM. A concentration selected from 1 μM to 5 μM, which is close to the calcium ion concentration in early endosomes in vivo, may also be preferred.

[0081] Whether the antigen-binding activity of an antigen-binding domain or an antibody comprising said domain changes depending on metal ion concentration (e.g., calcium ion concentration) conditions can be easily determined using known methods, such as those described in the context of Disclosure A of the present specification or WO2012 / 073992. For example, the antigen-binding activity of an antigen-binding domain or an antibody comprising said domain can be measured and compared under low and high calcium ion concentrations. In this case, it is preferable to keep the conditions other than the calcium ion concentration the same. Furthermore, conditions other than the calcium ion concentration when measuring antigen-binding activity can be appropriately selected by those skilled in the art. For example, measurements may be performed in HEPES buffer at 37°C, or using a BIACORE (GE Healthcare) or the like.

[0082] In one embodiment within the context of Disclosure A, the ion concentration-dependent antigen-binding domain, ion concentration-dependent antibody, ion concentration-dependent antigen-binding domain with an increased pI, or ion concentration-dependent antibody with an increased pI preferably has higher antigen-binding activity under high calcium ion concentrations than under low calcium ion concentrations. In such cases, the ratio of antigen-binding activity under low calcium ion concentrations to that under high calcium ion concentrations is not limited, but the ratio of the KD (dissociation constant) for the antigen under low calcium ion concentrations to the KD under high calcium ion concentrations, i.e., KD (3 μM Ca) / KD (2 mM Ca), is preferably 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 limited and may be any value, such as 400, 1,000, or 10,000.

[0083] The value of antigen-binding activity can be expressed as the dissociation constant (KD) when the antigen is soluble, or as the apparent dissociation constant (KD) when the antigen is membrane-type. KD and apparent KD can be measured by known methods, such as BIACORE (GE Healthcare), Scatchard plots, or a flow cytometer.

[0084] Alternatively, the dissociation rate constant (kd) may be used as an indicator of the binding activity ratio. When the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator of the antigen-binding activity ratio, the ratio of the dissociation rate constant (kd) for the antigen under low calcium ion concentration conditions to the dissociation rate constant (kd) under high calcium ion concentration conditions, i.e., kd (low calcium ion concentration conditions) / kd (high calcium ion concentration conditions), is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. There is no upper limit to the value of kd (low calcium ion concentration conditions) / kd (high calcium ion concentration conditions), and it may be any value, such as 50, 100, or 200.

[0085] When the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as the value of antigen-binding activity. When the antigen is a membrane-type antigen, the apparent dissociation rate constant (kd) can be used. The dissociation rate constant (kd) and apparent dissociation rate constant (kd) can be measured by known methods, for example, by BIACORE (GE Healthcare) or a flow cytometer.

[0086] In one embodiment, the method for producing or screening for a calcium ion concentration-dependent antigen-binding domain or calcium ion concentration-dependent antibody, or a library thereof, that has higher antigen-binding activity under a high calcium ion concentration condition than under a low calcium ion concentration condition, is not limited to, but examples thereof include the methods described in WO2012 / 073992 (e.g., paragraphs 0200 to 0213).

[0087] Such methods include, for example, (a) determining the antigen-binding activity of an antigen-binding domain or antibody under a low calcium ion concentration condition; (b) determining the antigen-binding activity of the antigen-binding domain or antibody under high calcium ion concentration conditions; and (c) selecting antigen-binding domains or antibodies whose antigen-binding activity at low calcium ion concentrations is lower than that at high calcium ion concentrations; The method may include:

[0088] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under conditions of a high calcium ion concentration; (b) incubating the antigen-binding domain or antibody bound to the antigen in step (a) under conditions of low calcium ion concentration; and (c) isolating the antigen-binding domain or antibody dissociated in step (b). The method may include:

[0089] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under conditions of a low calcium ion concentration; (b) selecting antigen-binding domains or antibodies that do not bind to the antigen or have low antigen-binding ability in step (a); (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to the antigen under conditions of a high calcium ion concentration; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c). The method may include:

[0090] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with a column onto which an antigen has been immobilized under conditions of high calcium ion concentration; (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under conditions of a low calcium ion concentration; and (c) isolating the antigen-binding domain or antibody eluted in step (b). The method may include:

[0091] Or, for example, (a) passing antigen-binding domains or antibodies, or a library thereof, through an antigen-immobilized column under conditions of low calcium ion concentration, and recovering antigen-binding domains or antibodies that do not bind to the column and are eluted; (b) allowing the antigen-binding domain or antibody recovered in step (a) to bind to an antigen under conditions of a high calcium ion concentration; and (c) isolating the antigen-binding domain or antibody that bound to the antigen in step (b). The method may include:

[0092] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under conditions of a high calcium ion concentration; (b) obtaining the antigen-binding domain or antibody bound to the antigen in step (a); (c) incubating the antigen-binding domain or antibody obtained in step (b) at a low calcium ion concentration; and (d) isolating the antigen-binding domains or antibodies of step (c) whose binding activity to the antigen is weaker than the standard selected in step (b). The method may include:

[0093] Each step in these various screening methods may be repeated multiple times or may be combined as appropriate to obtain the optimal molecule. The low calcium ion concentration and high calcium ion concentration conditions may be selected as appropriate from the conditions described above. This allows the desired calcium ion concentration-dependent antigen-binding domain or calcium ion concentration-dependent antibody to be obtained.

[0094] In the context of Disclosure A, in one embodiment, the starting antigen-binding domain or antibody may be a modified antigen-binding domain or antibody having an increased pI, for example, by modifying the charge of at least one amino acid residue that may be exposed on its surface. In an alternative embodiment, when an amino acid that alters the binding activity of the ion concentration-dependent antigen-binding domain is introduced into the sequence, a modification that increases the pI may be introduced in addition to modifying the charge of at least one amino acid residue that may be exposed on the surface of the antigen-binding domain or antibody.

[0095] Alternatively, in the context of Disclosure A, for example, existing antigen-binding domains or antibodies, existing libraries (such as phage libraries), hybridomas obtained by immunizing animals, or antibodies or libraries thereof produced from B cells derived from immunized animals may be used, or antigen-binding domains, antibodies, or libraries obtained by introducing natural or unnatural amino acid mutations (described below) capable of chelating calcium into these may be used (for example, libraries with an increased content of amino acids capable of chelating calcium, or libraries into which amino acids capable of chelating calcium have been introduced at specific sites).

[0096] In one embodiment within the context of Disclosure A, when the ion concentration is calcium ion concentration, the amino acid that alters the binding activity of the ion concentration-dependent antigen-binding domain or the ion concentration-dependent antigen-binding domain with an increased pI is not limited to any particular type, as long as it is an amino acid that can form a calcium-binding motif. For example, calcium binding motifs are well known to those skilled in the art (e.g. Springer et al. (Cell 102: 275-277 (2000)); Kawasaki et al. (Protein Prof. 2: 305-490 (1995)); Moncrief et al. (J. Mol. Evol. 30: 522-562 (1990)); Chauvaux et al. al.(Biochem. J. 265: 261-265 (1990));Bairoch et al.(FEBS Lett. 269: 454-456 (1990));Davis(New Biol. 2: 410-419 (1990));Schaefer et al.(Genomics 25: 638-643 (1995));Economou et al.(EMBO J. 9: 349-354 (1990)); Wurzburg et al. (Structure. 14(6): 1049-1058 (2006)). Therefore, when an antigen-binding domain has any calcium-binding motif of a C-type lectin, such as ASGPR, CD23, MBR, or DC-SIGN, the antigen-binding activity of the domain can be changed depending on calcium ion concentration conditions. In addition to the above, examples of such calcium-binding motifs include the calcium-binding motif set forth in SEQ ID NO: 7 (corresponding to "Vk5-2") contained in the antigen-binding domain.

[0097] In one embodiment within the context of Disclosure A, when the ion concentration is calcium ion concentration, an amino acid with metal chelating activity may be used as an amino acid that alters the binding activity of an ion concentration-dependent antigen-binding domain or an ion concentration-dependent antigen-binding domain with an increased pI. As examples of amino acids with metal chelating activity, any amino acid may be suitably used as long as it forms a calcium-binding motif, but specific examples include electron-donating amino acids. Suitable examples of such amino acids include, but are not limited to, Ser (S), Thr (T), Asn (N), Gln (Q), Asp (D), and Glu (E).

[0098] The position of such an amino acid having metal chelating activity in an antigen-binding domain is not limited to a specific position. In one embodiment, the amino acid may be located at any position in the heavy chain variable region and / or light chain variable region that may form the antigen-binding domain. For example, at least one amino acid residue that changes the antigen-binding activity of an antibody in a calcium ion concentration-dependent manner may be contained in the CDRs (one or more of CDR1, CDR2, and CDR3) and / or FRs (one or more of FR1, FR2, FR3, and FR4) of the heavy chain and / or light chain. The amino acid residue may be located, for example, at one or more of positions 95, 96, 100a, and 101 (Kabat numbering) in heavy chain CDR3, at one or more of positions 30, 31, and 32 (Kabat numbering) in light chain CDR1, at position 50 (Kabat numbering) in light chain CDR2, and / or at position 92 (Kabat numbering) in light chain CDR3. These amino acid residues may be located alone or in combination.

[0099] Troponin C, calmodulin, parvalbumin, myosin light chain, and the like are known to have multiple calcium-binding sites and are thought to have a common origin in molecular evolution. In one embodiment, one or more of the light chain CDR1, CDR2, and CDR3 can be designed to contain this binding motif. 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 receptor and mannose-binding receptor, an A domain contained in the LDL receptor, annexin, a thrombospondin type 3 domain, and an EGF-like domain can be used as appropriate.

[0100] In one embodiment, when the ion concentration is hydrogen ion concentration (pH), the term proton, i.e., the concentration of hydrogen atom nuclei, is treated synonymously with the term hydrogen exponent (pH). The activity of hydrogen ions in an aqueous solution is expressed as aH + When expressed as -log10aH, pH is + It is defined as follows. When the ionic strength in the aqueous solution is (for example, 10 -3 If it is lower than aH + is approximately equal to the hydrogen ion 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 -7In this case, a pH of 7 is neutral, an aqueous solution with a pH lower than 7 is acidic, and an aqueous solution with a pH higher than 7 is alkaline. Therefore, the hydrogen ion concentration conditions may be hydrogen ion concentration conditions or pH conditions that take into account the difference in the biological behavior of pH-dependent antibodies between high hydrogen ion concentrations (acidic pH range) and low hydrogen ion concentrations (neutral pH range). For example, in the context of Disclosure A, "the antigen-binding activity under a high proton concentration (acidic pH range) is lower than that under a low proton concentration (neutral pH range)" may mean that the antigen-binding activity of an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with an increased pI, or an ion concentration-dependent antibody with an increased pI is weaker at a pH selected from pH 4.0 to pH 6.5, preferably pH 4.5 to pH 6.5, more preferably pH 5.0 to pH 6.5, and even more preferably pH 5.5 to pH 6.5, than at a pH selected from pH 6.7 to pH 10.0, preferably pH 6.7 to pH 9.5, more preferably pH 7.0 to pH 9.0, and even more preferably pH 7.0 to pH 8.0. Preferably, the above expression may mean that the antigen-binding activity at the pH in the in vivo early endosome is weaker than the antigen-binding activity at the pH in the in vivo plasma, specifically, that the antigen-binding activity of an antibody at, for example, pH 5.8 is weaker than the antigen-binding activity at, for example, pH 7.4.

[0101] Whether the hydrogen ion concentration conditions affect the antigen-binding activity of an antigen-binding domain or an antibody comprising said domain can be easily determined using known methods, such as the assay methods described in this specification in the context of Disclosure A or WO2009 / 125825. For example, the antigen-binding activity of an antigen-binding domain or an antibody comprising said domain for an antigen of interest can be measured and compared under low and high hydrogen ion concentrations. In this case, it is preferable to keep the conditions other than the hydrogen ion concentration the same. Furthermore, conditions other than the hydrogen ion concentration when measuring antigen-binding activity can be appropriately selected by those skilled in the art. For example, measurements can be performed in HEPES buffer at 37°C, or using a BIACORE (GE Healthcare) or the like.

[0102] Within the scope of Disclosure A of this specification, unless otherwise specified by the context, the "neutral pH range" (also referred to as "low hydrogen ion concentration," "high pH," "neutral pH conditions," or "neutral pH") is not particularly limited to a particular value, but may be preferably selected from pH 6.7 to pH 10.0, pH 6.7 to pH 9.5, pH 7.0 to pH 9.0, or pH 7.0 to pH 8.0. Preferably, the neutral pH range is pH 7.4, which is close to the pH in plasma (blood) in vivo, but for convenience of measurement, for example, pH 7.0 may be used.

[0103] Within the scope of Disclosure A of the present specification, unless otherwise specified by the context, the "acidic pH range" (also referred to as "high hydrogen ion concentration," "low pH," "acidic pH conditions," or "acidic pH") is not particularly limited to a particular value, but may be preferably selected from pH 4.0 to pH 6.5, pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. Preferably, the acidic pH range is pH 5.8, which is close to the hydrogen ion concentration in early endosomes in vivo, but for convenience of measurement, for example, pH 6.0 may be used.

[0104] In one embodiment within the context of Disclosure A, when the ion concentration is hydrogen ion concentration, the antigen-binding activity of an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with an increased pI, or an ion concentration-dependent antibody with an increased pI is preferably higher under neutral pH conditions than under acidic pH conditions. In such cases, the ratio of the antigen-binding activity under neutral pH conditions to the antigen-binding activity under acidic pH conditions is not limited, but preferably, the ratio of the antigen dissociation constant (KD) under acidic pH conditions to the KD under neutral pH conditions, i.e., KD (acidic pH range) / KD (neutral pH range) (e.g., KD (pH 5.8) / KD (pH 7.4)), may be 2 or greater, 10 or greater, or 40 or greater. The upper limit of KD (acidic pH range) / KD (neutral pH range) is not limited and may be any value, such as 400, 1,000, or 10,000.

[0105] In an alternative embodiment, the dissociation rate constant (kd) may be used as an indicator of the binding activity ratio. When the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator of the binding activity ratio, the ratio of the dissociation rate constant (kd) at a high proton concentration to the dissociation rate constant (kd) at a low proton concentration for the antigen, i.e., the value of kd (acidic pH range) / kd (neutral pH range), may be 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the value of kd (acidic pH range) / kd (neutral pH range) is not limited and may be any value, such as 50, 100, or 200.

[0106] The value of antigen-binding activity can be expressed as the dissociation rate constant (kd) when the antigen is a soluble antigen, whereas such a value can be expressed as the apparent dissociation rate constant (apparent kd) when the antigen is a membrane-type antigen. The dissociation rate constant (kd) and the apparent dissociation rate constant (apparent kd) can be measured by known methods, such as using BIACORE (GE Healthcare) or a flow cytometer.

[0107] In one embodiment, methods for producing or screening for pH-dependent antigen-binding domains or pH-dependent antibodies that have higher antigen-binding activity under neutral pH conditions than under acidic pH conditions, or libraries thereof, include, but are not limited to, the methods described in WO2009 / 125825 (e.g., paragraphs 0158 to 0190), etc.

[0108] The method may, for example, (a) determining the antigen-binding activity of an antigen-binding domain or antibody under acidic pH conditions; (b) determining the antigen-binding activity of the antigen-binding domain or antibody under neutral pH conditions; and (c) selecting an antigen-binding domain or antibody whose antigen-binding activity under acidic pH conditions is lower than that under neutral pH conditions; The method may include:

[0109] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under neutral pH conditions; (b) incubating the antigen-binding domain or antibody bound to the antigen in step (a) under acidic pH conditions; and (c) isolating the antigen-binding domain or antibody dissociated in step (b). The method may include:

[0110] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under acidic pH conditions; (b) selecting antigen-binding domains or antibodies that do not bind to the antigen or have low antigen-binding ability in step (a); (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to the antigen under neutral pH conditions; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c). The method may include:

[0111] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with a column onto which an antigen has been immobilized under neutral pH conditions; (b) eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under acidic pH conditions; and (c) isolating the antigen-binding domain or antibody eluted in step (b). The method may include:

[0112] Or, for example, (a) passing antigen-binding domains or antibodies, or a library thereof, through an antigen-immobilized column under acidic pH conditions, and recovering antigen-binding domains or antibodies that do not bind to the column and are eluted; (b) allowing the antigen-binding domain or antibody recovered in step (a) to bind to an antigen under neutral pH conditions; and (c) isolating the antigen-binding domain or antibody that bound to the antigen in step (b). The method may include:

[0113] Or, for example, (a) contacting an antigen-binding domain or antibody, or a library thereof, with an antigen under neutral pH conditions; (b) obtaining the antigen-binding domain or antibody bound to the antigen in step (a); (c) incubating the antigen-binding domain or antibody obtained in step (b) under acidic pH conditions; and (d) isolating the antigen-binding domains or antibodies of step (c) whose binding activity to the antigen is weaker than the standard selected in step (b). The method may include:

[0114] Each step in these various screening methods may be repeated multiple times or may be combined. The acidic pH conditions and neutral pH conditions may be appropriately selected from those described above. This allows the desired pH-dependent antigen-binding domain or pH-dependent antibody to be obtained.

[0115] In the context of Disclosure A, in one embodiment, the starting antigen-binding domain or antibody may be a modified antigen-binding domain or antibody having an increased pI, for example, by modifying the charge of at least one amino acid residue that may be exposed on its surface. In an alternative embodiment, when an amino acid that alters the binding activity of the ion concentration-dependent antigen-binding domain is introduced into the sequence, a modification that increases the pI may be introduced in addition to modifying the charge of at least one amino acid residue that may be exposed on the surface of the antigen-binding domain or antibody.

[0116] Alternatively, in the context of Disclosure A, for example, existing antigen-binding domains or antibodies, existing libraries (such as phage libraries), hybridomas obtained by immunizing animals, or antibodies or libraries thereof produced from B cells derived from immunized animals may be used. Alternatively, antigen-binding domains, antibodies, or libraries obtained by introducing mutations (described below) of natural or unnatural amino acids with a side chain pKa of 4.0 to 8.0 into these may be used (e.g., libraries with an increased content of mutations of natural or unnatural amino acids with a side chain pKa of 4.0 to 8.0, or libraries in which mutations of natural or unnatural amino acids with a side chain pKa of 4.0 to 8.0 have been introduced at specific sites). Such preferred antigen-binding domains may have an amino acid sequence in which at least one amino acid residue is substituted and / or inserted with an amino acid with a side chain pKa of 4.0 to 8.0, as described, for example, in WO2009 / 125825.

[0117] In one embodiment within the context of Disclosure A, the site at which a mutation of an amino acid having a side chain pKa of 4.0 to 8.0 is introduced is not limited, and the mutation may be introduced at any site as long as the antigen-binding activity in the acidic pH range is weaker than 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). When the antibody has a variable region or CDR, the site may be within the variable region or CDR. The number of amino acids to be substituted or inserted can be determined appropriately by those skilled in the art and may be one or more. Furthermore, in addition to the above-mentioned substitution or insertion, other amino acids may be deleted, added, inserted, and / or substituted, or modified. Substitution or insertion with an amino acid having a side chain pKa of 4.0 to 8.0 may be carried out randomly by a scanning method such as histidine scanning, in which histidine is used in place of alanine in the alanine scanning method known to those skilled in the art, and / or antibodies with a higher KD (acidic pH range) / KD (neutral pH range) or kd (acidic pH range) / kd (neutral pH range) value compared to before mutation may be selected from antigen-binding domains or antibodies obtained by random substitution or insertion mutation with the amino acid, or from a library thereof.

[0118] Furthermore, antigen-binding domains or antibodies whose antigen-binding activity in the neutral pH range before and after these mutations is not significantly reduced, is not substantially reduced, is substantially the same, or is increased (in other words, antigen-binding domains or antibodies whose activity is maintained at at least 10% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more, or higher. When the binding activity of an antigen-binding domain or antibody is attenuated by the substitution or insertion of an amino acid with a pKa of 4.0 to 8.0, the binding activity may be restored or increased, for example, by substituting, deleting, adding, or inserting one or more amino acids at a site other than the substitution or insertion site.

[0119] In an alternative embodiment, the amino acid having a side chain pKa of 4.0 to 8.0 may be located at any position in the heavy chain variable region and / or light chain variable region that may form the antigen-binding domain. For example, at least one amino acid residue having a side chain pKa of 4.0 to 8.0 may be located in the CDR (one or more of CDR1, CDR2, and CDR3) and / or FR (one or more of FR1, FR2, FR3, and FR4) of the heavy chain and / or light chain. Such amino acid residues include, but are not limited to, amino acid residues at one or more of positions 24, 27, 28, 31, 32, and 34, as determined by Kabat numbering, in CDR1 of the light chain variable region; amino acid residues at one or more of positions 50, 51, 52, 53, 54, 55, and 56, as determined by Kabat numbering, in CDR2 of the light chain variable region; and / or amino acid residues at one or more of positions 89, 90, 91, 92, 93, 94, and 95A, as determined by Kabat numbering, in CDR3 of the light chain variable region. These amino acid residues may be included alone or in combination, as long as the antigen-binding activity of the antibody changes depending on the hydrogen ion concentration conditions.

[0120] In one embodiment within the scope of Disclosure A, any amino acid residue can be suitably used as an amino acid residue that changes the antigen-binding activity of an antigen-binding domain or antibody 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. Examples of such electron-donating amino acids include natural amino acids such as His (H) and Glu (E), as well as histidine analogs (US2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11 (17): 3761-3768 (2003)). Suitable examples of such amino acid residues include amino acids whose side chains have a pKa of 6.0 to 7.0, particularly His (H).

[0121] Within the scope of Disclosure A of this specification, unless expressly stated otherwise and unless contrary to the context, it is understood that the isoelectric point (pI) may be either the theoretical isoelectric point or the experimentally measured isoelectric point, and is also referred to as "pI".

[0122] For example, pI values can be experimentally determined by isoelectric focusing, while theoretical pI values can be calculated using gene and amino acid sequence analysis software (such as Genetyx).

[0123] In one embodiment, whether the pI of an antibody with an increased pI or an antibody of Disclosure A is increased compared to the antibody before modification (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (e.g., an antibody before modification, or an antibody before or in the process of being made into a library)) can be determined, in addition to or instead of the above-mentioned method, by conducting a pharmacokinetic test of the antibody using mouse, rat, rabbit, dog, monkey, or human plasma in combination with a method such as BIACORE, cell proliferation assay, ELISA, enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), or fluorescent immunoassay.

[0124] Within the scope of Disclosure A of the present specification, the term "surface-exposed amino acid residues" generally refers to amino acid residues located on the surface of a polypeptide constituting an antibody. An "amino acid residue located on the surface of a polypeptide" refers to an amino acid residue whose side chain can contact a solvent molecule (usually a water molecule). However, the entire side chain does not necessarily need to contact the solvent molecule; an amino acid residue can be defined as a "surface-exposed amino acid" if even a portion of its side chain contacts the solvent molecule. Furthermore, amino acid residues located on the surface of a polypeptide can also include amino acid residues that are located close to the surface of the antibody and thus can mutually influence the charge of other amino acid residues whose side chains are partially in contact with solvent molecules. For example, those skilled in the art can create homology models of polypeptides and antibodies by homology modeling using commercially available software. Alternatively, methods such as X-ray crystallography can be used. For example, surface-exposed amino acid residues can be determined using coordinates from a three-dimensional model of an antibody using a computer program such as the InsightII program (Accelrys). Surface-exposed sites can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. 55: 379-400 (1971)); Connolly (J. Appl. Cryst. 16: 548-558 (1983)). Determination of potentially surface-exposed sites can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. Software available for such purposes includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). If the algorithm requires user input of size parameters, the "size" of the probe used in the calculation can be set, for example, to a radius of about 1.4 Å or less. Furthermore, methods for determining surface-exposed regions and areas using software for personal computers are described in Pacios (Pacios, Comput. Chem. 18 (4): 377-386 (1994) and J. Mol. Model. 1: 46-53 (1995)).Based on the above information, appropriate amino acid residues to be located on the surface of the polypeptide that constitutes the antibody can be selected.

[0125] Methods for increasing the pI of a protein include, for example, reducing the number of amino acids with negative side chain charges (e.g., aspartic acid and glutamic acid) under neutral pH conditions and / or increasing the number of amino acids with positive side chain charges (e.g., arginine, lysine, and histidine). According to a theory well known to those skilled in the art, an amino acid residue with a negative side chain charge has a negative charge, expressed as -1, under pH conditions sufficiently higher than the pKa of the side chain. For example, the theoretical pKa of the aspartic acid side chain is 3.9, and under neutral pH conditions (e.g., in a solution of pH 7.0), the side chain has a negative charge, expressed as -1. Conversely, an amino acid residue with a positive side chain charge has a positive charge, expressed as +1, under pH conditions sufficiently lower than the pKa of the side chain. For example, the theoretical pKa of the arginine side chain is 12.5, and under neutral pH conditions (e.g., in a solution of pH 7.0), the side chain has a positive charge, expressed as +1. Under neutral pH conditions (e.g., in a solution of pH 7.0), amino acid residues without a side chain charge are known to include 15 naturally occurring amino acids, namely alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. It is understood that amino acids that alter the pI may also be unnatural amino acids.

[0126] Based on the above, one method for increasing the pI of a protein under neutral pH conditions (e.g., in a solution of pH 7.0) is to, for example, substitute an aspartic acid (residue) or glutamic acid (residue) (having a negative charge of -1 on the side chain) present in the amino acid sequence of the target protein with an amino acid (residue) that does not have a charge on the side chain, thereby imparting a charge change of +1 to the protein. Furthermore, for example, substituting an amino acid (residue) that does not have a charge on the side chain with arginine or lysine (having a positive charge of +1 on the side chain) can impart a charge change of +1 to the protein. Furthermore, substituting aspartic acid or glutamic acid (having a negative charge of -1 on the side chain) with arginine or lysine (having a positive charge of +1 on the side chain) can simultaneously impart a charge change of +2. Alternatively, the pI of a protein can be increased by adding or inserting amino acids with no charge in the side chain and / or amino acids with a positive charge in the side chain into the amino acid sequence of the protein, or by deleting amino acids with no charge in the side chain and / or amino acids with a negative charge in the side chain that are present in the amino acid sequence of the protein. Amino acid residues located at, for example, the N-terminus or C-terminus of a protein have charges derived from the main chain (the NH of the amino group at the N-terminus) in addition to the charges derived from the side chain. 3+ , and at the C-terminus, the carbonyl group COO - ) Therefore, the pI of a protein can be increased by adding, deleting, substituting, or inserting functional groups derived from these main chains.

[0127] Those skilled in the art will understand that the effect of altering the net charge or pI of a protein obtained by modifying one or more amino acids (residues) in an amino acid sequence with a focus on the presence or absence and degree of charge of the amino acids (residues) does not depend solely (or substantially) on the amino acid sequence constituting the antibody or the type of target antigen, but rather on the type and number of amino acid residues added, deleted, substituted, or inserted.

[0128] Antibodies that have been modified to have an increased pI by modifying at least one amino acid residue that may be exposed on the surface of the antibody ("pI-increased antibodies" or "pI-increased antibodies") may be more rapidly taken up into cells or may promote elimination of antigens from plasma, as described or suggested, for example, in WO2007 / 114319, WO2009 / 041643, WO2014 / 145159 or WO2012 / 016227.

[0129] Among the various antibody isotypes, for example, IgG antibodies have a sufficiently large molecular weight that their primary metabolic pathway is not renal excretion. IgG antibodies, which contain an Fc region as part of their molecule, are known to be recycled via a salvage pathway mediated by FcRn, thus possessing a long in vivo half-life. IgG antibodies are thought to be primarily metabolized via metabolic pathways in endothelial cells (He et al., J. Immunol. 160(2): 1029-1035 (1998)). Specifically, IgG antibodies nonspecifically internalized by endothelial cells are recycled by binding to FcRn, while IgG antibodies that do not bind are thought to be metabolized. The plasma half-life of IgG antibodies can be shortened if their Fc region is modified to reduce their FcRn-binding activity. On the other hand, it has been shown that the plasma half-life of an antibody with an increased pI is highly dependent on the pI, as described in, for example, WO2007 / 114319 and WO2009 / 041643. That is, the plasma half-life of the antibody with an increased pI described in the above documents was shortened without modifying the amino acid sequence constituting the Fc, which may result in the acquisition of immunogenicity. This suggests that techniques for increasing the pI can be widely applied to any type of antibody molecule, such as scFv, Fab, or Fc fusion protein, whose main metabolic pathway is renal excretion.

[0130] The pH concentration in biological fluids (e.g., plasma) is in the neutral pH range. Without being bound by any particular theory, it is believed that in biological fluids, an antibody with an increased pI has a higher net positive charge due to the increased pI, and as a result, is more strongly attracted to the endothelial cell surface, which has a net negative charge, through physicochemical Coulombic interactions than an antibody without an increased pI; the antibody binds to the surface through nonspecific binding and is internalized within the cells, resulting in a shortened plasma half-life of the antibody or increased antigen elimination from the plasma. Furthermore, increasing the pI of an antibody is believed to increase the intracellular uptake and / or intracellular permeability of the antibody (or antigen-antibody complex), which results in a lower plasma concentration of the antibody, a lower bioavailability of the antibody, and / or a shortened plasma half-life of the antibody; these phenomena are believed to occur widely in vivo, regardless of cell type, tissue type, organ type, etc. Furthermore, when an antibody forms a complex with an antigen and is taken up into cells, the pI of the antigen as well as the pI of the antibody can affect the increase or decrease in the amount of uptake into cells.

[0131] In one embodiment, examples of a method for producing or screening for an antibody with increased pI include the methods described in WO2007 / 114319 (e.g., paragraphs 0060 to 0087), WO2009 / 041643 (e.g., paragraphs 0115 to ), WO2014 / 145159, and WO2012 / 016227. (a) modifying a nucleic acid encoding an antibody containing at least one amino acid residue that can be exposed on the surface of the antibody so that the charge of the amino acid residue is modified to increase the pI of the antibody; (b) culturing the host cell so that the nucleic acid is expressed; and (c) recovering the antibody from the host cell culture The method may include:

[0132] Or, for example, (a') modifying a nucleic acid encoding an antibody containing at least one amino acid residue that can be exposed on the surface of the antibody so that the charge of the amino acid residue is modified; (b') culturing the host cell so that the nucleic acid is expressed; (c') recovering the antibody from the host cell culture; and (d') selecting (optionally by confirming or measuring) antibodies that have an increased pI compared to the antibody before modification; In this case, the starting antibody, the antibody before modification, or the reference antibody may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying amino acid residues, amino acids that change the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence.

[0133] Alternatively, the method may simply comprise the steps of culturing the host cells obtained in step (b) or (b') and recovering the antibody from the cell culture.

[0134] In an alternative embodiment, for example, 1. A method for producing a multispecific antibody comprising a first polypeptide and a second polypeptide, and optionally a third polypeptide and a fourth polypeptide, the method comprising: (A) modifying nucleic acids encoding the first polypeptide and / or the second polypeptide, and optionally the third polypeptide and / or the fourth polypeptide, such that any one or more of the polypeptides contains at least one amino acid residue that may be exposed on the surface of the polypeptide such that the charge of that amino acid residue is modified such that the pI of the antibody is increased; (B) culturing the host cell so that the nucleic acid is expressed; and (C) Recovering the multispecific antibody from the host cell culture The method may include:

[0135] Alternatively, the method may comprise, for example: (A') modifying nucleic acids encoding the first polypeptide and / or the second polypeptide, and optionally the third polypeptide and / or the fourth polypeptide, such that any one or more of the polypeptides contains at least one amino acid residue that may be exposed on the surface of the polypeptide such that the charge of that amino acid residue is modified; (B') culturing the host cell so that the nucleic acid is expressed (C') recovering the multispecific antibody from the host cell culture; and (D') A step of selecting (and optionally confirming) an antibody whose pI is increased compared to the antibody before modification. may include:

[0136] Here, for example, the starting antibody, the antibody before modification, or the reference antibody may be an ion concentration-dependent antibody. Alternatively, the sequence may contain amino acids that alter the binding activity of the ion concentration-dependent antigen-binding domain when amino acid residues are modified.

[0137] Alternatively, the method may simply comprise the steps of culturing the host cells obtained in step (B) or (B') and recovering the antibody from the cell culture. In such cases, the polypeptide in which the nucleic acid is modified may preferably be a homomultimer of a first polypeptide, a homomultimer of a second polypeptide, or a heteromultimer of the first and second polypeptides (and, optionally, a homomultimer of a third polypeptide, a homomultimer of a fourth polypeptide, or a heteromultimer of the third and fourth polypeptides).

[0138] In an alternative embodiment, for example, A method for producing a humanized or human antibody with a reduced plasma half-life, comprising: An antibody comprising a CDR selected from the group consisting of a human-derived CDR, a non-human-derived CDR, and a synthetic CDR; a human-derived FR; and a human constant region, (I) modifying at least one amino acid residue that can be exposed on the surface of at least one region selected from the group consisting of the CDR, the FR, and the constant region to an amino acid residue having a different charge from the amino acid residue present at the corresponding position before modification, so as to increase the pI of the antibody. The method may include:

[0139] Alternatively, for example, an antibody comprising a CDR selected from the group consisting of a human-derived CDR, a non-human animal-derived CDR, and a synthetic CDR; a human-derived FR; and a human constant region, (I') modifying at least one amino acid residue that can be exposed on the surface of at least one region selected from the group consisting of the CDR, the FR, and the constant region to an amino acid residue having a charge different from that of the amino acid residue present at the corresponding position before modification; and (II') A step of selecting (and optionally confirming) an antibody whose pI is increased compared to the antibody before modification. The method may include:

[0140] Here, for example, the starting antibody, the antibody before modification, or the reference antibody may be an ion concentration-dependent antibody. Alternatively, the sequence may contain amino acids that alter the binding activity of the ion concentration-dependent antigen-binding domain when amino acid residues are modified.

[0141] Alternatively, for example, existing antigen-binding domains or antibodies, existing libraries (such as phage libraries), antibodies or libraries thereof prepared from hybridomas obtained by immunizing animals or B cells derived from immunized animals, or libraries thereof, may be used; or antigen-binding domains or antibodies or libraries thereof with increased pI prepared by modifying at least one amino acid residue that can be exposed on the surface, for example, based on any of the above-mentioned embodiments.

[0142] In one embodiment of the antibody of Disclosure A, the pI value of the antibody before modification or alteration (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody), or a reference antibody or parent antibody (e.g., an antibody before modification, or an antibody before or in the process of being made into a library, etc.)) may preferably be increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 or more, or by at least 0.6, 0.7, 0.8, or 0.9 or more; and to significantly shorten the plasma half-life of the antibody, the pI value may be increased, for example, by at least 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 or more, or by at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 or more, or by 3.0 or more. Those skilled in the art can routinely determine the optimal pI value of an antibody of Disclosure A, taking into account the balance between pharmacological effect and toxicity, as well as, for example, the number of antigen-binding domains of the antibody and the pI of the antigen for a given purpose. Without being bound by any particular theory, in one embodiment, the antibody of Disclosure A has an ion concentration-dependent antigen-binding domain, which allows a single antibody molecule to repeatedly bind to multiple antigens by traveling between plasma and cellular endosomes. Furthermore, the increased pI results in an increased net positive charge of the antibody, which allows the antibody to be more rapidly internalized into cells. These properties may shorten the antibody's plasma half-life, increase the antibody's binding activity to the extracellular matrix, or promote antigen elimination from plasma. An optimal pI value may be determined to take advantage of these properties.

[0143] In one embodiment in the context of Disclosure A, the ion concentration-dependent antibody of Disclosure A with an increased pI preferably exhibits a reduced rate of antigen elimination from plasma (when the antibody is administered in vivo) compared to an antibody (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody), or a reference antibody or parent antibody (e.g., an antibody before modification, or an antibody before or in the process of being compiled into a library, etc.), which may be an ion concentration-dependent antibody) before at least one amino acid residue has been modified or altered to increase the pI. , for example, at least 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or more promotion, or the binding activity to extracellular matrix may preferably be increased, for example, by at least 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5-fold or more.

[0144] In one embodiment in the context of Disclosure A, the ion concentration-dependent antibody of Disclosure A having an increased pI preferably reduces, for example, the elimination of antigen from plasma (when the antibody is administered in vivo) by a small amount, compared to an antibody before the introduction of an ion concentration-dependent antigen-binding domain (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody), or a reference antibody or parent antibody (e.g., an antibody before modification, or an antibody before or in the process of being compiled into a library, etc.), which may be an antibody with an increased pI). The activity of the antibody may be enhanced by at least 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or more, or the binding activity to the extracellular matrix may be increased by, for example, at least 1.1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5-fold or more.

[0145] In one embodiment, the assay method for determining whether the binding activity of an antibody of Disclosure A to an extracellular matrix is increased compared to an antibody before modification or alteration (a natural antibody (e.g., a natural Ig antibody, which may be a natural IgG antibody), or a reference antibody or parent antibody (e.g., an antibody before modification, or an antibody before or in the process of library creation), which may be an ion concentration-dependent antibody or an antibody with an increased pI) is not limited. For example, an assay can be performed using an ELISA system that detects binding of an antibody to an extracellular matrix, in which an antibody is added to a plate on which an extracellular matrix has been solidified, and then a labeled antibody against that antibody is added. Alternatively, electrochemiluminescence (ECL), which can detect extracellular matrix binding ability with higher sensitivity, may be used, as described in Examples 1 to 4 of the present specification and WO2012 / 093704, etc., may be used. This method can be performed, for example, using an ECL system in which a mixture of an antibody and a ruthenium-antibody is added to a plate on which an extracellular matrix has been solidified, and binding of the antibody to the extracellular matrix is measured based on the electrochemiluminescence of ruthenium. The concentration of antibody added can be set arbitrarily, and can be increased to enhance the detection sensitivity of binding to the extracellular matrix. Such extracellular matrices may be derived from either animals or plants, as long as they contain glycoproteins such as collagen, proteoglycan, fibronectin, laminin, entactin, fibrin, and perlecan. Animal-derived matrices may be preferred. For example, extracellular matrices derived from animals such as humans, mice, rats, monkeys, rabbits, or dogs can be used. For example, natural extracellular matrices derived from humans may be used as an indicator of the plasma pharmacokinetics of antibodies in humans. Conditions for assessing antibody binding to the extracellular matrix are preferably in the neutral pH range around pH 7.4, which corresponds to physiological conditions, but the neutral range is not necessary; binding may also be assessed in the acidic pH range (e.g., around pH 6.0). Alternatively, when assessing antibody binding to the extracellular matrix, an assay may be performed by coexisting the antibody with an antigen molecule to which the antibody binds and assessing the binding activity of the antigen-antibody complex to the extracellular matrix.

[0146] In one embodiment, the antibody of Disclosure A can (substantially) maintain its antigen-binding activity compared to the antibody (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (e.g., an antibody before antibody modification, or an antibody before or during library construction, etc.)) before at least one amino acid residue is modified or altered to increase its pI. In such cases, "(substantially) maintaining its antigen-binding activity" can mean having at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and even more preferably 80%, 85%, 90%, or 95% or more of the binding activity of the antibody before modification or alteration. Alternatively, the antibody of Disclosure A may maintain binding activity to such an extent that its function is maintained when binding to an antigen. Thus, for example, the affinity measured under physiological conditions at 37°C may be 100 nM or less, preferably 50 nM or less, more preferably 10 nM or less, and even more preferably 1 nM or less.

[0147] In one embodiment of Disclosure A, "modification of at least one amino acid residue that can be exposed on the surface of the antibody" or expressions with the same meaning may mean that at least one amino acid residue that can be exposed on the surface of the antibody has been subjected to one or more of addition, deletion, substitution, and insertion. The modification may preferably include substitution of at least one amino acid residue.

[0148] For example, such amino acid residue substitutions may include, in the amino acid sequence of an antibody of interest, substitutions of amino acid residues with negatively charged side chains with amino acid residues with uncharged side chains, substitutions of amino acid residues with uncharged side chains with amino acid residues with positively charged side chains, and substitutions of amino acid residues with negatively charged side chains with amino acid residues with positively charged side chains, and these substitutions may be performed alone or in appropriate combinations. For example, such amino acid residue insertions or additions may include, in the amino acid sequence of an antibody of interest, insertions or additions of amino acids with uncharged side chains and / or insertions or additions of amino acids with positively charged side chains, and these may be performed alone or in appropriate combinations. For example, such amino acid residue deletions may include deletions of amino acid residues with uncharged side chains and / or deletions of amino acid residues with negatively charged side chains, and these may be performed alone or in appropriate combinations.

[0149] Those skilled in the art can appropriately combine one or more of these additions, deletions, substitutions, and insertions in the amino acid sequence of a target antibody. Since the net pI of the antibody of Disclosure A needs to be increased, modifications that result in a reduction in the local charge of amino acid residues may be performed. For example, if desired, modifications may be performed to (slightly) reduce the pI of an antibody whose pI is (too) increased. The local charge of an amino acid residue may also be reduced as a result of simultaneously or at different times modifying at least one amino acid residue for other purposes (e.g., increasing antibody stability or reducing immunogenicity). Such antibodies may be antibodies from a library produced for a specific purpose.

[0150] In one embodiment, among the amino acids (residues) used to modify at least one amino acid residue that may be exposed on the surface of an antibody, the naturally occurring amino acids are: the amino acid having a negative charge in the side chain is Glu (E) or Asp (D); the amino acid having no charge in the side chain is Ala (A), Asn (N), Cys (C), Gln (Q), Gly (G), His (H), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), or Val (V); The amino acid having a positive charge in the side chain is His (H), Lys (K) or Arg (R).

[0151] As described in detail in Examples 1 to 4 of the present specification, in a solution at neutral pH (e.g., pH 7.0), nearly 100% of lysine and arginine residues in an antibody are positively charged, whereas only about 9% of histidine residues in an antibody are positively charged, with the majority of the remaining residues being uncharged. Therefore, it is preferable to select Lys (K) or Arg (R) as an amino acid with a positive charge in the side chain.

[0152] In one embodiment, the antibody of Disclosure A preferably has a variable region and / or a constant region. Furthermore, the variable region may preferably have a heavy chain variable region and / or a light chain variable region, and / or may preferably have CDRs (e.g., one or more of CDR1, CDR2, and CDR3) and / or FRs (e.g., one or more of FR1, FR2, FR3, and FR4). The constant region may preferably have a heavy chain constant region and / or a light chain constant region, and the sequence and type thereof may be, for example, an IgG-type constant region (preferably a human IgG1-, human IgG2-, human IgG3-, or human IgG4-type constant region, a human κ-chain constant region, or a human λ-chain constant region). Modified variants of these constant regions may also be used.

[0153] In one embodiment, the modification of at least one amino acid residue that can be exposed on the antibody surface can be either a single amino acid modification or a combination of multiple amino acid modifications. A preferred method is to introduce multiple amino acid substitutions in combination at a site where the amino acid can be exposed on the antibody surface. Furthermore, although not limited thereto, it is preferred that such multiple amino acid substitutions are introduced at positions that are structurally close to each other. For example, when an amino acid that can be exposed on the surface of an antibody molecule (preferably, but not limited to, an amino acid having a negative charge in its side chain (e.g., Glu (E) or Asp (D)) is substituted with an amino acid having a positive charge in its side chain (e.g., Lys (K) or Arg (R)), or when an existing positively charged amino acid (e.g., Lys (K) or Arg (R)) is used, one or more amino acids conformationally adjacent to the amino acid (which may include amino acids buried within the antibody molecule in some cases) may also be substituted with positively charged amino acids, resulting in localized clustering of positive charges at conformationally adjacent positions. Here, the definition of "conformationally adjacent positions" is not particularly limited, but may refer to a state in which one or more amino acid substitutions have been introduced within a distance of, for example, 20 Å, preferably 15 Å, and more preferably 10 Å. Whether the target amino acid substitution site is exposed on the surface of the antibody molecule or whether a certain amino acid substitution site is adjacent to another amino acid substitution site or the above-mentioned existing amino acids can be determined by known methods such as X-ray crystal structure analysis.

[0154] In addition to the above methods, methods for providing multiple positive charges at positions that are structurally close to each other may also include a method using amino acids that are inherently positively charged in the constant region of native IgG. Arginines at positions 255, 292, 301, 344, 355, and 416 in the EU numbering system, and Lysines at positions 121, 133, 147, 205, 210, 213, 214, 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360, 370, 392, 409, 414, and 439 (EU numbering) By substituting positively charged amino acids at positions conformationally close to these positively charged amino acids, it is possible to impart multiple positive charges to conformationally close sites.

[0155] Furthermore, when the antibody of Disclosure A has a variable region (which may be modified), amino acid residues that are not masked upon antigen binding (i.e., can still be exposed on the surface) may be modified, and / or no amino acid modifications may be introduced at sites that are masked upon antigen binding, or amino acid modifications that do not (substantially) inhibit antigen binding may be made. Furthermore, when amino acid residues present in an ion concentration-dependent binding domain that can be exposed on the surface of the antibody molecule are modified, amino acids in the antigen-binding domain may be modified so as not to (substantially) attenuate the binding activity of amino acid residues that can change the antigen-binding activity of the antibody depending on ion concentration conditions (e.g., those in a calcium-binding motif, or histidine insertion site and / or histidine substitution site) in the antigen-binding domain, or amino acid residues may be modified at sites other than those that can change the antigen-binding activity of the antibody depending on the ion concentration conditions. On the other hand, if amino acid residues present in the ion concentration-binding domain that can be exposed on the surface of the antibody molecule have already been modified, the positions and types of amino acid residues that can change the antigen-binding activity of the antibody depending on the ion concentration conditions may be selected so that the pI of the antibody does not fall below an acceptable level. If the pI of the antibody falls below an acceptable level, the pI of the entire antibody may be increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody molecule.

[0156] Preferably, but not exclusively, a FR sequence with a high pI may be selected from a human germline FR sequence or a sequence of an equivalent region thereof, and the amino acids may optionally be modified.

[0157] When the antibody of Disclosure A has a constant region (which may be modified) having an FcγR-binding domain (which may be a binding domain for any FcγR isoform or allotype as described below) and / or an FcRn-binding domain, the site of modification of at least one amino acid residue that may be exposed on the surface of the constant region may, if desired, be an amino acid residue other than those in the FcγR-binding domain and / or the FcRn-binding domain. Alternatively, when selecting an amino acid residue in the FcγR-binding domain and / or the FcRn-binding domain as the modification site, it may be preferable to select a site that does not (substantially) affect the binding activity or affinity to FcγR and / or FcRn, or a site that does affect the binding activity or affinity but is biologically or pharmacologically acceptable.

[0158] In one embodiment, to prepare an antibody of Disclosure A having an increased pI by modifying at least one amino acid residue that may be exposed on the surface of the variable region (which may be modified), the site of modification of at least one amino acid residue may be, but is not limited to, any of the following, as represented by the Kabat numbering system: (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44 in the heavy chain variable region FR; 46th, 68th, 71st, 72nd, 73rd, 75th, 76th, 77th, 81st, 82nd, 82a, 82b, 83rd, 84th, 85th, 86th, 105th, 108th, 110th, and 112th; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region The amino acid at each position after modification can be selected from any of the amino acids listed above in terms of side chain charge, such as, but not limited to, Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N). In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acid positions are modified. In some embodiments, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of the above amino acid positions are modified.

[0159] In one embodiment, the following positions among those modified can be used in combination with other positions that may themselves have a sufficient effect to increase the pi of the antibody to help increase the pi of the antibody of Disclosure A. Such positions to help increase the pi can be selected, for example, from the group consisting of positions 27, 52, 56, 65, and 69 according to the Kabat numbering for the light chain variable region.

[0160] Furthermore, the site of modification of at least one amino acid residue in the CDR and / or FR may include, but is not limited to, (a) positions 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in the light chain variable region FR; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acid positions are modified. In some embodiments, 1 to 20, 1 to 15, 1 to 10, and 1 to 5 of the above amino acid positions are modified.

[0161] For example, when the site of modification of at least one amino acid residue is selected from the group including the above-mentioned group, the type of amino acid after modification in the heavy chain variable region may be, for example, (a) In the 8th place, 8K, 8R, 8Q, 8G, 8S, or 8N; (b) at position 13, 13K, 13R, 13Q, 13G, 13S, or 13N; (c) in the 15th place, 15K, 15R, 15Q, 15G, 15S, or 15N; (d) at the 16th position, 16K, 16R, 16Q, 16G, 16S, or 16N; (e) at 18th place, 18K, 18R, 18Q, 18G, 18S, or 18N; (f) at position 39, 39K, 39R, 39Q, 39G, 39S, or 39N; (g) at position 41, 41K, 41R, 41Q, 41G, 41S, or 41N; (h) at position 43, 43K, 43R, 43Q, 43G, 43S, or 43N; (i) at position 44, 44K, 44R, 44Q, 44G, 44S, or 44N; (j) at position 63, 63K, 63R, 63Q, 63G, 63S, or 63N; (k) at position 64, 64K, 64R, 64Q, 64G, 64S, or 64N; (l) at position 77, 77K, 77R, 77Q, 77G, 77S, or 77N; (m) At position 82, 82K, 82R, 82Q, 82G, 82S, or 82N; (n) at position 82a, 82aK, 82aR, 82aQ, 82aG, 82aS, or 82aN; (o) at position 82b, 82bK, 82bR, 82bQ, 82bG, 82bS, or 82bN; (p) At position 83, 83K, 83R, 83Q, 83G, 83S, or 83N; (q) At position 84, 84K, 84R, 84Q, 84G, 84S, or 84N; (r) At position 85, 85K, 85R, 85Q, 85G, 85S, or 85N; or (s) In the 105th place, 105K, 105R, 105Q, 105G, 105S, or 105N In some embodiments, any combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acid positions are modified. In some embodiments, any combination of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of the above amino acid positions are modified.

[0162] Non-limiting examples of combinations of altered amino acid positions in the heavy chain variable region include, for example, the following, represented by Kabat numbering: any two or more positions selected from the group consisting of positions 16, 43, 64, and 105; any two or more positions selected from the group consisting of positions 77, 82a, and 82b; 77th and 85th; 41st and 44th places; Positions 82a and 82b; 82nd and 82b; positions 82b and 83; or 63rd and 64th place and the amino acid at each position after modification can be selected from any of the above-mentioned amino acids, for example, Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N), in terms of the charge of the side chain, without being limited thereto.

[0163] Specific combinations can be, for example, 16Q / 43R / 64K / 105Q; 77R / 82aN / 82bR; 77R / 82aG / 82bR; 77R / 82aS / 82bR; 77R / 85G; 41R / 44R; 82aN / 82bR; 82aG / 82bR; 82aS / 82bR; 82K / 82bR; 82bR / 83R; 77R / 85R; or 63R / 64K.

[0164] Similarly, the types of amino acids after modification in the light chain variable region are, for example, (a) at position 16, 16K, 16R, 16Q, 16G, 16S, or 16N; (b) at position 18, 18K, 18R, 18Q, 18G, 18S, or 18N; (c) at position 24, 24K, 24R, 24Q, 24G, 24S, or 24N; (d) at 25th place, 25K, 25R, 25Q, 25G, 25S, or 25N; (e) at position 26, 26K, 26R, 26Q, 26G, 26S, or 26N; (f) at position 27, 27K, 27R, 27Q, 27G, 27S, or 27N; (g) at position 37, 37K, 37R, 37Q, 37G, 37S, or 37N; (h) at position 41, 41K, 41R, 41Q, 41G, 41S, or 41N; (i) at position 42, 42K, 42R, 42Q, 42G, 42S, or 42N; (j) at 45th place, 45K, 45R, 45Q, 45G, 45S, or 45N; (k) at position 52, 52K, 52R, 52Q, 52G, 52S, or 52N; (l) at position 53, 53K, 53R, 53Q, 53G, 53S, or 53N; (m) at position 54, 54K, 54R, 54Q, 54G, 54S, or 54N; (n) In the 55th place, 55K, 55R, 55Q, 55G, 55S, or 55N; (o) at position 56, 56K, 56R, 56Q, 56G, 56S, or 56N; (p) At position 65, 65K, 65R, 65Q, 65G, 65S, or 65N; (q) At position 69, 69K, 69R, 69Q, 69G, 69S, or 69N; (r) at position 74, 74K, 74R, 74Q, 74G, 74S, or 74N; (s) At position 76, 76K, 76R, 76Q, 76G, 76S, or 76N; (t) at position 77, 77K, 77R, 77Q, 77G, 77S, or 77N; (u) at position 79, 79K, 79R, 79Q, 79G, 79S, or 79N; and (v) at position 107, 107K, 107R, 107Q, 107G, 107S, or 107N In some embodiments, any combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the above amino acid positions are modified. In some embodiments, any combination of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of the above combinations of amino acid positions are modified.

[0165] Non-limiting examples of combinations of modified amino acid positions in the light chain variable region include, for example, the following, represented by Kabat numbering: 24th and 27th places; 25th and 26th places; 41st and 42nd places; 42nd and 76th; 52nd and 56th places; 65th and 79th; 74th and 77th; 76th and 79th; any two or more positions selected from the group consisting of positions 16, 24, and 27; any two or more positions selected from the group consisting of positions 24, 27, and 37; any two or more positions selected from the group consisting of positions 25, 26, and 37; any two or more positions selected from the group consisting of positions 27, 76, and 79; any two or more positions selected from the group consisting of positions 41, 74, and 77; any two or more positions selected from the group consisting of positions 41, 76, and 79; any two or more positions selected from the group consisting of positions 24, 27, 41, and 42; any two or more positions selected from the group consisting of positions 24, 27, 52, and 56; any two or more positions selected from the group consisting of positions 24, 27, 65, and 69; any two or more positions selected from the group consisting of positions 24, 27, 74, and 77; any two or more positions selected from the group consisting of positions 24, 27, 76, and 79; any two or more positions selected from the group consisting of positions 25, 26, 52, and 56; any two or more positions selected from the group consisting of positions 25, 26, 65, and 69; any two or more positions selected from the group consisting of positions 25, 26, 76, and 79; any two or more positions selected from the group consisting of positions 27, 41, 74, and 77; any two or more positions selected from the group consisting of positions 27, 41, 76, and 79; any two or more positions selected from the group consisting of positions 52, 56, 74, and 77; any two or more positions selected from the group consisting of positions 52, 56, 76, and 79; any two or more positions selected from the group consisting of positions 65, 69, 76, and 79; any two or more positions selected from the group consisting of positions 65, 69, 74, and 77; any two or more positions selected from the group consisting of positions 18, 24, 45, 79, and 107; any two or more positions selected from the group consisting of positions 27, 52, 56, 74, and 77; any two or more positions selected from the group consisting of positions 27, 52, 56, 76, and 79; any two or more positions selected from the group consisting of positions 27, 65, 69, 74, and 77; any two or more positions selected from the group consisting of positions 27, 65, 69, 76, and 79; any two or more positions selected from the group consisting of positions 41, 52, 56, 74, and 77; any two or more positions selected from the group consisting of positions 41, 52, 56, 76, and 79; any two or more positions selected from the group consisting of positions 41, 65, 69, 74, and 77; any two or more positions selected from the group consisting of positions 41, 65, 69, 76, and 79; any two or more positions selected from the group consisting of positions 24, 27, 41, 42, 65, and 69; any two or more positions selected from the group consisting of positions 24, 27, 52, 56, 65, and 69; any two or more positions selected from the group consisting of positions 24, 27, 65, 69, 74, and 77; any two or more positions selected from the group consisting of positions 24, 27, 65, 69, 76, and 79; any two or more positions selected from the group consisting of positions 24, 27, 41, 42, 74, and 77; any two or more positions selected from the group consisting of positions 24, 27, 52, 56, 74, and 77; any two or more positions selected from the group consisting of positions 24, 27, 41, 42, 76, and 79; any two or more positions selected from the group consisting of positions 24, 27, 52, 56, 76, and 79; any two or more positions selected from the group consisting of positions 24, 27, 74, 76, 77, and 79; any two or more positions selected from the group consisting of positions 52, 56, 65, 69, 74, and 77; or Any two or more positions selected from the group consisting of positions 52, 56, 65, 69, 76, and 79 and the amino acid at each position after modification can be selected from any of the above-mentioned amino acids, for example, Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N), in terms of the charge of the side chain, without being limited thereto.

[0166] Specific combinations can be, for example, 24R / 27Q; 24R / 27R; 24K / 27K; 25R / 26R; 25K / 26K; 41R / 42K; 42K / 76R; 52R / 56R; 65R / 79K; 74K / 77R; 76R / 79K; 16K / 24R / 27R; 24R / 27R / 37R; 25R / 26R / 37R; 27R / 76R / 79K; 41R / 74K / 77R; 41R / 76R / 79K; 24R / 27R / 41R / 42K; 24R / 27R / 52R / 56R; 24R / 27R / 52K / 56K; 24R / 27R / 65R / 69R; 24R / 27R / 74K / 77R; 24R / 27R / 76R / 79K; 25R / 26R / 52R / 56R; 25R / 26R / 52K / 56K; 25R / 26R / 65R / 69R; 25R / 26R / 76R / 79K; 27R / 41R / 74K / 77R; 27R / 41R / 76R / 79K; 52R / 56R / 74K / 77R; 52R / 56R / 76R / 79K; 65R / 69R / 76R / 79K; 65R / 69R / 74K / 77R; 18R / 24R / 45K / 79Q / 107K; 27R / 52R / 56R / 74K / 77R; 27R / 52R / 56R / 76R / 79K; 27R / 65R / 69R / 74K / 77R; 27R / 65R / 69R / 76R / 79K; 41R / 52R / 56R / 74K / 77R; 41R / 52R / 56R / 76R / 79K; 41R / 65R / 69R / 74K / 77R; 41R / 65R / 69R / 76R / 79K; 24R / 27R / 41R / 42K / 65R / 69R; 24R / 27R / 52R / 56R / 65R / 69R; 24R / 27R / 65R / 69R / 74K / 77R; 24R / 27R / 65R / 69R / 76R / 79K; 24R / 27R / 41R / 42K / 74K / 77R; 24R / 27R / 52R / 56R / 74K / 77R; 24R / 27R / 41R / 42K / 76R / 79K; 24R / 27R / 52R / 56R / 76R / 79K; 24R / 27R / 74K / 76R / 77R / 79K; 52R / 56R / 65R / 69R / 74K / 77R; or 52R / 56R / 65R / 69R / 76R / 79K.

[0167] It has already been explained or demonstrated based on theoretical grounds, homology modeling, or experimental techniques in WO2007 / 114319 or WO2009 / 041643 that the effect of increasing pI by modifying several amino acid residues in the variable region does not depend solely (or substantially) on the amino acid sequence constituting the antibody itself or the type of target antigen, but rather on the type and number of amino acid residues substituted. Furthermore, it has been demonstrated that a person skilled in the art can expect that the antigen-binding activity of an antibody against multiple types of antigens will be (substantially) maintained, or at least maintained with a high probability, even after modifying several amino acids.

[0168] For example, WO2009 / 041643 specifically discloses that, in the heavy chain FR of the humanized glypican 3 antibody represented by SEQ ID NO: 8, preferred surface-exposed amino acid residue modification sites are positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87, 89, 90, 107, 110, 112, and 114 (Kabat numbering). It also reports that the amino acid residue at position 97 (Kabat numbering) is preferred because it is surface-exposed in almost all antibodies. WO2009 / 041643 also discloses that preferred amino acid residues are positions 52, 54, 62, 63, 65, and 66 (Kabat numbering) in the heavy chain CDR of the antibody. It also indicates that the amino acid residues at positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, 44, 45, 46, 48, 49, 50, 54, 62, 65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90, 105, 108, 110, 111, and 112 (Kabat numbering) are preferred in the light chain FR of the humanized anti-glypican 3 antibody represented by SEQ ID NO: 9. It also indicates that the amino acid residues at positions 24, 27, 33, 55, and 59 (Kabat numbering) are preferred in the light chain CDR of the antibody. Furthermore, WO2009 / 041643 also discloses that preferred sites for modifying amino acid residues that can be exposed on the surface while maintaining antigen-binding activity are the amino acid residues at positions 31, 64, and 65, as determined by Kabat numbering, in the heavy chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 10. It also discloses that preferred sites for modifying amino acid residues that can be exposed on the surface while maintaining antigen-binding activity are the amino acid residues at positions 24, 27, 53, and 55, as determined by Kabat numbering, in the light chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 11. It also discloses that preferred sites for modifying amino acid residues that can be exposed on the surface while maintaining antigen-binding activity are the amino acid residue at position 31, as determined by Kabat numbering, in the heavy chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 12.It has also been shown that the amino acid residues at positions 24, 53, 54, and 55 (Kabat numbering) in the light chain CDR of the anti-human IL-6 receptor antibody represented by SEQ ID NO: 13 are preferred. WO 2009 / 041643 also shows that the amino acid residues at positions 61, 62, 64, and 65 (Kabat numbering) in the heavy chain CDR of the anti-human glypican 3 antibody represented by SEQ ID NO: 14 are preferred as sites at which surface-exposed amino acid residues can be modified while maintaining antigen-binding activity. It has also been shown that the amino acid residues at positions 24 and 27 (Kabat numbering) in the light chain CDR of the anti-human glypican 3 antibody represented by SEQ ID NO: 15 are preferred. It has also been shown that the amino acid residues at positions 61, 62, 64, and 65 (Kabat numbering) in the heavy chain CDR of the anti-human IL-31 receptor antibody represented by SEQ ID NO: 16 are preferred as sites at which surface-exposed amino acid residues can be modified while maintaining antigen-binding activity. WO 2009 / 041643 also discloses that the amino acid residues at positions 24 and 54 (Kabat numbering) in the light chain CDR of the anti-human IL-31 receptor antibody represented by SEQ ID NO: 17 are preferred. Similarly, WO 2007 / 114319 reports that the pI of antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and hB26-PF, which were prepared by modifying the charge of one or more amino acid residues that may be exposed on the surface, was verified by isoelectric focusing, and that the binding activity to the antigens factor IXa or factor X was comparable to that of the unmodified or unmodified antibodies. It also reported that administration of these antibodies to mice showed a high correlation between the pI of each antibody and its plasma clearance (CL), plasma residence time, and plasma half-life (T1 / 2). Furthermore, WO2007 / 114319 demonstrates that amino acid residues at positions 10, 12, 23, 39, 43, 97, and 105 in the variable region are preferred sites for modifying amino acid residues that may be exposed on the surface.

[0169] In an alternative or further embodiment, amino acid residues that may be exposed on the surface of the antibody constant region may be identified using known methods, such as, for example, X-ray crystal structure analysis or a homology model constructed by homology modeling from an antibody constant region (which is not limited to, but is preferably a human constant region, more preferably a human Ig constant region, and even more preferably a human IgG constant region), and the site of modification of at least one amino acid residue to create an antibody of Disclosure A with an increased pI may be determined. The site of modification of at least one amino acid residue that may be exposed on the surface of the constant region is preferably, but not limited to, positions 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 328, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 3 From the group consisting of positions 30, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 can be selected, preferably positions 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, The amino acid at each site after modification may be selected from the group consisting of positions 419, 421, 433, 434, and 443, or preferably from the group consisting of positions 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413. The amino acid at each site after modification can be selected from the above-mentioned amino acids, such as, but not limited to, Lys (K), Arg (R), Gln (Q), or Asn (N), in terms of the charge of the side chain.Therefore, for example, when at least one amino acid residue modification site is selected from the group including the above-mentioned groups, the type of amino acid after modification at each site may be, for example, as follows: At position 254, 254K, 254R, 254Q, or 254N; At position 258, 258K, 258R, 258Q, or 258N; At position 281, 281K, 281R, 281Q, or 281N; At position 282, 282K, 282R, 282Q, or 282N; At 285th place, 285K, 285R, 285Q, or 285N; At position 309, 309K, 309R, 309Q, or 309N; At position 311, 311K, 311R, 311Q, or 311N; At position 315, 315K, 315R, 315Q, or 315N; At position 327, 327K, 327R, 327Q, or 327N; At 330th position, 330K, 330R, 330Q, or 330N; At position 342, 342K, 342R, 342Q, or 342N; at position 311, 343K, 343R, 343Q, or 343N; At position 345, 345K, 345R, 345Q, or 345N; At position 356, 356K, 356R, 356Q, or 356N; At position 358, 358K, 358R, 358Q, or 358N; At position 359, 359K, 359R, 359Q, or 359N; At position 361, 361K, 361R, 361Q, or 361N; At position 362, 362K, 362R, 362Q, or 362N; At position 384, 384K, 384R, 384Q, or 384N; At position 385, 385K, 385R, 385Q, or 385N; At position 386, 386K, 386R, 386Q, or 386N; At position 387, 387K, 387R, 387Q, or 387N; At position 389, 389K, 389R, 389Q, or 389N; At 399th place, 399K, 399R, 399Q, or 399N; In the 400th place, 400K, 400R, 400Q, or 400N; In the 401st place, 401K, 401R, 401Q, or 401N; At 402nd place, 402K, 402R, 402Q, or 402N; At position 413, 413K, 413R, 413Q, or 413N; At position 418, 418K, 418R, 418Q, or 418N; At position 419, 419K, 419R, 419Q, or 419N; At position 421, 421K, 421R, 421Q, or 421N; At position 433, 433K, 433R, 433Q, or 433N; at position 434, 434K, 434R, 434Q, or 434N; and At 443rd place, 443K, 443R, 443Q, or 443N It could be.

[0170] In an alternative embodiment, the site of modification of at least one amino acid residue and the type of amino acid after modification may include 345R or 345K, and / or 430R, 430K, 430G, or 435T, as represented by EU numbering.

[0171] In one embodiment of the antibody of Disclosure A, at least one amino acid residue that can be exposed on the surface of the variable region (which may be modified) may be modified as described above, and at least one amino acid residue that can be exposed on the surface of the constant region (which may be modified) may be modified as described above, thereby increasing the net pI of the antibody.

[0172] Within the scope of Disclosures A and B described herein, when the antibody of Disclosure A or B is an IgG-type antibody or a molecule derived therefrom, the heavy chain constant region of the antibody may include an IgG1, IgG2, IgG3, or IgG4-type constant region. In Disclosure A or B, the heavy chain constant region may be, but is not limited to, a human heavy chain constant region. Several allotypes of human IgG are known to exist. That is, it has been reported that the amino acid sequences of human IgG constant regions vary slightly between individuals (Methods Mol. Biol. 882:635-80 (2012); Sequences of proteins of immunological interest, NIH Publication No. 91-3242). Examples include a human IgG1 constant region (SEQ ID NO: 18), a human IgG2 constant region (SEQ ID NO: 19), a human IgG3 constant region (SEQ ID NO: 20), and a human IgG4 constant region (SEQ ID NO: 21), respectively.

[0173] Among these, for example, allotypes called G1m1,17 and G1m3 are known for human IgG1. These allotypes differ in their amino acid sequences. Specifically, G1m1,17 has an aspartic acid at position 356 and a leucine at position 358 (EU numbering), whereas G1m3 has a glutamic acid at position 356 and a methionine at position 358. However, no reports have suggested significant differences in the essential antibody functions or properties among the reported allotypes. Therefore, those skilled in the art will readily understand that various studies have been conducted using specific allotypes, and that the results are not limited to the allotypes used to obtain these examples, and that similar effects are expected for all allotypes. For these reasons, within the scope of Disclosures A and B herein, when "human IgG1," "human IgG2," "human IgG3," or "human IgG4" is used, the allotype is not limited to a specific allotype, but includes all reported allotypes.

[0174] In alternative or further embodiments of Disclosure A or B, the antibody light chain constant region may comprise any constant region of the κ chain (IgK) or λ chain (IgL1, IgL2, IgL3, IgL6, or IgL7) type. Preferably, but not exclusively, the light chain constant region is a human light chain constant region. Multiple allotype sequences due to genetic polymorphisms for the human κ chain constant region and the human λ chain constant region have been reported, for example, in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242. Examples of such allotypes include the human κ chain constant region (SEQ ID NO: 22) and the human λ chain constant region (SEQ ID NO: 23). However, no reports have been made suggesting that the reported allotypes have significant differences in the essential antibody functions or properties. Therefore, when a specific allotype is mentioned within the scope of Disclosures A and B in this specification, a person skilled in the art can easily infer that the same effect can be expected for all allotypes (hereinafter, these are also collectively referred to as native (human) IgG (type) constant regions).

[0175] Furthermore, since the Fc region of a native IgG antibody constitutes a part of the constant region of a native IgG antibody, when the antibody of Disclosure A or B is, for example, an IgG type antibody or a molecule derived therefrom, the antibody may have an Fc region contained in the constant region of a native IgG (IgG1, IgG2, IgG3, or IgG4 type) (hereinafter, these are also collectively referred to as native (human) IgG (type) Fc region.) The Fc region of a native IgG can mean an Fc region consisting of the same amino acid sequence as an Fc region derived from IgG found in nature. Specific examples of the Fc region of native human IgG include the Fc region contained in the above-mentioned human IgG1 constant region (SEQ ID NO: 18), human IgG2 constant region (SEQ ID NO: 19), human IgG3 constant region (SEQ ID NO: 20), or human IgG4 constant region (SEQ ID NO: 21) (the Fc region of the IgG class can mean, for example, from cysteine at position 226 according to EU numbering to the C-terminus, or from proline at position 230 according to EU numbering to the C-terminus).

[0176] In one embodiment, the antibodies of Disclosures A and B may comprise variants in which one or more modifications selected from amino acid substitution, addition, deletion, or insertion have been made in the constant region (heavy chain constant region and / or light chain constant region) of a native (preferably human) IgG or the Fc region of a native (preferably human) IgG.

[0177] Within the scope of Disclosure A of the present specification, WO2013 / 081143 reports, for example, that ion concentration-dependent antibodies capable of forming multivalent immune complexes (multivalent complexes between antigens and antibodies) with multimeric antigens, and multispecific ion concentration-dependent antibodies or multiparatopic ion concentration-dependent antibodies capable of forming multivalent immune complexes (multivalent complexes between antigens and antibodies) by recognizing two or more epitopes present on a monomeric antigen, can bind more tightly to FcγR, FcRn, and complement receptors due to the avidity (the sum of the binding strengths between multiple epitopes and multiple paratopes) mediated by at least two or more multivalent constant regions (which may be modified) or Fc regions (which may be modified) contained in these antibody molecules, and that, as a result, the antibodies are taken up into cells more quickly. Therefore, when the above-described ion concentration-dependent antibody capable of forming a multivalent immune complex with a multimeric or monomeric antigen is modified to have an increased pI by modifying at least one amino acid residue that can be exposed on the antibody's surface, it can also be used as the antibody of Disclosure A (an ion concentration-dependent antibody with an increased pI). Those skilled in the art will recognize that an ion concentration-dependent antibody with an increased pI that can form a multivalent immune complex with a multimeric or monomeric antigen can be internalized more rapidly into cells compared with an ion concentration-dependent antibody with an increased pI that cannot form a multivalent immune complex. Furthermore, in one embodiment, the antibody of Disclosure A may have increased binding activity to FcRn and / or FcγR under neutral pH conditions. In such a case, those skilled in the art will understand that an ion concentration-dependent antibody with an increased pI that can form a multivalent immune complex with a multimeric or monomeric antigen can be internalized even more rapidly into cells.

[0178] In one embodiment, the antibody of Disclosure A may be a one-armed antibody (including any embodiment of the one-armed antibody described in WO2005 / 063816). Typically, a one-armed antibody is an antibody lacking one of the two Fab regions that a normal IgG antibody has, and can be prepared by, for example, but not limited to, the method described in WO2005 / 063816. For example, but not limited to, an IgG antibody whose heavy chain has a VH-CH1-Hinge-CH2-CH3 structure may have a one-armed antibody at the site N-terminal to the Hinge (e.g., VH or CH1). Cleavage of one of the Fab regions results in the antibody being expressed in a form containing an extra sequence, and cleavage of one of the Fab regions at a site C-terminal to the hinge (e.g., CH2) results in an incomplete Fc region. Therefore, although not limited thereto, from the viewpoint of antibody molecule stability, it is preferable to produce a single-armed antibody by cleaving one of the two Fab regions of an IgG antibody at the hinge region (hinge). It is more preferable that the cleaved heavy chain is linked to the uncleaved heavy chain via an intramolecular disulfide bond. It has been reported in WO 2005 / 063816 that such single-armed antibodies have increased stability compared to Fab molecules. By producing such single-armed antibodies, it is also possible to produce antibodies with increased or decreased pI. Furthermore, when an ion concentration-dependent antigen-binding domain is incorporated into a single-armed antibody with an increased pI, the plasma half-life of the antibody can be further shortened, or the uptake of the antibody into cells can be further promoted, or the elimination of the antigen from plasma can be further promoted, or the affinity of the antibody for the extracellular matrix can be further increased, compared to an increased pI antibody that does not have an ion concentration-dependent antigen-binding domain.

[0179] Without being bound by any particular theory, and without being particularly limited, one embodiment in which the effect of accelerating cellular uptake by a single-armed antibody can be expected is when the soluble antigen has a lower pI than the antibody. The net pI of a complex consisting of an antibody and an antigen can be calculated using known methods by treating the complex as a single molecule. In this case, the lower the pI of the soluble antigen, the lower the net pI of the complex, and the higher the pI of the soluble antigen, the higher the net pI of the complex. Furthermore, when a conventional IgG antibody molecule (having two Fabs) is bound to one soluble antigen with a low pI than when two soluble antigens with low pIs are bound, the net pI of the complex will be lower in the latter case. When such a conventional antibody is converted into a single-armed antibody, only one antigen can be bound to one antibody molecule, but this makes it possible to suppress the decrease in pI of the complex caused by the binding of a second antigen. In other words, if the pI of the soluble antigen is lower than that of the antibody, conversion to a single-armed antibody would increase the pI of the complex compared to a normal antibody, thereby accelerating its uptake into cells.

[0180] Furthermore, although not limited thereto, in a typical IgG antibody molecule (having two Fabs), when the pI of the Fab is lower than the pI of the Fc, conversion to a single-armed antibody increases the net pI of the complex consisting of the single-armed antibody and antigen. Furthermore, when such conversion to a single-armed antibody is performed, it is preferable to cleave one of the Fabs at the hinge region located at the boundary between the Fab and Fc, from the viewpoint of the stability of the single-armed antibody. In this case, selecting a site that increases the pI of the single-armed antibody to the desired level is expected to effectively increase the pI.

[0181] Therefore, by calculating the theoretical pI of an antibody (theoretical pI of Fc, theoretical pI of Fab) and the theoretical pI of a soluble antigen and predicting the relationship between the difference in these theoretical pI values, it will be understood by those skilled in the art that the pI of an antibody can be increased and the uptake of an antigen into cells can be accelerated, without relying solely (or substantially) on the amino acid sequence of the antibody itself or the type of soluble antigen.

[0182] In one embodiment, the antibody of Disclosure A or B may be a multispecific antibody, including, but not limited to, a bispecific antibody. The multispecific antibody comprises a first polypeptide and a second polypeptide. Here, "a multispecific antibody comprising a first polypeptide and a second polypeptide" refers to an antibody that binds to at least two or more different antigens or at least two or more different epitopes within the same antigen. The first and second polypeptides preferably comprise a heavy chain variable region, and more preferably, the variable region comprises a CDR and / or FR. In another embodiment, the first and second polypeptides each preferably comprise a heavy chain constant region. In yet another embodiment, the multispecific antibody may comprise a third polypeptide and a fourth polypeptide, each comprising a light chain variable region and preferably further comprising a light chain constant region. In such a case, the first to fourth polypeptides may associate together to form a multispecific antibody.

[0183] In one embodiment, when the antibody of Disclosure A is a multispecific antibody and the multispecific antibody comprises a heavy chain constant region, for example, an IgG2 or IgG4 sequence at position 137, an IgG1 or IgG2 or IgG4 sequence at position 196, an IgG2 or IgG4 sequence at position 203, an IgG2 sequence at position 214, an IgG1 or IgG3 or IgG4 sequence at position 217, an IgG1 or IgG3 or IgG4 sequence at position 233, an IgG4 sequence at position 268, an IgG2 or IgG3 or IgG4 sequence at position 274, an IgG1 or IgG2 or IgG4 sequence at position 276, an IgG4 sequence at position 355, an IgG3 sequence at position 392, an IgG4 sequence at position 419, or an IgG1 or IgG2 or IgG4 sequence at position 435 may be used to reduce its pI. On the other hand, to increase the pI, for example, an IgG1 or IgG3 sequence at position 137, an IgG3 sequence at position 196, an IgG1 or IgG3 sequence at position 203, an IgG1, IgG3, or IgG4 sequence at position 214, an IgG2 sequence at position 217, an IgG2 sequence at position 233, an IgG1, IgG2, or IgG3 sequence at position 268, an IgG1 sequence at position 274, an IgG3 sequence at position 276, an IgG1, IgG2, or IgG3 sequence at position 355, an IgG1, IgG2, or IgG4 sequence at position 392, an IgG1, IgG2, or IgG3 sequence at position 419, or an IgG3 sequence at position 435 may be used.

[0184] In one embodiment, when the antibody of Disclosure A has two heavy chain constant regions, the pIs of the two heavy chain constant regions may be the same or different. Such heavy chain constant regions may be heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, which originally have different pIs. Alternatively, a pI difference may be introduced between the two heavy chain constant regions. The site of modification of at least one amino acid residue to introduce a pI difference in the constant region may be any of the positions described above, or may be a site selected from the group consisting of positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (EU numbering) of the heavy chain constant region described in WO2009 / 041643. Alternatively, because pI differences arise when the sugar chains are removed from the heavy chain constant region, the sugar chains may be removed by modifying the amino acid residue at position 297, which is the glycosylation site.

[0185] In one embodiment, the antibody of Disclosure A or B may be a polyclonal or monoclonal antibody, preferably a mammalian monoclonal antibody. Monoclonal antibodies include those produced by hybridomas or those produced by host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques. The antibody of Disclosure A or B may be, for example, a chimeric antibody, a humanized antibody, or an antibody prepared by affinity maturation, or a molecule derived therefrom.

[0186] In one embodiment, the antibody of Disclosure A or B may be derived from any animal species (e.g., human; or non-human animal such as mouse, rat, hamster, rabbit, monkey, cynomolgus monkey, rhesus monkey, hamadryas baboon, chimpanzee, goat, sheep, dog, cow, or camel) or any bird, etc., and preferably the antibody is derived from a human, monkey, or mouse.

[0187] In one embodiment, the antibody of Disclosure A or B may be an Ig-type antibody, preferably an IgG-type antibody.

[0188] Within the scope of Disclosures A and B herein, an Fc receptor (also referred to as "FcR") refers to a receptor protein capable of binding to the Fc region or Fc region variant of an immunoglobulin (antibody) or a molecule derived therefrom. Within the scope of Disclosure A herein, for example, Fc receptors for IgG, IgA, IgE, and IgM are known as FcγR, FcαR, FcεR, and FcμR, respectively. Within the scope of Disclosures A and B herein, an Fc receptor may be, for example, FcRn (also referred to as "neonatal Fc receptor").

[0189] Within the scope of Disclosure A of the present specification, "FcγR" may refer to a receptor protein capable of binding to the Fc region or Fc region variant of an IgG1, IgG2, IgG3, or IgG4 antibody or a molecule derived therefrom, and substantially encompasses any one or more, or all, members of the family of proteins encoded by the FcγR gene. In humans, this family includes FcγRI (CD64) including the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as all unidentified human FcγRs and FcγR isoforms and allotypes, but are not limited to these. Furthermore, splicing variants of human FcγRIIb (hFcγRIIb) have been reported, namely FcγRIIb1 and FcγRIIb2. Furthermore, a splicing variant called FcγRIIb3 has also been reported (Brooks et al., J. Exp. Med., 170: 1369-1385 (1989)). In addition to these, hFcγRIIb includes all splicing variants registered in NCBI as NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, hFcγRIIb encompasses not only all previously reported genetic polymorphisms, such as FcγRIIb (Arthritis Rheum, 48: 3242-3252 (2003), Kono et al., Hum. Mol. Genet, 14: 2881-2892 (2005), Kyogoku et al., Arthritis Rheum. 46(5):1242-1254 (2002)), but also all genetic polymorphisms that will be reported in the future.

[0190] FcγR may be derived from any organism, including, but not limited to, those derived from humans, mice, rats, rabbits, or monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs and FcγR isoforms and allotypes. Suitable examples of such FcγRs include human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), or FcγRIIIB (CD16). Since FcγRs exist in vivo as membrane proteins, they may be artificially converted to soluble forms before use in experimental systems.

[0191] As an example, as cited in WO2014 / 163101, The polynucleotide sequence and amino acid sequence of FcγRI may be the sequences set forth in NM_000566.3 and NP_000557.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIA may be the sequences set forth in BC020823.1 and AAH20823.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIB may be the sequences set forth in BC146678.1 and AAI46679.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIIA may be the sequences set forth in BC033678.1 and AAH33678.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIIB may be the sequences set forth in BC128562.1 and AAI28563.1, respectively (RefSeq accession numbers are shown).

[0192] FcγRIIa has two genetic polymorphisms in which the amino acid at position 131 of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med. 172: 19-25, 1990).

[0193] In FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes FcγRIIIa (including allotypes V158 and F158), the α chain that binds to the Fc region of IgG is linked to a common γ chain containing ITAMs that transduce activation signals intracellularly. FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPI-anchored protein. On the other hand, the cytoplasmic domain of FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIc, contains ITAMs. These receptors are expressed on many immune cells, such as macrophages, mast cells, and antigen-presenting cells. The activation signals transmitted by these receptors upon binding to the Fc region of IgG promote the phagocytic activity of macrophages, the production of inflammatory cytokines, the degranulation of mast cells, and the enhanced function of antigen-presenting cells. FcγRs capable of transmitting such activation signals are also referred to as activating FcγRs within the scope of Disclosures A and B of the present specification.

[0194] 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 activity and the ability to produce inflammatory cytokines. FcγRs capable of transmitting inhibitory signals in this way are also referred to as inhibitory Fcγ receptors within the scope of Disclosures A and B of the present specification.

[0195] Within the scope of Disclosure A of the present specification, whether the binding activity of an antibody or Fc region (variant) to various FcγRs is increased, (substantially) maintained, or decreased compared to the antibody or Fc region (variant) before modification can be assessed by methods known to those skilled in the art. Such methods are not particularly limited, and may include those described in the present Examples, or, for example, BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010), which utilizes the surface plasmon resonance (SPR) phenomenon. Alternatively, for example, ELISA, fluorescence-activated cell sorting (FACS), or an ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) may be used. For these assays, the extracellular domain of human FcγR may be used as a soluble antigen (e.g., WO2013 / 047752).

[0196] Acidic or neutral pH conditions are suitable for measuring the binding activity of an FcγR-binding domain contained in an antibody or Fc region (variant) to FcγR. The binding activity (binding affinity) of an FcγR-binding domain to FcγR can be evaluated at any temperature between 10°C and 50°C, for example. A preferred temperature for determining the binding activity (binding affinity) of a human FcγR-binding domain to FcγR is 15°C to 40°C, for example. 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 activity (binding affinity) of an FcγR-binding domain to FcγR. The temperature of 25°C is a non-limiting example.

[0197] In one embodiment, when an antibody of Disclosure A or B has a constant region (which may be modified), the constant region may have an Fc region or a variant Fc region (preferably a human Fc region or a variant human Fc region), and preferably has an FcγR-binding domain within the scope of Disclosure A, and an FcRn-binding domain within the scope of Disclosures A and B described herein.

[0198] In one embodiment, when the antibody of Disclosure A has binding activity to FcγR, it may have an FcγR-binding domain, preferably a human FcγR-binding domain. The FcγR-binding domain is not particularly limited as long as the antibody has binding activity or affinity for FcγR at acidic pH and / or neutral pH, or may be a domain that has activity to directly or indirectly bind to FcγR.

[0199] In one embodiment, when an antibody of Disclosure A has FcγR-binding activity, the antibody preferably has increased FcγR-binding activity under neutral pH conditions compared to a reference antibody comprising a natural IgG constant region. Here, although not limited thereto, it is preferable, from the perspective of comparing the FcγR-binding activities of the antibody of Disclosure A and the reference antibody comprising a natural IgG constant region, that they have identical amino acid sequences in other regions (e.g., variable regions), preferably excluding the constant region of the antibody of Disclosure A in which one or more amino acid residues have been altered.

[0200] In one embodiment, when an antibody of Disclosure A has FcγR-binding activity or its FcγR-binding activity is enhanced under neutral pH conditions (e.g., pH 7.4), without being bound by theory, it is believed that the antibody possesses an ion concentration-dependent antigen-binding domain, thereby allowing a single antibody molecule to repeatedly bind to multiple antigens by traveling between plasma and intracellular endosomes; the antibody as a whole has an increased pI and an increased positive charge, allowing it to be rapidly internalized within cells; and the antibody's enhanced FcγR-binding activity under neutral pH conditions allows it to be rapidly internalized within cells. As a result, the antibody of Disclosure A is believed to be advantageous because it can further shorten the antibody's plasma half-life, further increase the antibody's binding activity to the extracellular matrix, or further promote antigen elimination from plasma. Those skilled in the art can routinely determine the optimal antibody pI value to utilize these properties.

[0201] In one embodiment, an FcγR-binding domain having higher FcγR-binding activity than that of the Fc region or constant region of a native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain can be prepared by modifying amino acid residues in the Fc region or constant region of native human IgG (see WO2013 / 047752). Furthermore, any domain with an FcγR-binding structure can be used as the FcγR-binding domain. In this case, the FcγR-binding domain can be prepared without the need to introduce amino acid modifications, or additional modifications can be introduced to enhance affinity for FcγR. Examples of such FcγR-binding domains include FcγRIIIa-binding Fab fragment antibodies, camel-derived single-domain antibodies, and single-chain Fv antibodies described in Schlapschly et al. (Protein Eng. Des. Sel. 22(3):175-188 (2009)), Behar et al. (Protein Eng. Des. Sel. 21(1):1-10 (2008)), and Kipriyanov et al. (J. Immunol. 169(1):137-144 (2002)), as well as the FcγRI-binding cyclic peptide described in Bonetto et al., FASEB J. 23(2):575-585 (2009). Whether the FcγR-binding activity of an FcγR-binding domain is higher than the FcγR-binding activity of the Fc region or constant region of native human IgG in which the sugar chain attached to position 297 (EU numbering) is a fucose-containing sugar chain can be appropriately evaluated using the methods described above.

[0202] In one embodiment of Disclosure A, examples of the starting FcγR-binding domain preferably include the Fc region of (human) IgG or the constant region of (human) IgG. Any Fc region or constant region can be used as the starting Fc region or constant region, as long as the modified starting Fc region or constant region can bind to human FcγR in the neutral pH range. Furthermore, an Fc region or constant region obtained by further modifying a starting Fc region or constant region that has already had amino acid residue modifications added thereto can also be preferably used as the Fc region or constant region in Disclosure A. The starting Fc region or constant region may refer to the polypeptide itself, a composition containing the starting Fc region or constant region, or an amino acid sequence encoding the starting Fc region or constant region. The starting Fc region or constant region may include a known Fc region or constant region produced by recombinant technology. The source of the starting Fc region or constant region is not limited, and it may be obtained from any non-human animal or human. Furthermore, the starting FcγR-binding domain can be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. Preferably, the starting Fc region or starting constant region may be obtained from human IgG1, but is not limited to a particular IgG class. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the starting FcγR-binding domain. Similarly, within the scope of Disclosure A of the present specification, Fc regions or constant regions of IgG classes or subclasses from any organism can preferably be used as the starting Fc region or starting constant region.Examples of modified or altered native IgGs are described in known literature (e.g., Strohl, Curr. Opin. Biotechnol. 20 (6): 685-691 (2009), Presta, Curr. Opin. Immunol. 20 (4): 460-470 (2008), Davis et al., Protein Eng. Des. Sel. 23 (4): 195-202 (2010), WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338), but are not limited thereto.

[0203] In one embodiment, examples of amino acid residues in the starting FcγR-binding domain, starting Fc region, or starting constant region may include one or more mutations, such as substitution of an amino acid residue different from that of the starting Fc region or starting constant region, insertion of one or more amino acid residues relative to the amino acid residues of the starting Fc region or starting constant region, or deletion of one or more amino acid residues from the amino acid residues of the starting Fc region or starting constant region. Preferably, the amino acid sequence of the modified Fc region or constant region may comprise at least a portion of an Fc region or constant region that does not occur in nature. Such variants necessarily have less than 100% sequence identity or similarity to the starting Fc region or starting constant region. For example, the variants have an amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, even more preferably about 90% to less than 100%, and most preferably about 95% to less than 100% to the amino acid sequence of the starting Fc region or starting constant region. In a non-limiting example, at least one amino acid differs between the starting Fc region or starting constant region and the modified Fc region or constant region of Disclosure A.

[0204] In one embodiment, an Fc region or constant region having FcγR-binding activity in the acidic and / or neutral pH range, which may be included in an antibody of Disclosure A, can be obtained by any method. Specifically, a modified Fc region or constant region having FcγR-binding activity in the neutral pH range can be obtained by modifying the amino acids of a human IgG antibody that can be used as a starting Fc region or starting constant region. Examples of Fc regions of IgG antibodies or constant regions of IgG-type antibodies suitable for modification include the Fc region or constant region of human IgG (IgG1, IgG2, IgG3, or IgG4, or variants thereof) and naturally occurring mutants thereof. Regarding the Fc region or constant region of human IgG1, human IgG2, human IgG3, or human IgG4 antibodies, several allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these may be used in Disclosure A. In particular, with regard to the sequence of human IgG1, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM.

[0205] In further embodiments within the scope of Disclosure A, modifications to other amino acids are not limited, as long as the modified product has FcγR-binding activity in the neutral pH range. Such amino acid modification positions are described in, for example, WO2007 / 024249, WO2007 / 021841, WO2006 / 031370, WO2000 / 042072, WO2004 / 029207, WO2004 / 099249, WO2006 / 105338, WO2007 / 041635, WO2008 / 092117, WO2005 / 070963, WO20 06 / 020114, WO2006 / 116260, WO2006 / 023403, WO2013 / 047752, WO2006 / 019447, WO2012 / 115241, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101, WO2013 / 118858, and WO2014 / 030750.

[0206] Examples of amino acid modification sites in the constant region or Fc region to increase FcγR-binding activity in the neutral pH range include those at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318 235th, 236th, 237th, 238th, 239th, 240th, 241st, 243rd, 244th, 245th, 246th, 247th, 249th, 250th, 251st, 254th, 255th, 256th 258th, 260th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 27th 8th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 288th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 311th, 313th, 315th, 317th, 318th, 320th, 322nd, 323rd, 324th, 325th , 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440. Such modification of amino acid residues can enhance FcγR binding under neutral pH conditions in the Fc region or constant region of an IgG antibody. WO2013 / 047752 describes, for example, the following positions, represented by EU numbering, as preferred modifications in the IgG constant region or Fc region: amino acid at position 221 to either Lys or Tyr; the amino acid at position 222 to one of Phe, Trp, Glu, and Tyr; the amino acid at position 223 to one of Phe, Trp, Glu, and Lys; the amino acid at position 224 to one of Phe, Trp, Glu, and Tyr; the amino acid at position 225 to either Glu, Lys, or Trp; the amino acid at position 227 to one of Glu, Gly, Lys, and Tyr; the amino acid at position 228 to one of Glu, Gly, Lys, and Tyr; the amino acid at position 230 to one of Ala, Glu, Gly, and Tyr; the amino acid at position 231 to one of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 232 to one of Glu, Gly, Lys, and Tyr; the amino acid at position 233 to one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 to one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 to one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 to one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 to one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 238 to one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 239 to one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 240 to one of Ala, Ile, Met, and Thr; the amino acid at position 241 to one of Asp, Glu, Leu, Arg, Trp, and Tyr; the amino acid at position 243 to one of Glu, Leu, Gln, Arg, Trp, and Tyr; amino acid at position 244 to His; amino acid at position 245 to Ala; the amino acid at position 246 to one of Asp, Glu, His, and Tyr; the amino acid at position 247 to one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; the amino acid at position 249 to one of Glu, His, Gln, and Tyr; the amino acid at position 250 to either Glu or Gln; amino acid at position 251 to Phe; amino acid at position 254 to either Phe, Met, or Tyr; the amino acid at position 255 to one of Glu, Leu, and Tyr; the amino acid at position 256 to either Ala, Met, or Pro; the amino acid at position 258 to one of Asp, Glu, His, Ser, and Tyr; the amino acid at position 260 to one of Asp, Glu, His, and Tyr; the amino acid at position 262 to one of Ala, Glu, Phe, Ile, and Thr; the amino acid at position 263 to one of Ala, Ile, Met, and Thr; the amino acid at position 264 to one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 265 to one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 266 to one of Ala, Ile, Met, and Thr; the amino acid at position 267 to one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 268 to one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp; the amino acid at position 269 to one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 270 to one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 to one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 272 to one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; amino acid at position 273 to either Phe or Ile; the amino acid at position 274 to one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; amino acid at position 275 to either Leu or Trp; the amino acid at position 276 to one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 278 to one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; amino acid at position 279 to Ala; the amino acid at position 280 to one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid at position 281 to one of Asp, Lys, Pro, and Tyr; the amino acid at position 282 to one of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 283 to any of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid at position 284 to one of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid at position 285 to one of Asp, Glu, Lys, Gln, Trp, and Tyr; the amino acid at position 286 to one of Glu, Gly, Pro, and Tyr; the amino acid at position 288 to one of Asn, Asp, Glu, and Tyr; the amino acid at position 290 to one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid at position 291 to one of Asp, Glu, Gly, His, Ile, Gln, and Thr; the amino acid at position 292 to any of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid at position 293 to one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 294 to one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 to one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 296 to one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val; the amino acid at position 297 to one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 298 to one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 299 to one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 300 to one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; the amino acid at position 301 to one of Asp, Glu, His, and Tyr; amino acid at position 302 to Ile; the amino acid at position 303 to either Asp, Gly, or Tyr; the amino acid at position 304 to one of Asp, His, Leu, Asn, and Thr; the amino acid at position 305 to one of Glu, Ile, Thr, and Tyr; the amino acid at position 311 to any of Ala, Asp, Asn, Thr, Val, and Tyr; amino acid at position 313 to Phe; amino acid at position 315 to Leu; amino acid at position 317 to either Glu or Gln; the amino acid at position 318 to one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 to one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 322 to any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; amino acid at position 323 to Ile; the amino acid at position 324 to one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 325 to one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 326 to one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 327 to one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 328 to one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 329 to one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 330 to one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 to one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 332 to one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 333 to any of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 to any of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; the amino acid at position 335 to one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 to one of Glu, Lys, and Tyr; the amino acid at position 337 to either Glu, His, or Asn; the amino acid at position 339 to any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr; amino acid at position 376 to either Ala or Val; amino acid at position 377 to either Gly or Lys; amino acid at position 378 to Asp; amino acid at position 379 to Asn; the amino acid at position 380 to either Ala, Asn, or Ser; amino acid at position 382 to either Ala or Ile; amino acid at position 385 to Glu; amino acid at position 392 to Thr; amino acid at position 396 to Leu; amino acid at position 421 to Lys; amino acid at position 427 to Asn; amino acid at position 428 to either Phe or Leu; amino acid at position 429 to Met; amino acid at position 434 to Trp; the amino acid at position 436 to Ile; and Amino acid at position 440 is changed to one of Gly, His, Ile, Leu, and Tyr. The present disclosure also provides modifications of one or more amino acid residues selected from the group consisting of: (a) a modified amino acid sequence; (b) a modified amino acid sequence; (c) a modified amino acid sequence; (d) a modified amino acid sequence; (e) a modified amino acid sequence; (f) a modified amino acid sequence; (g) a modified amino acid sequence; (h) a modified amino acid sequence; (i) a modified amino acid sequence; (ii) a modified amino acid sequence; (iii) a modified amino acid sequence; (iv) a modified amino acid sequence; (v) a modified amino acid sequence; (vi ...

[0207] In one embodiment, the binding activity of the antibody of Disclosure A (the FcγR-binding domain thereof) to one or more (human) FcγRs, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, may be higher than the binding activity of a native IgG (the Fc region or constant region thereof) or a reference antibody comprising the starting Fc region or starting constant region to FcγR. For example, the FcγR-binding activity of the antibody of Disclosure A (the FcγR-binding domain thereof) is 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, particularly preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, compared to the FcγR-binding activity of the reference antibody. or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, or 195% or more, or may be 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 7.5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, or 100 times or more compared to the FcγR-binding activity of the reference antibody.

[0208] In a further embodiment, the degree of increase in binding activity to inhibitory FcγR (FcγRIIb-1 and / or FcγRIIb-2) (in the neutral pH range) may be higher than the degree of increase in binding activity to activating FcγR (FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including allotype V158, FcγRIIIa including allotype F158, FcγRIIIb including allotype FcγRIIIb-NA1, FcγRIIIb including allotype FcγRIIIb-NA2, FcγRIIa including allotype H131, or FcγRIIa including allotype R131).

[0209] In one embodiment, the antibody of Disclosure A may have binding activity to FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

[0210] In one embodiment, examples of suitable FcγR-binding domains in Disclosure A include FcγR-binding domains whose binding activity to a specific FcγR is higher than its binding activity to other FcγRs (FcγR-binding domains with selective FcγR-binding activity). When an antibody (or an Fc region is used as an FcγR-binding domain) is used, one antibody molecule can bind to only one FcγR molecule. Therefore, one antibody molecule bound to an inhibitory FcγR cannot bind to other activating FcγRs, and one antibody molecule bound to an activating FcγR cannot bind to other activating FcγRs or inhibitory FcγRs.

[0211] As described above, preferred examples of activating FcγR include FcγRI (CD64), such as FcγRIa, FcγRIb, or FcγRIc; and FcγRIII (CD16), such as FcγRIIIa (e.g., allotype V158 or F158), or FcγRIIIb (e.g., allotype FcγRIIIb-NA1 or FcγRIIIb-NA2). On the other hand, preferred examples of inhibitory FcγR include FcγRIIb (e.g., FcγRIIb-1 or FcγRIIb-2).

[0212] In one embodiment, an FcγR-binding domain having higher binding activity to inhibitory FcγRs than to activating FcγRs may be used as the selective FcγR-binding domain contained in the antibody of Disclosure A. For example, the selective FcγR-binding domain may be an FcγR-binding domain having higher binding activity to FcγRIIb (such as FcγRIIb-1 and / or FcγRIIb-2) than to any one or more activating FcγRs selected from the group consisting of FcγRI (CD64) such as FcγRIa, FcγRIb, or FcγRIc; FcγRIII (CD16) such as FcγRIIIa (e.g., allotype V158 or F158) or FcγRIIIb (e.g., FcγRIIIb-NA1 or FcγRIIIb-NA2); and FcγRIIa (including allotype H131 or R131), and FcγRII (CD32) such as FcγRIIc.

[0213] Furthermore, whether an FcγR-binding domain has selective binding activity can be determined by comparing the binding activity to each FcγR determined by the methods described above, for example, by comparing the values (ratios) obtained by dividing the KD value for activating FcγR by the KD value for inhibitory FcγR, i.e., by comparing the FcγR selectivity indexes represented by the following formula 1: [Formula 1] FcγR selectivity index = KD value for activating FcγR / KD value for inhibitory FcγR

[0214] In Formula 1, the KD value for activating FcγR refers to the KD value for one or more of FcγRIa; FcγRIb; FcγRIc; FcγRIIIa containing allotypes V158 and / or F158; FcγRIIIb containing FcγRIIIb-NA1 and / or FcγRIIIb-NA2; FcγRIIa containing allotypes H131 and / or R131; and FcγRIIc, while the KD value for inhibitory FcγR refers to the KD value for FcγRIIb-1 and / or FcγRIIb-2. The activating FcγR and inhibitory FcγR used to measure the KD value may be selected from any combination. For example, the value (ratio) obtained by dividing the KD value for FcγRIIa containing allotype H131 by the KD value for FcγRIIb-1 and / or FcγRIIb-2 may be used, but is not limited to these.

[0215] The FcγR selectivity index is, for example, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 3 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 10 0 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more , 410 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 or more, 500 or more, 520 or more, 540 or more, 560 or more, 580 or more, 600 or more, 620 or more, 640 or more, 660 or more, 680 or more, 700 or more, 720 or more, 740 or more, 760 or more, 780 or more, 800 or more, 820 or more, 840 or more, 860 or more, 880 or more, 900 or more, 92 It can be, but is not limited to, 0 or more, 940 or more, 960 or more, 980 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, 8000 or more, 8500 or more, 9000 or more, 9500 or more, 10000 or more, or 100,000 or more.

[0216] In one embodiment, an Fc region variant or constant region variant (an antibody comprising the variant) in which the amino acid at position 238 or 328 according to EU numbering of human IgG (IgG1, IgG2, IgG3, or IgG4) is Asp or Glu, respectively, is used, particularly as described in WO2013 / 125667, WO2012 / 115241, and W2013 / 047752, including FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including allotype V158, and allota Since these antibodies have higher binding activity to FcγRIIb-1 and / or FcγRIIb-2 than to FcγRIIIa comprising allotype F158, FcγRIIIb comprising allotype FcγRIIIb-NA1, FcγRIIIb comprising allotype FcγRIIIb-NA2, FcγRIIa comprising allotype H131, FcγRIIa comprising allotype R131, and / or FcγRIIc, they can be preferably used as antibodies of Disclosure A comprising an Fc region variant or constant region variant. In such an embodiment, the antibody of Disclosure A has binding activity to all activating FcγRs and FcγRIIb (selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa in the present specification), and its binding activity to FcγRIIb is maintained or enhanced and / or its binding activity to all activating FcγRs is decreased, compared to a reference antibody comprising the constant region of a native IgG or the Fc region of a native IgG.

[0217] In one embodiment of the antibodies of Disclosure A comprising a variant Fc region or constant region, their binding activity to FcγRIIb may be maintained or increased, and their binding activity to FcγRIIa (H type) and FcγRIIa (R type) may be decreased, compared to the activity of a reference antibody having a native IgG constant region or Fc region. Such antibodies may have increased selectivity for FcγRIIb over FcγRIIa.

[0218] Within the scope of Disclosure A in the present specification, the degree of "reduced binding activity to all activating FcγRs" is not limited to, but may include, but is not limited to, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 68 ... %, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% 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, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

[0219] Within the scope of Disclosure A of the present specification, the degree to which "the binding activity to FcγRIIb is maintained or increased" or "the binding activity to FcγRIIb maintained or increased" is not limited to, but includes 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or more, 118% or more, 119% or more, 120% or more, 121% or more, 122% or more, 123% or more, 124% or more, 125% or more, 126% or more, 127% or more, 128% or more, 129% or more, 130% or more, 131% or more, 132% or more, 133% or more, 134% or more, 135% or more, 136% or more, 137% or more, 138% or more, 139 % or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 112% or more, 114% or more, 116% or more, 118% or more, 120% or more, 122% or more, 12 4% or more, 126% or more, 128% or more, 130% or more, 132% or more, 134% or more, 136% or more, 138% or more, 140% or more, 142% or more, 144% or more, 146% or more, 148% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 2x or more, 3x or more, 4x or more, 5x or more fold or more, 6 fold or more, 7 fold or more, 8 fold or more, 9 fold or more, 10 fold or more, 20 fold or more, 30 fold or more, 40 fold or more, 50 fold or more, 60 fold or more, 70 fold or more, 80 fold or more, 90 fold or more, 100 fold or more, 200 fold or more, 300 fold or more, 400 fold or more, 500 fold or more, 600 fold or more, 700 fold or more, 800 fold or more, 900 fold or more, 1000 fold or more, 10,000 fold or more, or 100,000 fold or more.

[0220] Within the scope of Disclosure A in the present specification, the degree to which the "binding activity to FcγRIIa (H type) and FcγRIIa (R type) is reduced" or the "reduced binding activity to FcγRIIa (H type) and FcγRIIa (R type)" includes, but is not limited to, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 79% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 79% or less, 79% or less, 8 ...9% or less, 98% or less, 97% or less, 96% or less, It can be 6% or less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% 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, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

[0221] Within the scope of Disclosure A of the present specification, alterations that increase binding selectivity for FcγRIIb over FcγRIIa (R type) may be preferred, and alterations that increase binding selectivity for FcγRIIb over FcγRIIa (H type) may be more preferred. Preferred amino acid substitutions for such alterations are those represented by EU numbering, as reported in WO2013 / 047752, for example. (a) modification to replace Gly with Trp at position 237; (b) modification to replace Gly with Phe at position 237; (c) modification to replace Pro with Phe at position 238; (d) modification to replace Asn at position 325 with Met; (e) a modification to replace Ser with Ile at position 267; (f) modification to replace Leu at position 328 with Asp; (g) modification to replace Ser with Val at position 267; (h) modification to replace Leu at position 328 with Trp; (i) a modification to replace Ser with Gln at position 267; (j) a modification to replace Ser with Met at position 267; (k) a modification to replace Gly with Asp at position 236; (l) modification to replace Ala with Asn at position 327; (m) modification of Asn at position 325 with Ser; (n) a modification to replace Leu with Tyr at position 235; (o) modification to replace Val with Met at position 266; (p) modification to replace Leu with Tyr at position 328; (q) modification to replace Leu with Trp at position 235; (r) modification to replace Leu with Phe at position 235; (s) modification to replace Ser with Gly at position 239; (t) modification to replace Ala with Glu at position 327; (u) modification to replace Ala with Gly at position 327; (v) a modification to replace Pro with Leu at position 238; (w) modification to replace Ser with Leu at position 239; (x) modification of Leu at position 328 with Thr; (y) modification to replace Leu with Ser at position 328; (z) modification to replace Leu with Met at position 328; (aa) modification to replace Pro with Trp at position 331; (ab) modification to replace Pro with Tyr at position 331; (ac) modification to replace Pro with Phe at position 331; (ad) modification to replace Ala with Asp at position 327; (ae) modification to replace Leu with Phe at position 328; (af) modification to replace Pro with Leu at position 271; (ag) modification to replace Ser with Glu at position 267; (ah) modification to replace Leu with Ala at position 328; (ai) modification to replace Leu with Ile at position 328; (aj) modification to replace Leu with Gln at position 328; (ak) modification to replace Leu with Val at position 328; (al) an alteration to replace Lys with Trp at position 326; (am) modification to replace Lys with Arg at position 334; (an) modification to replace His with Gly at position 268; (ao) modification of position 268 by substituting His with Asn; (ap) modification to replace Ser with Val at position 324; (aq) modification to replace Val with Leu at position 266; (ar) modification to replace Pro with Gly at position 271; (as) modification to replace Ile with Phe at position 332; (at) modification to replace Ser with Ile at position 324; (au) modification to replace Glu with Pro at position 333; (av) modification to replace Tyr with Asp at position 300; (aw) modification to replace Ser with Asp at position 337; (ax) modification to replace Tyr with Gln at position 300; (ay) modification to replace Thr with Asp at position 335; (az) modification to replace Ser with Asn at position 239; (ba) modification to replace Lys with Leu at position 326; (bb) a modification to replace Lys with Ile at position 326; (bc) modification to replace Ser with Glu at position 239; (bd) modification to replace Lys with Phe at position 326; (be) modification to replace Lys with Val at position 326; (bf) modification to replace Lys with Tyr at position 326; (bg) modification to replace Ser with Asp at position 267; (bh) modification to replace Lys with Pro at position 326; (bi) modification to replace Lys with His at position 326; (bj) modification to replace Lys with Ala at position 334; (bk) modification to replace Lys with Trp at position 334; (bl) modification to replace His with Gln at position 268; (bm) modification to replace Lys with Gln at position 326; (bn) modification to replace Lys with Glu at position 326; (bo) an alteration to replace Lys with Met at position 326; (bp) modification to replace Val with Ile at position 266; (bq) modification to replace Lys with Glu at position 334; (br) modification to replace Tyr with Glu at position 300; (bs) modification to replace Lys with Met at position 334; (bt) modification to replace Lys with Val at position 334; (bu) modification to replace Lys with Thr at position 334; (bv) modification to replace Lys with Ser at position 334; (bw) modification to replace Lys with His at position 334; (bx) modification to replace Lys with Phe at position 334; (by) substitution of Lys with Gln at position 334; (bz) modification to replace Lys with Pro at position 334; (ca) modification to replace Lys with Tyr at position 334; (cb) modification to replace Lys with Ile at position 334; (cc) modification to replace Gln with Leu at position 295; (cd) modification to replace Lys with Leu at position 334; (ce) modification of Lys at position 334 with Asn; (cf) modification to replace His with Ala at position 268; (cg) modification to replace Ser with Asp at position 239; (ch) modification to replace Ser with Ala at position 267; (ci) modification to replace Leu with Trp at position 234; (cj) modification to replace Leu at position 234 with Tyr; (ck) modification to replace Gly with Ala at position 237; (cl) modification to replace Gly with Asp at position 237; (cm) modification to replace Gly with Glu at position 237; (cn) modification to replace Gly with Leu at position 237; (co) modification to replace Gly with Met at position 237; (cp) modification to replace Gly with Tyr at position 237; (cq) modification to replace Ala with Lys at position 330; (cr) modification to replace Ala with Arg at position 330; (cs) modification to replace Glu with Asp at position 233; (ct) modification to replace His with Asp at position 268; (cu) modification to replace His with Glu at position 268; (cv) modification to replace Lys with Asp at position 326; (cw) modification to replace Lys with Ser at position 326; (cx) modification to replace Lys with Thr at position 326; (cy) modification to replace Val with Ile at position 323; (cz) modification to replace Val with Leu at position 323; (da) modification to replace Val with Met at position 323; (db) modification to replace Tyr with Asp at position 296; (dc) modification to replace Lys with Ala at position 326; (dd) a modification to replace Lys with Asn at position 326; (de) Ala at position 330 replaced with Met It could be.

[0222] The above alterations may be made at only one site or in combination of two or more sites. Preferred examples of such alterations include those described in Tables 14-15, 17-24, and 26-28 of WO2013 / 047752, such as variants of human constant regions or human Fc regions in which the amino acid at position 238 (EU numbering) in human IgG (IgG1, IgG2, IgG3, or IgG4) is Asp and the amino acid at position 271 (EU numbering) is Gly, and further, one or more of positions 233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396 (EU numbering) may be substituted. In such cases, the variant may be represented by EU numbering: Asp, ranked 233rd; Tyr, ranked 234th; Asp, ranked 237th; Ile, ranked 264th; Glu at position 265; either Phe, Met, or Leu at position 266; any of Ala, Glu, Gly, or Gln at position 267; Either Asp or Glu at position 268 Asp, ranked 269th; at position 272, any of Asp, Phe, Ile, Met, Asn, or Gln; Asp, ranked 296th; Either Ala or Asp at position 326, Gly, ranked 327th; Either Lys or Arg at position 330; Ser, ranked 331st; Thr, ranked 332nd; either Thr, Lys, or Arg at position 333, and Any of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr at position 396 Examples of human Fc region variants include, but are not limited to, variants of human constant regions or human Fc regions comprising one or more of:

[0223] In an alternative embodiment, an antibody of Disclosure A comprising a variant Fc region or a variant constant region may have maintained or increased binding activity to FcγRIIb and decreased binding activity to FcγRIIa (H type) and FcγRIIa (R type) compared to a reference antibody comprising the constant region or Fc region of a native IgG. As reported in WO2014 / 030728, preferred amino acid substitution sites for such variants may be, for example, amino acid position 238 (EU numbering), and at least one amino acid position selected from the group consisting of positions 233, 234, 235, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409, and 419 (EU numbering).

[0224] More preferably, the variant may have Asp at position 238 (EU numbering), and may also have Asp at position 233, Tyr at position 234, Phe at position 235, Asp at position 237, Ile at position 264, Glu at position 265, Phe, Leu or Met at position 266, Ala, Glu, Gly or Gln at position 267, Asp, Gln or Glu at position 268, Asp at position 269, Gly at position 271, Asp, Phe, Ile, Met, Asn, Pro or Gln at position 272, Gln at position 274, Asp or Phe at position 296, Ala or Asp at position 326, It may have at least one amino acid selected from the group consisting of Gly at position 327, Lys, Arg or Ser at position 330, Ser at position 331, Lys, Arg, Ser or Thr at position 332, Lys, Arg, Ser or Thr at position 333, Arg, Ser or Thr at position 334, Ala or Gln at position 355, Glu at position 356, Met at position 358, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr at position 396, Arg at position 409, and Glu at position 419.

[0225] In an alternative embodiment, the antibody of Disclosure A comprising a variant Fc region or constant region may maintain its binding activity to FcγRIIb and have reduced binding activity to all activating FcγRs, particularly FcγRIIa (R type), compared to a reference antibody comprising a native IgG constant region or Fc region. Preferred amino acid substitution sites for such variants may be, as reported in WO2014 / 163101, at least one amino acid position selected from positions 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 (EU numbering), in addition to amino acid position 238 (EU numbering). More preferably, the variant may have Asp at position 238, as represented by EU numbering, and at least one amino acid selected from the group consisting of Phe at position 235, Gln or Asp at position 237, Met or Leu at position 241, Pro at position 268, Met or Val at position 295, Glu, His, Asn or Asp at position 296, Ala or Met at position 298, Ile at position 323, Asn or His at position 324, and His or Tyr at position 330.

[0226] Within the scope of Disclosure A in the present specification, the degree to which the "binding activity to FcγRIIb is maintained" is not limited to, but may include, but is not limited to, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 175% or more, or more than twice the normal range.

[0227] Within the scope of Disclosure A in the present specification, the degree to which "binding activity to all activating FcγRs, in particular FcγRIIa (R type) is reduced" is not limited to, but includes 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% 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, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

[0228] WO2014 / 030750 also reports variants of the mouse constant region and Fc region. In one embodiment, the antibody of Disclosure A or B may contain such variants.

[0229] Within the scope of Disclosures A and B herein, unlike FcγR, which belongs to the immunoglobulin superfamily, "FcRn," particularly human FcRn, is structurally similar to polypeptides of the major histocompatibility complex (MHC) class I, exhibiting 22% to 29% sequence identity to MHC class I molecules (Ghetie et al., Immunol. Today 18 (12), 592-598 (1997)). 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 contains 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 of the antibody Fc region (Raghavan et al., (Immunity 1: 303-315 (1994)).

[0230] FcRn is expressed in the maternal placenta and 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 a variety of other tissues and endothelial cell lines in various species. FcRn 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. Typically, FcRn binding to IgG molecules is strictly pH-dependent; optimal binding is observed in the acidic pH range below 7.0.

[0231] As an example, the polynucleotide sequence and amino acid sequence of human FcRn may be derived from the precursors described in NM_004107.4 and NP_004098.1 (including the signal sequence), respectively (RefSeq accession numbers are shown in parentheses).

[0232] This precursor forms a complex with human β2-microglobulin in vivo. Therefore, for appropriate use in various experimental systems, soluble human FcRn capable of forming a complex with human β2-microglobulin may be produced using known recombinant expression techniques. Such soluble human FcRn may be used to evaluate antibodies or Fc region variants for their FcRn-binding activity. In Disclosure A or B, FcRn is not particularly limited as long as it is in a form that can bind to the FcRn-binding domain, but human FcRn is preferred.

[0233] Within the scope of Disclosures A and B herein, when an antibody or Fc region variant has FcRn-binding activity, it may have an "FcRn-binding domain," preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited as long as the antibody has binding activity or affinity for FcRn at acidic and / or neutral pH, or may be a domain that has activity that directly or indirectly binds to FcRn. Examples of such domains include, but are not limited to, the Fc region of an IgG immunoglobulin, albumin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides, and anti-FcRn scaffolding molecules that have FcRn-binding activity directly, as well as molecules that bind to IgG or albumin that have FcRn-binding activity indirectly. In Disclosures A and B, a domain that has FcRn-binding activity in the acidic and / or neutral pH range may be used. If the domain originally has FcRn-binding activity in the acidic and / or neutral pH range, it can be used without further modification. If the domain has no or weak FcRn-binding activity in the acidic and / or neutral pH range, amino acid residues in the FcRn-binding domain of an antibody or Fc region variant may be altered to confer FcRn-binding activity in the acidic and / or neutral pH range. Alternatively, amino acids in a domain that originally has FcRn-binding activity in the acidic and / or neutral pH range may be altered to further enhance its FcRn-binding activity. The desired amino acid alteration in the FcRn-binding domain can be identified by comparing the FcRn-binding activity in the acidic and / or neutral pH range before and after the amino acid alteration.

[0234] In some cases, the FcRn-binding domain is preferably a region that directly binds to FcRn. Preferred examples of the FcRn-binding domain include antibody constant regions and Fc regions. However, regions capable of binding to polypeptides with FcRn-binding activity, such as albumin or IgG, can indirectly bind to FcRn via albumin or IgG. Therefore, the FcRn-binding domain may also be a region that binds to polypeptides with albumin or IgG-binding activity. Although not limited thereto, to promote antigen elimination from plasma, an FcRn-binding domain with high FcRn-binding activity at neutral pH is preferred. Furthermore, to improve the plasma retention of an antibody, an FcRn-binding domain with high FcRn-binding activity at acidic pH is preferred. For example, an FcRn-binding domain that originally has high FcRn-binding activity at neutral or acidic pH can be selected. Alternatively, FcRn-binding activity at neutral or acidic pH may be conferred by modifying amino acids in an antibody or Fc region variant. Alternatively, the existing FcRn-binding activity at neutral or acidic pH may be increased.

[0235] Within the scope of Disclosures A and B herein, whether the FcRn-binding activity of an antibody or Fc region (variant) is increased, (substantially) maintained, or decreased compared to the antibody or Fc region (variant) before modification may be assessed using known methods, such as those described in the Examples herein, as well as, for example, BIACORE, Scatchard plots, and flow cytometry (see WO 2013 / 046722 ). The extracellular domain of human FcRn may be used as a soluble antigen in these assays. Conditions other than pH for measuring the FcRn-binding activity of an antibody or Fc region (variant) can be appropriately selected by those skilled in the art. For example, as described in WO 2009 / 125825 , the assay can be performed using MES buffer at 37°C. The FcRn-binding activity of an antibody or Fc region (variant) may be measured, for example, by flowing FcRn as an analyte over a chip to which an antibody has been immobilized.

[0236] The binding activity of an antibody or Fc region (variant) to FcRn can be evaluated based on the dissociation constant (KD), apparent dissociation constant (apparent KD), dissociation rate (kd), and apparent dissociation rate (apparent kd).

[0237] Acidic or neutral pH conditions can be used as appropriate for measuring the binding activity of an FcRn-binding domain contained in an antibody or Fc region (variant) to FcRn. The binding activity (binding affinity) of an FcRn-binding domain to FcRn can be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to determine the binding activity (binding affinity) of a human FcRn-binding domain to 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 be used. 25°C is a non-limiting example of such a temperature.

[0238] In one embodiment, when an antibody of Disclosure A or B has binding activity to FcRn, it may have an FcRn-binding domain, preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited as long as the antibody has binding activity or affinity for FcRn at acidic and / or neutral pH, and may be a domain that has activity to directly or indirectly bind to FcRn. In a specific embodiment, the antibody of Disclosure A or B preferably has enhanced FcRn-binding activity under neutral pH conditions, as compared to, for example, a reference antibody comprising a native IgG constant region (see WO 2013 / 046722). It may be preferable, without limitation, from the perspective of comparing the FcRn-binding activities of the antibody of Disclosure A or B to the reference antibody comprising a native IgG constant region, that the amino acid sequences of other regions (e.g., variable regions) are identical to those of the antibody of Disclosure A or B, preferably the constant region, in which one or more amino acid residues have been modified.

[0239] In one embodiment within the scope of Disclosure A described herein, when an antibody of Disclosure A has enhanced FcRn-binding activity under neutral pH conditions, without being bound by any particular theory, the antibody of Disclosure A may combine any two or more of the following characteristics: (1) the antibody has an ion concentration-dependent antigen-binding domain, thereby trafficking between plasma and intracellular endosomes and repeatedly binding to multiple antigens as a single antibody molecule; (2) the antibody has an increased pI, resulting in a more positive overall antibody charge, which allows the antibody to be rapidly internalized within cells; and (3) the antibody has increased FcRn-binding activity under neutral pH conditions, which allows the antibody to be rapidly internalized within cells. As a result, the antibody's plasma half-life can be further shortened, or its binding ability to the extracellular matrix can be further increased, or antigen elimination from plasma can be further promoted. Those skilled in the art can determine the optimal pI value for the antibody of Disclosure A to take advantage of these characteristics.

[0240] Within the scope of disclosures A and B of the present specification, according to Yeung et al. (J. Immunol. 182:7663-7671 (2009)), the human FcRn-binding activity of native human IgG1 is KD 1.7 μM in the acidic pH range (pH 6.0), but the activity is barely detectable in the neutral pH range. Therefore, in order to increase FcRn-binding activity in the neutral pH range, it is preferable to use, as the antibody of Disclosure A or B, for example, an antibody or variant constant region or variant Fc region whose human FcRn-binding activity in the acidic pH range is 20 μM or stronger (KD) and whose human FcRn-binding activity in the neutral pH range is equivalent to or stronger than that of native human IgG; preferably, an antibody or variant constant region or variant Fc region whose human FcRn-binding activity in the acidic pH range is 2.0 μM or stronger (KD) and whose human FcRn-binding activity in the neutral pH range is 40 μM or stronger; and more preferably, an antibody or variant constant region or variant Fc region whose human FcRn-binding activity in the acidic pH range is 0.5 μM or stronger (KD) and whose human FcRn-binding activity in the neutral pH range is 15 μM or stronger (KD). The above KD values are determined by the method described in Yeung et al. (J. Immunol. 182: 7663-7671 (2009)) (by immobilizing the antibody on a chip and flowing human FcRn as the analyte).

[0241] Any domain with any structure that binds to FcRn can be used as an FcRn-binding domain within the scope of Disclosures A and B. In this case, the FcRn-binding domain can be produced without the need for introducing amino acid modifications, and further modifications may be introduced to increase affinity for FcRn.

[0242] Within the scope of Disclosures A and B herein, examples of starting FcRn-binding domains include the Fc region or constant region of (human) IgG. Any Fc region or constant region can be used as the starting Fc region or constant region, as long as the variant of the starting Fc region or constant region can bind to FcRn in the acidic and / or neutral pH ranges. Alternatively, an Fc region or constant region obtained by further modifying an Fc region or constant region that already contains amino acid residue modifications can also be suitably used. The starting Fc region or constant region can include known Fc regions produced by recombinant means. Depending on the context, the starting Fc region or constant region can refer to the polypeptide itself, a composition containing the starting Fc region or constant region, or an amino acid sequence encoding the starting Fc region or constant region. The origin of the starting Fc region or constant region is not limited, and it can be obtained from any non-human animal or human. Furthermore, the starting FcRn-binding domain can be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, and humans. The starting Fc region or starting constant region may be obtained from human IgG1, but is not limited to any particular IgG class. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as appropriate as the starting FcRn-binding domain, and that the Fc region or constant region of an IgG class or subclass from any organism can be used as the starting Fc region or starting constant region. Examples of native or modified IgG variants are described, for example, in Strohl (Curr. Opin. Biotechnol. 20 (6), 685-691 (2009)), Presta (Curr. Opin. Immunol. 20 (4), 460-470 (2008)), Davis et al., (Protein Eng. Des. Sel. 23 (4), 195-202 (2010)), WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338.

[0243] Within the scope of Disclosures A and B herein, examples of amino acid residues in the starting FcRn-binding domain, starting Fc region, or starting constant region may include one or more mutations, such as substitution mutations with amino acid residues different from those in the starting Fc region or starting constant region; insertion of one or more amino acid residues into the amino acid residues in the starting Fc region or starting constant region; or deletion of one or more amino acid residues from the amino acid residues in the starting Fc region or starting constant region. Preferably, the amino acid sequence of the modified Fc region or constant region is an amino acid sequence comprising at least a portion of an Fc region or constant region that does not occur in nature. Such variants necessarily have less than 100% sequence identity or similarity to the starting Fc region or starting constant region. For example, the variant has an amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, even more preferably about 90% to less than 100%, and even more preferably about 95% to less than 100% with respect to the amino acid sequence of the starting Fc region or starting constant region. In a non-limiting example, at least one amino acid differs between the starting Fc region or starting constant region and the modified Fc region or constant region of Disclosure A or B.

[0244] Within the scope of Disclosures A and B herein, an Fc region or constant region having FcRn-binding activity in the acidic and / or neutral pH range may be obtained by any method. Specifically, modified Fc regions or constant regions having FcRn-binding activity in the acidic and / or neutral pH range can be obtained by modifying amino acids in a human IgG antibody that can be used as a starting Fc region or starting constant region. Examples of IgG antibody Fc regions or constant regions suitable for modification include the Fc regions or constant regions of human IgG (IgG1, IgG2, IgG3, and IgG4, and variants thereof), and naturally occurring mutants thereof are also included in the IgG Fc regions or constant regions. Multiple allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, for the Fc regions or constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of these may be used in Disclosure A or B. In particular, in the sequence of human IgG1, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM.

[0245] In one embodiment of Disclosure A or B, the modification of other amino acids is not particularly limited, as long as the resulting variant has FcRn-binding activity in the acidic pH range and / or neutral pH range, preferably in the neutral pH range. Amino acid modification sites for increasing FcRn-binding activity under neutral pH conditions are described, for example, in WO2013 / 046722. Examples of such modification sites include, as described in WO2013 / 046722, 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 300, and 301, as defined by EU numbering, in the Fc region or constant region of a human IgG antibody. Examples of such positions include one or more selected from the group consisting of positions 312, 315 to 320, 324, 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. WO 2013 / 046722 also describes preferred modifications in the constant region or Fc region, such as modifying one or more amino acids selected from the group consisting of: Pro for amino acid 256, Lys for amino acid 280, Thr for amino acid 339, His for amino acid 385, Leu for amino acid 428, and Trp, Tyr, Phe, Ala, or His for amino acid 434 (EU numbering). The number of amino acids to be modified is not particularly limited; a single amino acid position or two or more positions may be modified. These amino acid residue modifications can enhance FcRn binding under neutral pH conditions in the Fc region or constant region of an IgG antibody. These amino acid residue modifications may also be incorporated into the antibodies of Disclosure A or B, as appropriate.

[0246] In further or alternative embodiments, amino acid modification sites may be used as appropriate to increase FcRn-binding activity under acidic pH conditions. Among such modification sites, one or more modification sites that can increase FcRn-binding even in the neutral pH range can be suitably used in Disclosure A or B. Such modification sites are reported in, for example, WO2011 / 122011, WO2013 / 046722, WO2013 / 046704, and WO2013 / 046722. Amino acid sites that allow such modifications in the constant region or Fc region of a human IgG antibody and the types of amino acids after modification are reported in Table 1 of WO2013 / 046722. Furthermore, WO2013 / 046722 discloses that particularly preferred sites for modification in the constant region or Fc region include, for example, positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 352, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383 WO2013 / 046722 also lists one or more amino acid positions selected from the group consisting of positions 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436. Modifications at these amino acid residue positions can also enhance the binding of the FcRn-binding domain to human FcRn in the neutral pH range. WO2013 / 046722 also lists, for example, the following positions, represented by EU numbering, as preferred modifications in the IgG-type constant region or Fc region: (a) amino acid at position 237 to Met; (b) amino acid at position 238 to Ala; (c) amino acid at position 239 to Lys; (d) amino acid at position 248 to Ile; (e) the amino acid at position 250 to any of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, and Tyr; (f) amino acid at position 252 to one of Phe, Trp, and Tyr; (g) amino acid at position 254 to Thr; (h) amino acid at position 255 to Glu; (i) the amino acid at position 256 to either Asp, Glu, or Gln; (j) the amino acid at position 257 to any of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, and Val; (k) amino acid at position 258 to His; (l) amino acid at position 265 to Ala; (m) amino acid at position 270 to Phe; (n) amino acid at position 286 to either Ala or Glu; (o) amino acid at position 289 to His; (p) amino acid at position 297 to Ala; (q) amino acid at position 298 to Gly; (r) amino acid at position 303 to Ala; (s) amino acid at position 305 to Ala; (t) the amino acid at position 307 to any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; (u) the amino acid at position 308 to any of Ala, Phe, Ile, Leu, Met, Pro, Gln, and Thr; (v) the amino acid at position 309 to any of Ala, Asp, Glu, Pro, and Arg; (w) amino acid at position 311 to any of Ala, His, and Ile; (x) amino acid at position 312 to either Ala or His; (y) amino acid at position 314 to either Lys or Arg; (z) amino acid at position 315 to either Ala or His; (aa) amino acid at position 317 to Ala; (ab) amino acid at position 325 to Gly; (ac) amino acid at position 332 to Val; (ad) amino acid at position 334 to Leu; (ae) amino acid at position 360 to His; (af) amino acid at position 376 to Ala; (ag) amino acid at position 380 to Ala; (ah) amino acid at position 382 to Ala; (ai) amino acid at position 384 to Ala; (aj) amino acid at position 385 to either Asp or His; (ak) amino acid at position 386 to Pro; (al) amino acid at position 387 to Glu; (am) amino acid at position 389 to either Ala or Ser; (an) amino acid at position 424 to Ala; (ao) the amino acid at position 428 to any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; (ap) amino acid at position 433 to Lys; (aq) amino acid at position 434 to Ala, Phe, His, Ser, Trp, and Tyr; and (ar) Amino acid at position 436 to His The present disclosure includes modifications of one or more amino acid residues selected from the group consisting of: (a) a nucleotide sequence selected from the group consisting of (b) a nucleotide sequence selected from the group consisting of (c) a nucleotide sequence selected from the group consisting of (d) a nucleotide sequence selected from the group consisting of (e) a nucleotide sequence selected from the group consisting of (f) a nucleotide sequence selected from the group consisting of (g ...

[0247] In one embodiment, the FcRn-binding activity of the FcRn-binding domain of the antibody of Disclosure A or B is increased compared to the FcRn-binding activity of a reference antibody comprising the Fc region or constant region of a native IgG or the starting Fc region or starting constant region. In other words, the FcRn-binding activity of the Fc region variant or constant region variant of Disclosure A or B, or an antibody comprising such a variant, is higher than the FcRn-binding activity of the reference antibody. This means that the FcRn-binding activity of the antibody of Disclosure A or B may be, for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, more preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold or more, or 5-fold or more higher than the FcRn-binding activity of the reference antibody.

[0248] In one embodiment, the amino acid sequence to be modified in the antibody of Disclosure A or B preferably comprises a human sequence (a sequence found in a natural antibody of human origin) to avoid increasing the immunogenicity of the antibody when administered in vivo (preferably to a human body). Alternatively, after modification, mutations may be introduced at sites other than the amino acid modification site where one or more FRs (FR1, FR2, FR3, and FR4) are replaced with human sequences. Methods for replacing FRs with human sequences are known in the art and include, but are not limited to, those reported in Ono et al., Mol. Immunol. 36(6): 387-395 (1999). Humanization methods are also known in the art and include, but are not limited to, those reported in Methods 36(1): 43-60 (2005).

[0249] In one embodiment, the framework region sequences (also referred to as "FR sequences") of the heavy and / or light chain variable regions of an antibody of Disclosure A or B may comprise human germline framework sequences. If the framework sequences are entirely human germline sequences, the antibody can be expected to provoke little or no immunogenic response when administered to humans (e.g., for the purpose of treating or preventing a disease).

[0250] The FR sequence may preferably include a fully human FR sequence, such as those shown in V-Base (vbase.mrc-cpe.cam.ac.uk / ). These FR sequences may be used as appropriate in Disclosure A or B. Germline sequences may be classified based on their similarity (Tomlinson et al. (J. Mol. Biol. 227: 776-798 (1992)); Williams et al. (Eur. J. Immunol. 23: 1456-1461 (1993)); and Cox et al. (Nat. Genetics 7: 162-168 (1994))). Furthermore, suitable germline sequences may be selected from Vκ, which are classified into seven subgroups, Vλ, which are classified into ten subgroups, and VH, which are classified into seven subgroups.

[0251] Fully human VH sequences include, for example, VH1 subgroups (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, and VH1-69), VH2 subgroups (e.g., VH2-5, VH2-26, and VH2-70), VH3 subgroups (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-34, VH3-35, VH3-36, VH3-37, VH3-39, VH3-40, VH3-41, VH3-42, VH3-43, VH3-44, VH3-45, VH3-46, VH3-58, and VH3-69), VH4 subgroups (e.g., VH4-5, VH4-60, VH4-61, VH4-62, VH4-63, VH4-64, VH4-65, VH4-66, VH4-67, VH4-68, VH4-69, VH4-70, VH4-71, VH4-72, VH4-73, VH4-74, VH4-75, VH4-76, VH4-77, VH4-78, VH4-79, VH4-80, VH4-81, VH4-82, VH4-83, VH4-84, VH4-85, VH4-86, VH4-87 Preferred examples of the VH sequences include VH sequences from the VH4 subgroup (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, and VH4-61), the VH5 subgroup (VH5-51), the VH6 subgroup (VH6-1), and the VH7 subgroup (VH7-4 and VH7-81). These sequences are described, for example, in Matsuda et al. (J. Exp. Med. 188: 1973-1975 (1998)), and those skilled in the art can appropriately design antibodies based on this sequence information. Other fully human FR sequences or sequences of regions equivalent thereto can also be suitably used.

[0252] Examples of fully human Vκ sequences include A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, and O18, which belong to the Vk1 subgroup; A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, which belong to the Vk2 subgroup; A11, A27, L2, L6, L10, L16, L20, and L25, which belong to the Vk3 subgroup; B3, which belongs to the Vk4 subgroup; B2 (also referred to as "Vk5-2"), which belongs to the Vk5 subgroup; and A10, A14, and A26, which belong to the Vk6 subgroup (Kawasaki et al. (Eur. J. Immunol. 31: 1017-1028 (2001); Hoppe Seyler (Biol. Chem. 374: 1001-1022 (1993)); Brensing-Kuppers et al. (Gene 191: 173-181 (1997)) may be mentioned as suitable examples.

[0253] Examples of fully human Vλ sequences include 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, and V2-1, V2-6, V2-7, V2-8, V2-11, and V2-12, which are classified into the VL2 subgroup. Preferred examples include 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. 7:250-261 (1997))).

[0254] These FR sequences usually differ from each other by one or more amino acid residues. These FR sequences can be used to modify the amino acid residues of antibodies. Furthermore, examples of fully human FR sequences that can be used for modification include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (see, for example, Kabat et al. (1991) supra; Wu et al. (J. Exp. Med. 132, 211-250 (1970))).

[0255] Within the scope of Disclosures A and B herein, the term "flexible residue" refers to variations in amino acid residues at positions where the light chain variable region or heavy chain variable region exhibits high amino acid diversity when comparing the amino acid sequences of known and / or naturally occurring antibodies or antigen-binding domains, with several different amino acids. Positions exhibiting high amino acid diversity are generally located within CDRs. In determining such highly diverse positions in known and / or naturally occurring antibodies, data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md.) (1987 and 1991) can be useful. Additionally, several databases on the Internet (vbase.mrc-cpe.cam.ac.uk / , bioinf.org.uk / abs / index.html) provide a collection of numerous human light chain and heavy chain sequences and their arrangements. Information on these sequences and arrangements is useful for determining the positions of flexible residues. For example, but not limited to, if the amino acid residues at a particular position have a variation of preferably 2 to 20, 3 to 19, 4 to 18, 5 to 17, 6 to 16, 7 to 15, 8 to 14, 9 to 13, or 10 to 12 amino acid residues, that position can be determined to exhibit (high) diversity.

[0256] In one embodiment, when an antibody of Disclosure A or B comprises all or a portion of a light chain variable region and / or a heavy chain variable region, it can be understood that the antibody may optionally contain one or more flexible residues. For example, a heavy chain and / or light chain variable region sequence selected as an FR sequence originally containing amino acid residues that change the antigen-binding activity of the antibody depending on ion concentration (hydrogen ion concentration or calcium ion concentration) can be designed to contain other amino acid residues in addition to those amino acid residues. In this case, as long as the antigen-binding activity of the antibody of Disclosure A or B changes depending on ion concentration, for example, the number and positions of the flexible residues can be determined without being limited to a specific embodiment. That is, at least one flexible residue can 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 that can be introduced into the light chain variable region sequence (Vk5-2 described above) include one or more amino acid residue positions listed in Table 1 or Table 2. Similarly, for example, in an ion concentration-dependent antibody or an antibody that does not have the ion concentration dependency, which comprises all or part of a light chain variable region and / or a heavy chain variable region, when at least one amino acid residue that can be exposed on the surface of the antibody is modified to increase the pI, a flexible residue can also be introduced as appropriate.

[0257] [Table 1] (Positions are expressed in Kabat numbering.)

[0258] [Table 2] (Positions are expressed in Kabat numbering.)

[0259] In one embodiment, when a chimeric antibody is humanized, one or more amino acid residues that can be exposed on the surface of the chimeric antibody are modified to increase the pI of the chimeric antibody, thereby producing a humanized antibody of Disclosure A or B having a reduced plasma half-life compared to a chimeric antibody that does not have such modifications. The modification of the amino acid residues that can be exposed on the surface of the humanized antibody may be performed before or simultaneously with antibody humanization. Alternatively, a humanized antibody may be used as a starting material, and the pI of the humanized antibody may be further modified by modifying its amino acid residues that can be exposed on the surface.

[0260] Adams et al. (Cancer Immunol. Immunother. 55(6): 717-727 (2006)) reported that the plasma pharmacokinetics of the humanized antibodies trastuzumab (antigen: HER2), bevacizumab (antigen: VEGF), and pertuzumab (antigen: HER2), which were humanized using the same human antibody FR sequence, were nearly equivalent. In particular, it can be understood that the plasma pharmacokinetics are nearly equivalent when humanization is performed using the same FR sequence. In one embodiment of Disclosure A, in addition to the humanization process, the antigen concentration in plasma is reduced by increasing the pI of the antibody through modification of amino ac...

Claims

[Claim 1] The invention described herein.

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