Antibodies comprising ion-concentration-dependent antigen-binding domains, fc region variants, il-8-binding antibodies, and uses thereof

Abstract

One non-exclusive aspect provides molecules further improved from antibodies capable of binding antigen in an ion-concentration-dependent manner. An alternative non-exclusive aspect provides safe and more favorable Fc region variants with reduced binding to pre-existing ADAs. An alternative non-exclusive aspect provides novel IL-8 antibodies superior as drugs.

Description

[0001] This application is a divisional application of Chinese Application No. 201680005280.2, filed on February 4, 2016, with the title "Antibodies Comprising Ion Concentration-Dependent Antigen Binding Domains, Fc Region Variants, IL-8-Binding Antibodies, and Uses Thereof." TECHNICAL FIELD

[0002] Cross-Reference to Related Applications

[0003] This application is related to and claims priority to Japanese Priority Patent Application No. 2015-021371, filed February 5, 2015, and Japanese Priority Patent Application No. 2015-185254, filed September 18, 2015. The contents of these priority applications are incorporated by reference in their entirety. TECHNICAL FIELD

[0005] In one non-exclusive aspect, the present disclosure relates to antibodies comprising an antigen binding domain whose antigen binding activity changes depending on ion concentration conditions, and pharmaceutical compositions containing the antibodies. Nucleic acids encoding these antibodies and host cells containing the nucleic acids are also provided, as are methods of using and producing the antibodies and pharmaceutical compositions. In another non-exclusive aspect, the present disclosure provides Fc region variants and antibodies containing the variants, and pharmaceutical compositions containing the Fc region variants and antibodies. Nucleic acids encoding the Fc region variants and antibodies, and host cells containing the nucleic acids, as well as methods of using and producing the Fc region variants and antibodies and pharmaceutical compositions are also provided. In a third non-exclusive aspect, the present disclosure provides anti-IL-8 antibodies, pharmaceutical compositions containing the antibodies, nucleic acids encoding the antibodies, and host cells containing the nucleic acids. Methods of producing and using the IL-8 antibodies and pharmaceutical compositions in the treatment of, for example, IL-8-related disorders are also provided. BACKGROUND

[0006] Antibodies are attracting attention as drugs because they are highly stable in plasma and have fewer side effects. Many IgG-type therapeutic antibodies are on the market, and even now many therapeutic antibodies are under development (Reichert et al., Nat. Biotechnol. 23: 1073-1078 (2005) (NPL 1); Pavlou et al., Eur. J. Pharm. Biopharm. 59(3): 389-396 (2005) (NPL 2)). At the same time, various technologies for a second generation of therapeutic antibodies are being developed; including technologies for 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) (NPL 3)). The dose of a therapeutic antibody is usually very high, and thus development of a therapeutic antibody encounters problems such as difficulty in producing a subcutaneous preparation and high production cost. Methods for improving the pharmacokinetics, pharmacodynamics, and antigen binding properties of a therapeutic antibody provide a way to reduce the dose and production cost associated with a therapeutic antibody.

[0007] Substitution of amino acid residues in the constant region provides a method for improving the pharmacokinetics of an antibody (Hinton et al., J. Immunol. 176(1): 346-356 (2006) (NPL 4); Ghetie et al., Nat. Biotechnol. 15(7): 637-640 (1997) (NPL 5)). Affinity maturation technology provides a method for enhancing the antigen neutralizing ability of an antibody (Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24): 8466-8471 (2005) (NPL 6); Wu et al., J. Mol. Biol. 368: 652 (2007) (NPL 7)), and one or more mutations can be introduced in one or more amino acid residues in the CDRs and / or framework regions of the variable domain of an antibody to increase antigen binding activity. Improving the antigen binding properties of an antibody can improve the in vitro biological activity or reduce the dose of an antibody, and can further improve in vivo efficacy (Wu et al., J. Mol. Biol. 368: 652-665 (2007) (NPL 8)).

[0008] The amount of antigen that can be neutralized by one antibody molecule depends on the affinity of the antibody to the antigen; and thus, it is possible to neutralize an antigen with a small amount of antibody by increasing the affinity. The affinity of an antibody to an antigen can be increased using various known methods (see, for example, Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24): 8466-8471 (2005) (NPL 6)). Furthermore, it is theoretically possible to neutralize one antigen molecule (2 antigens when the antibody is bivalent) with one antibody molecule if it can covalently bind to the antigen such that the affinity becomes infinite. Nonetheless, one of the limitations in the development of therapeutic antibodies to date is that one antibody molecule generally binds to and neutralizes only one antigen molecule (2 antigens when the antibody is bivalent). It has recently 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 a "pH-dependent-binding antibody") enables one antibody molecule to bind to and neutralize multiple antigen molecules (see, for example, WO 2009 / 125825 (PTL 1); Igawa et al., Nat. Biotechnol. 28: 1203-1207 (2010) (NPL 9)). The pH-dependent antibody strongly binds to an antigen under neutral pH conditions in the blood plasma, and dissociates from the antigen under acidic pH conditions in the endosome of a cell. After dissociation from the antigen, the antibody is recycled to the blood plasma through FcRn and is then free to bind to and neutralize another antigen molecule; and thus one pH-dependent antibody can repeatedly bind to and neutralize multiple antigen molecules.

[0009] It has recently been reported that the antibody recycling property can be achieved by focusing on the difference in the concentration of calcium (Ca) ions between the blood plasma and the endosome, and using an antibody that has an antigen-antibody interaction that is dependent on calcium (hereinafter also referred to as a "calcium ion concentration-dependent antibody") (WO 2012 / 073992 (PTL 2)). (Hereinafter, the pH-dependent antibody and the "calcium ion concentration-dependent antibody" are collectively referred to as a "pH / Ca concentration-dependent antibody".)

[0010] IgG antibodies have a long retention in the blood plasma by binding to FcRn. The binding between IgG antibodies and FcRn is strong under acidic pH conditions (e.g., pH 5.8), but almost none under neutral pH conditions (e.g., pH 7.4). IgG antibodies are non-specifically taken into cells, and are returned to the cell surface by binding to FcRn in the endosome under acidic pH conditions in the endosome. IgG is then dissociated from FcRn under neutral pH conditions in the blood plasma.

[0011] It has been reported that a pH-dependent antibody modified to increase its FcRn binding at neutral pH conditions has the ability to repeatedly bind and remove antigen molecules from plasma; and thus use of such an antibody allows removal of antigen from plasma (WO2011 / 122011 (PTL3)). According to this report, a pH-dependent antibody modified to increase its FcRn binding at neutral pH conditions (e.g., pH 7.4) can further accelerate removal of antigen compared to a pH-dependent antibody comprising the Fc region of a native IgG antibody (WO2011 / 122011 (PTL3)).

[0012] Meanwhile, when a mutation is introduced into the Fc region of an IgG antibody to eliminate its binding to FcRn at acidic pH conditions, it can no longer be recycled from endosomes into plasma, which significantly weakens the retention of the antibody in plasma. In turn, a method of increasing FcRn binding at acidic pH conditions has been reported as a method for improving the plasma retention of an IgG antibody. Introduction of an amino acid modification into the Fc region of an IgG antibody to increase its FcRn binding at acidic pH conditions can enhance the efficiency of recycling from endosomes into plasma, which in turn results in improvement of plasma retention. For example, it has been reported that modification of M252Y / S254T / T256E (YTE; Dall'Acqua et al., J. Biol. Chem. 281:23514-235249 (2006) (NPL 10)), M428L / N434S (LS; Zalevsky et al., Nat. Biotechnol. 28: 157-159 (2010)) (NPL 11), and N434H (Zheng et al., Clin. Pharm. & Ther. 89(2):283-290 (2011) (NPL 12)) results in increased antibody half-life relative to native IgG1.

[0013] However, in addition to considering that immunogenicity or the incidence of aggregates can be worse in antibodies containing such Fc region variants whose FcRn binding increases at neutral pH conditions or at acidic pH conditions, it has been further reported that binding to anti-drug antibodies (hereinafter also referred to as "pre-existing ADAs") (e.g., rheumatoid factor) that are present in a patient prior to administration of a therapeutic antibody is increased (WO2013 / 046722 (PTL4), WO2013 / 046704 (PTL5)). WO2013 / 046704 (PTL5) reports that an Fc region variant containing specific mutations (represented by modification of two residues according to EU numbering of Q438R / S440E) increases binding to FcRn at acidic pH conditions and also shows a significant decrease in binding to rheumatoid factor, as compared to unmodified native Fc. However, WO2013 / 046704 (PTL5) does not specifically demonstrate that an antibody having this Fc region variant has better plasma retention than a native Fc region.

[0014] Accordingly, a safe and more advantageous Fc region variant having further improved plasma retention that does not show binding to pre-existing ADAs is needed.

[0015] Antibody-dependent cellular cytotoxicity (hereinafter shown as "ADCC"), complement-dependent cellular cytotoxicity (hereinafter shown as "CDC"), and antibody-dependent cellular phagocytosis (ADCP) (which is phagocytosis of a target cell mediated by an IgG antibody) are reported as effector functions of IgG antibodies. In order for an IgG antibody to mediate ADCC activity or ADCP activity, the Fc region of the IgG antibody must bind to an antibody receptor (referred to herein as "Fcy receptor", "FcgR", "Fcy receptor", or "FcyR" in the scope of the disclosure described in the publication A) present on the surface of an effector cell such as a killer cell, a natural killer cell, or an activated macrophage. In humans, FcyRIa, FcyRIIa, FcyRIIb, FcyRIIIa, and FcyRIIIb isoforms are reported as FcyR family proteins, and their respective allotypes have also been reported (Jefferis et al., Immunol. Lett. 82: 57-65 (2002) (NPL 13)). The balance of the respective affinities of an antibody for an activating receptor including FcyRIa, FcyRIIa, FcyRIIIa, or FcyRIIIb and for an inhibitory receptor including FcyRIIb is an important element in optimizing effector functions of an antibody.

[0016] Various techniques to increase or improve the activity of therapeutic antibodies against antigens have been reported to date. For example, the activity of antibody binding to activating FcyR(s) plays an important role in the cytotoxicity of antibodies, and thus, antibodies targeting membrane-type antigens and having increased cytotoxicity due to enhanced activating FcyR(s) binding have been developed. See, for example, WO2000 / 042072 (PTL6); WO2006 / 019447 (PTL7); Lazar et al., Proc. Nat. Acad. Sci. USA. 103:4005-4010 (2006) (NPL14); Shinkawa et al., J. Biol. Chem. 278, 3466-3473 (2003) (NPL15); Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998) (NPL16); Clynes et al., Nat. Med. 6:443-446 (2000) (NPL17)). Similarly, the binding activity to inhibitory FcyR (FcyRIIb in humans) plays an important role in immunosuppressive activity, agonist activity, and thus, there have been studies on antibodies targeting membrane-type antigens having increased inhibitory FcyR-binding activity (Li et al., Proc. Nat. Acad. Sci. USA. 109(27):10966-10971 (2012) (NPL18)). In addition, the effects of FcyR binding of antibodies binding to soluble antigens have been mainly examined from the perspective of side effects (Scappaticci et al., J. Natl. Cancer Inst. 99(16):1232-1239 (2007) (NPL19)). For example, when an antibody having increased FcyRIIb binding is used as a drug, one can expect a reduced risk of anti-drug antibody production (Desai et al., J. Immunol. 178(10):6217-6226 (2007) (NPL20)).

[0017] Recently, it has been reported that the introduction of amino acid modifications into the Fc region of IgG antibodies to increase the activity of antibodies targeting soluble antigens to bind to activating and / or inhibitory FcyR can further accelerate the removal of antigens from serum (WO2012 / 115241 (PTL8), WO2013 / 047752 (PTL9), WO2013 / 125667 (PTL10), WO2014 / 030728 (PTL11)). In addition, Fc region variants have been identified that show little change in their FcyRIIb-binding activity from the native IgG antibody Fc region, but have reduced activity to other activating FcyRs (WO2014 / 163101 (PTL12)).

[0018] Plasma retention of soluble antigens is very short compared to antibodies having an FcRn-mediated recycling mechanism, and thus soluble antigens can exhibit increased plasma retention and plasma concentration by binding to antibodies having such a recycling mechanism (e.g., antibodies that do not have the characteristics of pH / Ca concentration-dependent antibodies). Thus, for example, when a soluble antigen in plasma has multiple types of physiological functions, even if one type of physiological function is blocked due to antibody binding, the plasma concentration of the antigen can make the pathological symptoms caused by other physiological functions due to the increased plasma retention and / or plasma concentration of the antigen by antibody binding worse. In this case, in addition to the above-described exemplary modification of antibodies to accelerate antigen removal, methods using the formation of multivalent immune complexes from multiple pH / Ca concentration-dependent antibodies and multiple antigens, and increasing the binding to FcRn, FcyR(s), complement receptors have been reported (WO2013 / 081143 (PTL13)).

[0019] Even when the Fc region is not modified, it has been reported that by modifying one or more amino acid residues to change the charge of one or more amino acid residues that can be exposed on the surface of the variable region of the antibody, to increase or decrease the isoelectric point (pi) of the antibody, the half-life of the antibody in the blood can be controlled without substantially reducing the antigen binding activity of the antibody, regardless of the type of antigen or antibody (WO2007 / 114319 (PTL14): technique of substituting amino acids mainly in FR; WO2009 / 041643 (PTL15): technique of substituting amino acids mainly in CDR). These documents suggest that it is possible to extend the plasma half-life of the antibody by lowering the pi of the antibody, and conversely to shorten the plasma half-life of the antibody by increasing the pi of the antibody.

[0020] Regarding the modification of the charge of the amino acid residues in the constant region of the antibody, it has been reported that the uptake of antigens in cells can be facilitated by modifying the charge of a particular one or more amino acid residues, particularly in the CH3 domain, thereby increasing the pi of the antibody, and it is also described that the modification preferably does not interfere with the binding to FcRn (WO2014 / 145159 (PTL16)). It has also been reported that modifying the charge of the amino acid residues in the constant region (mainly the CH1 domain) of the antibody to lower the pi can extend the half-life of the antibody in the plasma, and in combination with mutations of amino acid residues that increase FcRn binding, can enhance its binding to FcRn and extend the plasma half-life of the antibody (WO2012 / 016227 (PTL17)).

[0021] Meanwhile, when the modification technique designed to increase or decrease the pi of an antibody is combined with a technique other than the modification technique to increase or decrease the binding to FcRn or FcyR(s), it is unclear whether there is an effect in terms of promoting the plasma retention or removal of an antigen from plasma of the antibody.

[0022] Extracellular matrix (ECM) is a structure that covers cells in vivo, and is mainly composed of glycoproteins such as collagen, proteoglycans, fibronectin, and laminin. The role of ECM in vivo is to create a microenvironment for cell survival, and ECM is important in various functions performed by cells, such as cell proliferation and cell adhesion.

[0023] It has been reported that ECM is involved in the in vivo kinetics of a protein administered to a living body. The blood concentration of a VEGF-Trap molecule, which is a fusion protein between a VEGF receptor and Fc, was measured when administered subcutaneously (Holash et al., Proc. Natl. Acad. Sci., 99(17): 11393-11398 (2002) (NPL 21)). The plasma concentration of the VEGF-Trap molecule having a high pi was low when administered subcutaneously, and thus its bioavailability was low. The modified VEGF-Trap molecule whose pi was reduced by amino acid substitution had a higher plasma concentration, and its bioavailability was likely to be improved. Furthermore, the change in bioavailability is related to the strength of binding to ECM, and thus it became apparent that the bioavailability of the VEGF-Trap molecule when administered subcutaneously depends on the strength of its binding to ECM at the subcutaneous site.

[0024] WO2012 / 093704 (PTL 18) reports that there is an inverse correlation between antibody binding to ECM and plasma retention, and thus an antibody molecule that does not bind to ECM has better plasma retention when compared to an antibody that binds to ECM.

[0025] Thus, a technique to reduce extracellular matrix binding for the purpose of improving in vivo bioavailability and plasma retention of a protein has been reported. In contrast, the advantage of increasing the binding of an antibody to ECM has not been identified to date.

[0026] Human IL-8 (interleukin 8) is a member of the chemokine family, which is 72 or 77 amino acid residues in length. The term "chemokine" is a general term for a family of proteins having 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 according to the arrangement of cysteines. IL-8 is classified as a CXC chemokine, and is also called CXCL8.

[0027] IL-8 exists in solution as a monomer or a homodimer. The IL-8 monomer contains an anti-parallel beta sheet and has a structure in which the C-terminal alpha helix penetrates and covers the beta sheet. The IL-8 monomer, in the case of the 72 amino acid form of IL-8, includes two disulfide bond crosslinks between cysteine 7 and cysteine 34 and between cysteine 9 and cysteine 50. The IL-8 homodimer is stabilized by non-covalent interactions between the beta sheets of the two monomers, as there is no covalent bonding between the molecules of the homodimer.

[0028] IL-8 expression is induced in various cells such as peripheral blood mononuclear cells, 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) (NPL 22)).

[0029] In normal tissues, chemokines are generally not detectable or only weakly detectable, but are strongly detectable at sites of inflammation and participate in inducing inflammation by promoting lymphocyte infiltration into the site of inflamed tissue. IL-8 is a proinflammatory chemokine known to activate neutrophils, promote expression of cell adhesion molecules, and enhance neutrophil adhesion to vascular endothelial cells. IL-8 also has neutrophil chemotactic ability and produces IL-8 at damaged tissue to promote chemotaxis of neutrophils adhering to vascular endothelial cells into the tissue and induce inflammation with neutrophil infiltration. IL-8 is also known to be a potent angiogenic factor for endothelial cells and participates in promoting tumor angiogenesis.

[0030] Inflammatory diseases associated with elevated (e.g., excess) IL-8 levels include inflammatory diseases of the skin such as inflammatory keratosis (e.g., psoriasis), atopic dermatitis, contact dermatitis; chronic inflammatory conditions (which 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, 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, oral aphthae, chorditis, and inflammation associated with the use of artificial organs and / or artificial blood vessels. Elevated (e.g., excess) 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 renal cancer; sepsis resulting from infection; cystic fibrosis; and pulmonary fibrosis. (See, e.g., Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014) (NPL 22), which is incorporated herein by reference in its entirety).

[0031] For many of these diseases, human anti-IL-8 antibodies with high affinity have been developed as pharmaceutical compositions (Desai et al., J. Immunol. 178(10):6217-6226 (2007) (NPL 23)), however, they have not been brought to market. To date, only one pharmaceutical composition containing an IL-8 antibody is available, which is a murine anti-IL-8 antibody as an external medicine for psoriasis. New anti-IL-8 antibodies for treating diseases are desired.

[0032] [LIST OF CITATIONS]

[0033] [PATENT LITERATURE]

[0034] [PTL 1] WO2009 / 125825

[0035] [PTL 2] WO2012 / 073992

[0036] [PTL3] WO2011 / 122011

[0037] [PTL4] WO2013 / 046722

[0038] [PTL5] WO2013 / 046704

[0039] [PTL6] WO2000 / 042072

[0040] [PTL7] WO2006 / 019447

[0041] [PTL8] WO2012 / 115241

[0042] [PTL9] WO2013 / 047752

[0043] [PTL10] WO2013 / 125667

[0044] [PTL11] WO2014 / 030728

[0045] [PTL12] WO2014 / 163101

[0046] [PTL13] WO2013 / 081143

[0047] [PTL14] WO2007 / 114319

[0048] [PTL15] WO2009 / 041643

[0049] [PTL16] WO2014 / 145159

[0050] [PTL17] WO2012 / 016227

[0051] [PTL18] WO2012 / 093704

[0052] [Non-patent literature]

[0053] [NPL1] Reichert et al., Nat. Biotechnol. 23: 1073-1078 (2005)

[0054] [NPL2] Pavlou et al., Eur. J. Pharm. Biopharm. 59(3): 389-396 (2005)

[0055] [NPL3] Kim et al., Mol. Cells 20(1): 17-29 (2005)

[0056] [NPL4] Hinton et al., J. Immunol. 176(1): 346-356 (2006)

[0057] [NPL5] Ghetie et al., Nat. Biotechnol. 15(7): 637-640 (1997)

[0058] [NPL6] Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24): 8466-8471 (2005)

[0059] [NPL7] Wu et al., J. Mol. Biol. 368: 652 (2007)

[0060] [NPL8] Wu et al., J. Mol. Biol. 368: 652-665 (2007)

[0061] [NPL9] Igawa et al., Nat. Biotechnol. 28: 1203-1207 (2010)

[0062] [NPL10] Dall'Acqua et al., J. Biol. Chem. 281: 23514-235249 (2006)

[0063] [NPL11] Zalevsky et al., Nat. Biotechnol. 28: 157-159 (2010)

[0064] [NPL12] Zheng et al., Clin. Pharm. & Ther. 89(2): 283-290 (2011)

[0065] [NPL13] Jefferis et al., Immunol. Lett. 82: 57-65 (2002)

[0066] [NPL14] Lazar et al., Proc. Nat. Acad. Sci. USA. 103: 4005-4010 (2006)

[0067] [NPL15] Shinkawa et al., J. Biol. Chem. 278, 3466-3473 (2003)

[0068] [NPL16] Clynes et al., Proc. Natl. Acad. Sci. USA 95: 652-656 (1998)

[0069] [NPL 17] Clynes et al., Nat. Med. 6:443-446 (2000)

[0070] [NPL 18] Li et al., Proc. Nat. Acad. Sci. USA. 109(27): 10966-10971 (2012)

[0071] [NPL 19] Scappaticci et al., J. Natl. Cancer Inst. 99(16): 1232-1239 (2007)

[0072] [NPL 20] Desai et al., J. Immunol. 178(10): 6217-6226 (2007)

[0073] [NPL 21] Holash et al., Proc. Natl. Acad. Sci., 99(17): 11393-11398 (2002)

[0074] [NPL 22] Russo et al., Exp. Rev. Clin. Immunol. 10(5): 593-619 (2014)

[0075] [NPL 23] Desai et al., J. Immunol. 178(10): 6217-6226 (2007) SUMMARY

[0076] In one non-exclusive aspect, it is a non-limiting object of embodiments of the disclosure A to provide molecules with improved pharmacokinetic properties of the relative antibody, such as improved antibody half-life and / or ion-concentration-dependent antigen-binding properties of the antigen clearance from the plasma.

[0077] In one non-exclusive aspect, it is a non-limiting object of embodiments of the disclosure B to provide safe and more beneficial Fc region variants with increased half-life and reduced binding to pre-existing anti-drug antibodies (ADAs).

[0078] In one non-exclusive aspect, it is a non-limiting object of embodiments of the disclosure to provide anti-IL-8 antibodies having pH-dependent binding affinity for IL-8. Other embodiments relate to anti-IL-8 antibodies that have a rapid effect of removing IL-8 when administered to a subject, as compared to a reference antibody. In another embodiment, the disclosure relates to anti-IL-8 antibodies that are able to stably retain their IL-8-neutralizing activity when administered to a subject. In some embodiments, the anti-IL-8 antibodies exhibit reduced immunogenicity. In other embodiments, the disclosure relates to methods of generating and using the above-described anti-IL-8 antibodies. It is a further alternative non-limiting object of the disclosure to provide novel anti-IL-8 antibodies that can be included in pharmaceutical compositions.

[0079] In one non-exclusive aspect, within the scope of the disclosure A as provided herein, the inventors have surprisingly found that the ability of an ion-concentration-dependent antibody (which is 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 remove an antigen from plasma can be facilitated by modifying at least one amino acid residue exposed on the surface of the antibody so as to increase its isoelectric point (pi). In another non-exclusive aspect, the inventors have found that ion-concentration-dependent antibodies having an increased pi can further increase the extracellular matrix binding of the antibody. Thus, without being bound by a particular theory, the inventors have found that the removal of an antigen from plasma can be increased by increasing the binding of the antibody to the extracellular matrix.

[0080] In one non-exclusive aspect, within the scope of the disclosure B as provided herein, the inventors have conducted an in-depth study of safe and more favorable Fc region variants that do not exhibit binding to anti-drug antibodies (pre-existing ADAs) and can further improve plasma retention. As a result, the inventors have surprisingly found that an Fc region variant comprising substitution with Ala (A) of amino acid at position 434 according to EU numbering and two specific residue mutations (designated by Q438R / S440E according to EU numbering) as a combination of amino acid residue mutations is preferable for maintaining a significant reduction in binding to rheumatoid factor, along with achieving plasma retention of the antibody.

[0081] In one non-exclusive aspect, within the scope of the disclosure C as provided herein, the present inventors developed a number of pH-dependent anti-IL-8 antibodies (anti-IL-8 antibodies that bind to IL-8 in a pH-dependent manner). From the results of various verifications, the present inventors identified pH-dependent anti-IL-8 antibodies that have a rapid IL-8-removing effect when administered to a subject, as compared to a reference antibody. In some embodiments, the disclosure C relates to pH-dependent anti-IL-8 antibodies that can stably maintain their IL-8-neutralizing activity. In other non-limiting embodiments, the pH-dependent anti-IL-8 antibodies have reduced immunogenicity and excellent expression levels.

[0082] Further, within the scope of the disclosure C, the present inventors successfully obtained anti-IL-8 antibodies comprising an Fc region whose FcRn-binding affinity is increased at an acidic pH, as compared to that of a native Fc region. In an alternative aspect, the present inventors successfully obtained anti-IL-8 antibodies comprising an Fc region whose binding affinity to a pre-existing ADA is reduced, as compared to that of a native Fc region. In an alternative aspect, the present inventors successfully obtained anti-IL-8 antibodies comprising an Fc region whose plasma half-life is increased, as compared to that of a native Fc region. In an alternative aspect, the present inventors successfully obtained pH-dependent anti-IL-8 antibodies comprising an Fc region whose binding affinity to an effector receptor is reduced, as compared to that of a naturally occurring Fc region. In a different aspect, the present inventors identified nucleic acids encoding the above-described anti-IL-8 antibodies. In another aspect, the present inventors also obtained hosts comprising the above-described nucleic acids. In another aspect, the present inventors developed methods for producing the above-described anti-IL-8 antibodies, which comprise culturing the above-described hosts. In another aspect, the present inventors developed methods for promoting the removal of IL-8 from a subject, as compared to a reference antibody, which comprise administering the above-described anti-IL-8 antibodies to the subject.

[0083] In one embodiment, the disclosure A relates to, but is not limited to,

[0084] [1] An antibody comprising an antigen-binding domain whose antigen-binding activity is changed depending on ion concentration conditions, wherein its isoelectric point (pI) is increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody;

[0085] [2] The antibody of [1], wherein the antigen is a soluble antigen;

[0086] [3] The antibody of [1] or [2], wherein the antigen-binding domain is a domain whose antigen-binding activity is higher under high ion concentration conditions than under low ion concentration conditions;

[0087] [4] The antibody according to any one of [1] to [3], wherein the ion concentration is hydrogen ion concentration (pH) or calcium ion concentration;

[0088] [5] The antibody according to [4], wherein the ratio of KD at an acidic pH range to that at a neutral pH range, KD (acidic pH range) / KD (neutral pH range), is 2 or more for the antigen;

[0089] [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;

[0090] [7] The antibody according to any one of [1] to [6], which is capable of promoting removal of the antigen from plasma as compared to the antibody before the modification;

[0091] [8] The antibody according to any one of [1] to [7], wherein the extracellular matrix binding activity is enhanced as compared to the antibody before the modification;

[0092] [9] The antibody according to any one of [1] to [8], wherein the amino acid residue modification is substitution of an amino acid residue;

[0093]

[10] The antibody according to any one of [1] to [9], wherein the amino acid residue modification is selected from the group consisting of:

[0094] (a) substitution of a negatively charged amino acid residue with an uncharged amino acid residue;

[0095] (b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue; and

[0096] (c) substitution of an uncharged amino acid residue with a positively charged amino acid residue;

[0097]

[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 an amino acid residue modification in the variable region and / or the constant region;

[0098]

[12] The antibody according to

[11] , wherein the variable region comprises one or more complementarity determining region(s) (CDR(s)) and / or one or more framework region(s) (FR(s));

[0099]

[13] The antibody according to

[12] , wherein the variable region comprises a heavy chain variable region and / or a light chain variable region, and at least one amino acid residue is modified at a position in the CDR or FR selected from the group consisting of:

[0100] According to the numbering of Kabat

[0101] (a) 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;

[0102] (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDR of the heavy chain variable region;

[0103] (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

[0104] (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region;

[0105]

[14] The antibody of

[13] , wherein at least one amino acid residue is modified in a position selected from the group consisting of:

[0106] (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;

[0107] (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDR of the heavy chain variable region;

[0108] (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

[0109] (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region;

[0110]

[15] The antibody of any one of

[11] to

[14] , wherein at least one amino acid residue is modified in the constant region in a position selected from the group consisting of: position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 according to EU numbering;

[0111]

[16] The antibody of

[15] , wherein at least one amino acid residue is modified in the constant region in a position selected from the group consisting of: position 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, 419, 421, 433, 434, and 443;

[0112]

[17] The antibody of

[16] , wherein at least one amino acid residue is modified in the constant region in a position selected from the group consisting of: position 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413 according to EU numbering;

[0113]

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

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

[0114]

[19] The antibody of

[18] , wherein the FcγR is FcγRIIb;

[0115]

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

[17] , wherein the constant region has binding activity for one or more activating FcγRs selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa, and for FcγRIIb, and the FcγRIIb-binding activity is maintained or enhanced and the binding activity for the activating FcγRs is reduced compared to a reference antibody which differs only in that its constant region is that of a native IgG;

[0116]

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

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

[0117]

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

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

[0118]

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

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

[0119]

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

[23] ;

[0120]

[25] The pharmaceutical composition of

[24] for use in facilitating removal of an antigen from plasma;

[0121]

[26] The pharmaceutical composition of

[24] or

[25] for use in enhancing binding of an antibody to extracellular matrix;

[0122]

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

[23] ;

[0123]

[28] A vector comprising the nucleic acid of

[27] ;

[0124]

[29] A host cell comprising the vector of

[28] ;

[0125]

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

[29] and collecting the antibody from the cell culture;

[0126] [30A] A method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, wherein the method comprises modifying at least one amino acid residue that can be exposed on the surface of the antibody so as to increase the isoelectric point (pi);

[0127] [30B] The method of [30A], wherein the at least one amino acid residue is modified at a position selected from the group consisting of

[0128] (I) a position in a CDR or FR selected from the group consisting of: according to Kabat numbering, (a) 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 a FR of a heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65, and 97 in a CDR of a 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 a FR of a light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of a light chain variable region; or

[0129] (II) a position in a constant region selected from the group consisting of: according to EU numbering, positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443;

[0130]

[31] The method of [30A] or [30B], wherein the amino acid residue modification comprises a modification selected from the group consisting of:

[0131] (a) substitution of a negatively charged amino acid residue with an uncharged amino acid residue;

[0132] (b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue;

[0133] (c) substitution of an uncharged amino acid residue with a positively charged amino acid residue; and

[0134] (d) substitution or insertion with histidine in a CDR or FR.

[0135]

[32] The method of

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

[0136] compared to a reference antibody,

[0137] (a) selecting an antibody that is capable of promoting removal of an antigen from plasma;

[0138] (b) selecting an antibody that has enhanced binding activity to extracellular matrix;

[0139] (c) selecting an antibody that has enhanced FcyR-binding activity at neutral pH conditions (e.g., pH 7.4);

[0140] (d) selecting an antibody that has enhanced FcyRIIb-binding activity at neutral pH conditions (e.g., pH 7.4);

[0141] (e) selecting an antibody that has retained or enhanced FcyRIIb-binding activity and reduced binding activity to one or more activating FcyRs selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb, and FcyRIIa;

[0142] (f) selecting an antibody that has enhanced FcRn-binding activity at neutral pH conditions (e.g., pH 7.4);

[0143] (g) selecting an antibody that has an increased isoelectric point (pi);

[0144] (h) identifying the isoelectric point (pi) of a collected antibody, and subsequently selecting an antibody that has an increased isoelectric point (pi); and

[0145] (i) selecting an antibody whose antigen-binding activity is altered or increased depending on the ionic concentration conditions.

[0146] In an alternative embodiment, the disclosure A relates to, but is not limited to:

[0147] [A1] 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] is modified;

[0148] [A2] the antibody of [A1], further having a heavy chain variable region and / or a light chain variable region, wherein the variable region has one or more CDRs and / or one or more FRs, and wherein at least one amino acid residue in the CDRs and / or FRs selected from the group of modification sites identical to the modification sites in the group defined in

[13] or

[14] is modified;

[0149] [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 which is identical to the modification sites in the group defined in

[15] or

[16] is modified to increase its pi;

[0150] [A4] The antibody of [A3], further having a heavy chain variable region and / or a light chain variable region, wherein the variable region has one or more CDRs and / or one or more FRs, and wherein at least one amino acid residue in the CDRs and / or FRs selected from the group of modification sites which is identical to the modification sites in the group defined in

[13] or

[14] is modified;

[0151] [A5] An antibody comprising an antigen binding domain whose antigen binding activity changes depending on the ion concentration conditions, wherein the antibody has a constant region, and wherein at least one amino acid residue in the constant region selected from the group of modification sites which is identical to the modification sites in the group defined in

[15] or

[16] is modified;

[0152] [A6] The antibody of [A5], further having a heavy chain variable region and / or a light chain variable region, wherein the variable region has one or more CDRs and / or one or more FRs, and wherein at least one amino acid residue in the CDRs and / or FRs selected from the group of modification sites which is identical to the modification sites in the group defined in

[13] or

[14] is modified;

[0153] [A7] Use of the antibody of any one of [1] to

[23] and [Al] to [A6] in the manufacture of a medicament for facilitating removal of an antigen from plasma;

[0154] [A8] Use of the antibody of any one of [1] to

[23] and [Al] to [A6] in the manufacture of a medicament for increasing extracellular matrix binding;

[0155] [A9] Use of the antibody of any one of [1] to

[23] and [Al] to [A6] for removal of an antigen from plasma; and

[0156] [A10] Use of the antibody of any one of [1] to

[23] and [Al] to [A6] for increasing extracellular matrix binding.

[0157] [A11] An antibody obtained by the method of any one of

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

[31] ,

[32] .

[0158] According to various embodiments, the disclosure A includes a combination (partly or entirely) of one or more elements described in any one of the above [1] to

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

[31] ,

[32] , and [A1] to [A11], as long as the combination is technically not contradictory to common general knowledge in the field. For example, in some embodiments, the disclosure A includes a method for producing an antibody comprising a modification of an antigen-binding domain that promotes removal of an antigen from plasma compared to before the modification of the antibody, wherein the method comprises:

[0159] (a) modifying at least one amino acid residue that can be exposed on the surface of the antibody, the amino acid residue being at a position in:

[0160] (I) a position selected from the group consisting of: (a) 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; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDR 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, according to the Kabat numbering; or

[0161] (II) a position selected from the group consisting of: positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 according to the EU numbering, in the constant region;

[0162] (b) modifying the antigen-binding domain in such a way that the resulting antigen-binding activity changes depending on the ion concentration conditions, wherein the (a) and (b) can be performed simultaneously or sequentially;

[0163] (c) culturing the host cell to express the nucleic acid encoding the modified antibody; and

[0164] (d) collecting the modified antibody from the host cell culture.

[0165] In another embodiment, the method optionally further comprises one or more of the following:

[0166] compared to the antibody prior to modification,

[0167] (e) selecting an antibody that is capable of promoting removal of the antigen from the plasma;

[0168] (f) selecting an antibody that has enhanced binding activity to the extracellular matrix;

[0169] (g) selecting an antibody that has enhanced FcyR-binding activity at neutral pH conditions (e.g., pH 7.4);

[0170] (h) selecting an antibody that has enhanced FcyRIIb-binding activity at neutral pH conditions (e.g., pH 7.4);

[0171] (i) selecting an antibody that has retained or enhanced FcyRIIb-binding activity and reduced binding activity to one or more activating FcyRs, preferably selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb, and FcyRIIa;

[0172] (j) selecting an antibody that has enhanced FcRn-binding activity at neutral pH conditions (e.g., pH 7.4);

[0173] (k) selecting an antibody that has an increased isoelectric point (pi);

[0174] (l) confirming the isoelectric point (pi) of the collected antibody, and subsequently selecting an antibody that has an increased isoelectric point (pi); and

[0175] (m) selecting an antibody whose antigen binding activity is altered or increased as a function of ionic concentration conditions.

[0176] Another embodiment of the disclosure relates, for example, but not limited to, to:

[0177] [D1] A method for producing a modified antibody having an extended or reduced half-life in plasma compared to the antibody prior to modification, wherein the method comprises:

[0178] (a) modifying a nucleic acid encoding a pre-modified antibody to change the charge of at least one amino acid residue at a position selected from the group consisting of: position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 according to EU numbering;

[0179] (b) culturing the host cell to express the nucleic acid; and

[0180] (c) collecting the antibody from the host cell culture; or

[0181] [D2] A method for extending or reducing the half-life of an antibody in plasma, wherein the method comprises modifying at least one amino acid residue at a position selected from the group consisting of: position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 according to EU numbering.

[0182] In an embodiment, Disclosure B relates to, for example, but not limited to:

[0183]

[33] An Fc region variant comprising an FcRn-binding domain, wherein the FcRn-binding domain comprises an Ala at position 434; a Glu, Arg, Ser, or Lys at position 438; and a Glu, Asp, or Gin at position 440 according to EU numbering;

[0184]

[34] The Fc region variant of

[33] , wherein the FcRn-binding domain comprises an Ala at position 434; an Arg or Lys at position 438; and a Glu or Asp at position 440 according to EU numbering;

[0185]

[35] The Fc region variant of

[33] or

[34] , wherein the FcRn-binding domain further comprises an He or Leu at position 428; and / or an He, Leu, Val, Thr, or Phe at position 436, according to EU numbering;

[0186]

[36] The Fc region variant of

[35] , wherein the FcRn-binding domain comprises a Leu at position 428; and / or a Val or Thr at position 436, according to EU numbering.

[0187]

[37] The Fc region variant of any one of

[33] to

[36] , wherein the FcRn-binding domain comprises a combination of amino acid substitutions selected from the group consisting of: 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 / S440E; N434A / R435H / F436V / Q438K / S440D; M428L / N434A / Q438R / S440E; M428L / N434A / Q438R / S440D; M428L / N434A / Q438K / S440E; M428L / N434A / Q438K / S440D; M428L / N434A / Y436T / Q438R / S440E; M428L / N434A / Y436T / Q438R / S440D; M428L / N434A / Y436T / Q438K / S440E; M428L / N434A / Y436T / Q438K / S440D; M428L / N434A / Y436V / Q438R / S440E; M428L / N434A / Y436V / Q438R / S440D; M428L / N434A / Y436V / Q438K / S440E; M428L / N434A / Y436V / Q438K / S440D; L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; according to EU numbering.and L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E;

[0188]

[38] The Fc region variant of

[37] , wherein the FcRn-binding domain comprises a combination of amino acid substitutions selected from the group consisting of:

[0189] According to EU numbering, N434A / Q438R / S440E; N434A / Y436T / Q438R / S440E; N434A / Y436V / Q438R / S440E; M428L / N434A / Q438R / S440E; M428L / N434A / Y436T / Q438R / S440E; M428L / N434A / Y436V / Q438R / S440E; L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E;

[0190]

[39] The Fc region variant of any one of

[33] to

[38] , wherein its FcRn-binding activity is enhanced at acidic pH conditions (e.g., pH 5.8) compared to an Fc region of a native IgG;

[0191]

[40] The Fc region variant of any one of

[33] to

[39] , wherein its binding activity to an anti-drug antibody (ADA) is not significantly enhanced at neutral pH conditions compared to an Fc region of a native IgG;

[0192]

[41] The Fc region variant of

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

[0193]

[42] The Fc region variant of any one of

[33] to

[41] , wherein its plasma clearance (CL) is decreased, plasma retention time is increased, or plasma half-life (t½) is increased compared to an Fc region of a native IgG;

[0194]

[43] The Fc region variant of any one of

[33] to

[42] , wherein its plasma retention is increased compared to a reference Fc region variant comprising a combination of amino acid substitutions N434Y / Y436V / Q438R / S440E according to EU numbering;

[0195]

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

[33] to

[43] ;

[0196]

[45] The antibody of

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

[0197]

[46] A pharmaceutical composition comprising the antibody of

[44] or

[45] ;

[0198]

[47] The pharmaceutical composition of

[46] for use in increasing retention of an antibody in plasma;

[0199]

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

[33] to

[43] or the antibody of

[44] or

[45] ;

[0200]

[49] A vector comprising the nucleic acid of

[48] ;

[0201]

[50] A host cell comprising the vector of

[49] ;

[0202]

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

[50] , and subsequently collecting the Fc region variant or the antibody comprising the variant from the cell culture;

[0203]

[52] The method of

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

[0204] (a) selecting an Fc region variant having enhanced FcRn-binding activity at acidic pH conditions compared to the Fc region of a native IgG;

[0205] (b) selecting an Fc region variant whose binding activity to an anti-drug antibody (ADA) is not significantly enhanced at neutral pH conditions compared to the Fc region of a native IgG;

[0206] (c) selecting an Fc region variant having increased plasma retention compared to the Fc region of a native IgG; and

[0207] (d) selecting an antibody comprising an Fc region variant capable of facilitating removal of an antigen from plasma compared to a reference antibody comprising the Fc region of a native IgG; and

[0208]

[53] A method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the variant, wherein the method comprises substituting amino acids in such a way that the resulting Fc region variant or the antibody comprising the variant comprises Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gin at position 440 according to EU numbering.

[0209] In one embodiment, the disclosure B relates to, for example but not limited to:

[0210] [B1] Use of an Fc region variant of any one of

[33] to

[43] or an antibody of

[44] or

[45] in the manufacture of a medicament for increasing retention in plasma.

[0211] [B2] Use of an Fc region variant of any one of

[33] to

[43] or an antibody of

[44] or

[45] in the manufacture of a medicament for not substantially increasing binding activity to anti-drug antibodies (ADAs) under neutral pH conditions compared to an Fc region of a native IgG.

[0212] [B3] Use of an Fc region variant of any one of

[33] to

[43] or an antibody of

[44] or

[45] for increasing retention in plasma.

[0213] [B4] Use of an Fc region variant of any one of

[33] to

[43] or an antibody of

[44] or

[45] for not substantially increasing binding activity to anti-drug antibodies (ADAs) under neutral pH conditions compared to an Fc region of a native IgG; and

[0214] [B5] An Fc region variant or an antibody comprising the variant obtained by the method of any one of

[51] ,

[52] , and

[53] .

[0215] According to various embodiments, the disclosure B includes a combination (partially or entirely) of one or more elements described above in any one of

[33] to

[53] and [B1] to [B5], as long as the combination is not contradictory to common general knowledge in the art. For example, in some embodiments, the disclosure B includes an Fc region variant comprising an FcRn-binding domain, wherein the FcRn-binding domain can include:

[0216] (a) Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering;

[0217] (b) Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering;

[0218] (c) lie or Leu at position 428; Ala at position 434; lie, Leu, Val, Thr, or Phe at position 436; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering;

[0219] (d) an He or Leu at position 428; an Ala at position 434; an He, Leu, Val, Thr, or Phe at position 436; an Arg or Lys at position 438; and an Glu or Asp at position 440, according to EU numbering;

[0220] (e) a Leu at position 428; an Ala at position 434; a Val or Thr at position 436; an Glu, Arg, Ser, or Lys at position 438; and an Glu, Asp, or Gin at position 440, according to EU numbering; or

[0221] (f) a Leu at position 428; an Ala at position 434; a Val or Thr at position 436; an Arg or Lys at position 438; and an Glu or Asp at position 440, according to EU numbering.

[0222] In one embodiment, the disclosure C relates to, for example, but not limited to:

[0223]

[54] An isolated anti-IL-8 antibody that binds human IL-8 comprising at least one amino acid substitution in at least one of (a) to (f) below and binds IL-8 in a pH-dependent manner:

[0224] (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67;

[0225] (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68;

[0226] (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 69;

[0227] (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70;

[0228] (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 71; and

[0229] (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72;

[0230]

[55] The anti-IL-8 antibody of

[54] , comprising amino acid substitutions of a tyrosine at position 9 of the amino acid sequence of SEQ ID NO: 68, an arginine at position 11 of the amino acid sequence of SEQ ID NO: 68, and a tyrosine at position 3 of the amino acid sequence of SEQ ID NO: 69;

[0231]

[56] The anti-IL-8 antibody of

[54] or

[55] , 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;

[0232]

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

[54] to

[56] , comprising 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;

[0233]

[58] The anti-IL-8 antibody of 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;

[0234]

[59] The anti-IL-8 antibody of 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;

[0235]

[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;

[0236]

[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 (a) to (f):

[0237] (a) an Fc region having increased binding affinity to FcRn relative to the binding affinity of a native Fc region to FcRn;

[0238] (b) an Fc region having decreased binding affinity to pre-existing ADAs relative to the binding affinity of a native Fc region to pre-existing ADAs;

[0239] (c) an Fc region having increased plasma half-life relative to the plasma half-life of a native Fc region;

[0240] (d) an Fc region having decreased plasma clearance relative to the plasma clearance of a native Fc region; and

[0241] (e) an Fc region having reduced binding affinity to an effector receptor relative to a native Fc region;

[0242] (f) increased binding to extracellular matrix.

[0243]

[62] The anti-IL-8 antibody of

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

[0244]

[63] The anti-IL-8 antibody of

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

[0245]

[64] The anti-IL-8 antibody of

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

[0246]

[65] The anti-IL-8 antibody of

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

[0247]

[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;

[0248]

[67] An anti-IL-8 antibody comprising 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;

[0249]

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

[54] to

[67] ;

[0250]

[69] A vector comprising the nucleic acid of

[68] ;

[0251]

[70] A host cell comprising the vector of

[69] ;

[0252]

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

[70] ;

[0253]

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

[71] , comprising isolating the antibody from culture supernatant;

[0254]

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

[54] to

[67] , and a pharmaceutically acceptable carrier;

[0255]

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

[54] to

[67] for use in a pharmaceutical composition;

[0256]

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

[54] to

[67] for use in treating a condition in which excess IL-8 is present;

[0257]

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

[54] to

[67] in the manufacture of a medicament for a condition in which excess IL-8 is present;

[0258]

[77] A method for treating a patient having a condition in which excess IL-8 is present, comprising administering to the individual the anti-IL-8 antibody of any one of

[54] to

[67] ;

[0259]

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

[54] to

[67] ;

[0260]

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

[54] to

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

[0261]

[80] A method for producing an anti-IL-8 antibody comprising a variable region having pH-dependent IL-8-binding activity, wherein the method comprises:

[0262] (a) assessing binding of an anti-IL-8 antibody to extracellular matrix,

[0263] (b) selecting an anti-IL-8 antibody having strong binding to extracellular matrix,

[0264] (c) culturing a host comprising a vector comprising nucleic acid encoding the antibody, and

[0265] (d) isolating the antibody from the culture solution.

[0266] In an alternative embodiment, the disclosure C relates to:

[0267] The use of an anti-IL-8 antibody described in any one of

[54] to

[67] in the manufacture of a pharmaceutical composition for inhibiting accumulation of biologically active IL-8;

[0268] The use of an anti-IL-8 antibody described in any one of

[54] to

[67] for inhibiting accumulation of biologically active IL-8;

[0269] The use of an anti-IL-8 antibody described in any one of

[54] to

[67] in the manufacture of a pharmaceutical composition for inhibiting angiogenesis;

[0270] The use of an anti-IL-8 antibody described in any one of

[54] to

[67] for inhibiting angiogenesis;

[0271] The use of an anti-IL-8 antibody described in any one of

[54] to

[67] in the manufacture of a pharmaceutical composition for inhibiting promotion of neutrophil migration;

[0272] The use of an anti-IL-8 antibody described in any one of

[54] to

[67] for inhibiting promotion of neutrophil migration;

[0273] An anti-IL-8 antibody described in any one of

[54] to

[67] for use in inhibiting accumulation of biologically active IL-8;

[0274] A method for inhibiting accumulation of biologically active IL-8, wherein the method comprises administering to a subject an anti-IL-8 antibody described in any one of

[54] to

[67] ;

[0275] A pharmaceutical composition for inhibiting accumulation of biologically active IL-8, comprising an anti-IL-8 antibody described in any one of

[54] to

[67] ;

[0276] An anti-IL-8 antibody described in any one of

[54] to

[67] for use in inhibiting angiogenesis;

[0277] A method for inhibiting angiogenesis in a subject, wherein the method comprises administering to the subject an anti-IL-8 antibody described in any one of

[54] to

[67] ;

[0278] A pharmaceutical composition for inhibiting angiogenesis, comprising an anti-IL-8 antibody described in any one of

[54] to

[67] ;

[0279] An anti-IL-8 antibody described in any one of

[54] to

[67] for use in inhibiting promotion of neutrophil migration;

[0280] [C14] A method for inhibiting promotion of neutrophil migration in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody as described in any one of

[54] to

[67] ;

[0281] [C15] A pharmaceutical composition for use in inhibiting promotion of neutrophil migration, comprising an anti-IL-8 antibody as described in any one of

[54] to

[67] ;

[0282] [C16] An anti-IL-8 antibody as described in any one of

[54] to

[67] for use in treating a condition in which there is excess IL-8;

[0283] [C17] Use of an anti-IL-8 antibody as described in any one of

[54] to

[67] in the manufacture of a pharmaceutical composition for treating a condition in which there is excess IL-8;

[0284] [C18] Use of an anti-IL-8 antibody as described in any one of

[54] to

[67] for treating a condition in which there is excess IL-8;

[0285] [C19] A method of treating a condition in which there is excess IL-8 in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody as described in any one of

[54] to

[67] ;

[0286] [C20] A pharmaceutical composition for use in treating a condition in which there is excess IL-8, comprising an anti-IL-8 antibody as described in any one of

[54] to

[67] ;

[0287] [C21] An anti-IL-8 antibody as described in any one of

[54] to

[67] for use in promoting removal of IL-8;

[0288] [C22] Use of an anti-IL-8 antibody as described in any one of

[54] to

[67] in the manufacture of a pharmaceutical composition for promoting removal of IL-8;

[0289] [C23] Use of an anti-IL-8 antibody as described in any one of

[54] to

[67] for promoting removal of IL-8;

[0290] [C24] A method for promoting removal of IL-8 in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody as described in any one of

[54] to

[67] ; and

[0291] [C25] A pharmaceutical composition for use in promoting removal of IL-8, comprising an anti-IL-8 antibody as described in any one of

[54] to

[67] .

[0292] [C26] An anti-IL-8 antibody comprising an Fc region comprising one or more amino acid substitutions at one or more positions selected from the group consisting of: positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438, and 440, according to EU numbering.

[0293] [C27] The anti-IL-8 antibody of [C26], comprising an Fc region having at least one property selected from the following (a) to (f):

[0294] (a) increased binding affinity of the Fc region for the FcRn at acidic pH relative to the binding affinity of the native Fc region for the FcRn;

[0295] (b) decreased binding affinity of the Fc region for pre-existing ADAs relative to the binding affinity of the native Fc region for pre-existing ADAs;

[0296] (c) increased plasma half-life of the Fc region relative to the plasma half-life of the native Fc region;

[0297] (d) decreased plasma clearance of the Fc region relative to the plasma clearance of the native Fc region;

[0298] (e) decreased binding affinity of the Fc region for effector receptors relative to the binding affinity of the native Fc region for effector receptors; and

[0299] (f) increased binding to extracellular matrix.

[0300] [C28] The anti-IL-8 antibody of [C26] or [C27], comprising an Fc region comprising one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E, according to EU numbering.

[0301] [C29] The anti-IL-8 antibody of [C28], comprising an Fc region comprising 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, according to EU numbering.

[0302] [C30] The anti-IL-8 antibody of [C26], 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.

[0303] [C31] The anti-IL-8 antibody of [C26], comprising 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.

[0304] [C32] An isolated nucleic acid encoding the anti-IL-8 antibody of any one of [C26] to [C31].

[0305] [C33] A vector comprising the nucleic acid of [C32].

[0306] [C34] A host cell comprising the vector of [C33].

[0307] [C35] A method for producing an anti-IL-8 antibody, comprising culturing the host cell of [C34].

[0308] [C36] A method for producing the anti-IL-8 antibody of any one of [C26] to [C31], further comprising isolating the antibody from the host cell culture.

[0309] [C37] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of [C26] to [C31] and a pharmaceutically acceptable carrier.

[0310] [C38] A method for treating a patient having a disorder in which excess IL-8 is present, the method comprising administering to the individual the anti-IL-8 antibody of any one of [C26] to [C31].

[0311] [C39] A method for promoting removal of IL-8 from an individual, the method comprising administering to the individual the anti-IL-8 antibody of any one of [C26] to [C31].

[0312] According to various embodiments, the disclosure C includes a combination (partially or entirely) of one or more elements described in any one of

[54] to

[80] and [C1] to [C39] above, as long as the combination is technically possible with ordinary skill in the art. BRIEF DESCRIPTION OF DRAWINGS

[0313] [ Figure 1 ]

[0314] Figure 1Changes in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with an antibody that binds human IL-6 receptor in a pH-dependent manner and whose constant region is that of a native IgGl (low_pl-IgGl), or an antibody in which the pi of the variable region in the antibody has been increased (high_pl-IgGl).

[0315] [ Figure 2 ]

[0316] Figure 2 Changes in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with an antibody that binds human IL-6 receptor in a pH-dependent manner and whose constant region is that of a native IgGl (low_pl-IgGl), or an antibody in which the pi of the variable region in the antibody has been increased (high_pl-IgGl).

[0317] [ Figure 3 ]

[0318] Figure 3 Changes in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with an antibody that binds human IL-6 receptor in a pH-dependent manner and whose constant region is that of a native IgGl (low_pl-IgGl), or an antibody in which the pi of the variable region in the antibody has been increased (high_pl-IgGl).

[0319] [ Figure 4 ]

[0320] Figure 4 Changes in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with an antibody that binds human IL-6 receptor in a pH-dependent manner and whose constant region is that of a native IgGl (low_pl-IgGl), or an antibody in which the pi of the variable region in the antibody has been increased (high_pl-IgGl).

[0321] [ Figure 5 ]

[0322] Figure 5The degree of extracellular matrix binding of each of three types of antibodies with different pi that bind to human IL-6 receptor in a pH-dependent manner (low_pi-IgGl, mid_pi-IgGl, and high_pi-IgGl) and two types of antibodies with different pi that do not bind to human IL-6 receptor in a pH-dependent manner (low_pi(NPH)-IgGl and high_pi(NPH)-IgGl) is shown. "NPH" means non-pH dependent within the scope of the disclosure A described herein.

[0323] [ Figure 6 ]

[0324] Figure 6 The relative value of the degree of soluble human FcyRIIb binding (measured by BIACORE (registered trademark)) of antibodies comprising Fc region variants whose respective pi is increased by modifying one amino acid residue in the constant region of the Ab1H-P600 antibody that binds to IgE in a pH-dependent manner is shown, with the value of Ab1H-P600 set to 1.00.

[0325] [ Figure 7 ]

[0326] Figure 7 The relative value of the rate of uptake of cells of a cell line expressing hFcyRIIb by antibodies comprising Fc region variants whose respective pi is increased by modifying one amino acid residue in the constant region of Ab1H-P600 is shown, each evaluated with the value of Ab1H-P600 set to 1.00.

[0327] [ Figure 8 ]

[0328] Figure 8 The degree of binding of Fv4-IgGl (which has the Fc region of native human IgGl) to rheumatoid factor in the serum of each RA patient is shown.

[0329] [ Figure 9 ]

[0330] Figure 9 The degree of binding of Fv4-YTE (which comprises an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient is shown.

[0331] [ Figure 10 ]

[0332] Figure 10 The degree of binding of Fv4-LS (which comprises an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient is shown.

[0333] [ Figure 11 ]

[0334] Figure 11 The extent of binding of rheumatoid factor in serum of each RA patient by Fv4-N434H, which comprises an Fc region variant with increased FcRn binding, is shown.

[0335] [ Figure 12 ]

[0336] Figure 12 The extent of binding of rheumatoid factor in serum of each RA patient by Fv4-F1847m, which comprises an Fc region variant with increased FcRn binding, is shown.

[0337] [ Figure 13 ]

[0338] Figure 13 The extent of binding of rheumatoid factor in serum of each RA patient by Fv4-F1848m, which comprises an Fc region variant with increased FcRn binding, is shown.

[0339] [ Figure 14 ]

[0340] Figure 14 The extent of binding of rheumatoid factor in serum of each RA patient by Fv4-F1886m, which comprises an Fc region variant with increased FcRn binding, is shown.

[0341] [ Figure 15 ]

[0342] Figure 15 The extent of binding of rheumatoid factor in serum of each RA patient by Fv4-F1889m, which comprises an Fc region variant with increased FcRn binding, is shown.

[0343] [ Figure 16 ]

[0344] Figure 16 The extent of binding of rheumatoid factor in serum of each RA patient by Fv4-F1927m, which comprises an Fc region variant with increased FcRn binding, is shown.

[0345] [ Figure 17 ]

[0346] Figure 17 The extent of binding of rheumatoid factor in serum of each RA patient by Fv4-F1168m, which comprises an Fc region variant with increased FcRn binding, is shown.

[0347] [ Figure 18 ]

[0348] Figure 18Average values for binding of rheumatoid factor in serum of RA patients by Fv4-IgGl, which has the Fc region of native human IgGl and each antibody comprises a new Fc region variant in which the Fc region has increased binding to each FcRn.

[0349] [ Figure 19 ]

[0350] Figure 19 Changes in plasma concentration of each anti-human IgE antibody in cynomolgus monkeys when administered with OHB-IgGl, which is an anti-human IgE antibody and has the Fc region of native human IgGl, and each antibody comprises a new Fc region variant in which each Fc region comprises an Fc region variant with increased binding to FcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m).

[0351] [ Figure 20 ]

[0352] Figure 20 Changes in plasma concentration of anti-human IL-6 receptor antibody in human FcRn transgenic mice when administered with Fv4-IgGl, which is an anti-human IL-6 receptor antibody and has the Fc region of native human IgGl, or Fv4-F1718, which has increased antibody binding to FcRn at acidic pH conditions.

[0353] [ Figure 21 ]

[0354] Figure 21 Sensorgrams obtained for IL-8 binding of H998 / L63 and Hr9 measured with Biacore at pH 7.4 and pH 5.8.

[0355] [ Figure 22 ]

[0356] Figure 22 Changes in human IL-8 concentration in mouse plasma when H998 / L63 or H89 / L118 was administered to mice at 2 mg / kg in a mixture with human IL-8.

[0357] [ Figure 23 ]

[0358] Figure 23 Changes in human IL-8 concentration in mouse plasma when H89 / L118 was administered to mice at 2 mg / kg or 8 mg / kg in a mixture with human IL-8.

[0359] [Figure 24 ]

[0360] Figure 24 shows the change in human IL-8 concentration in mouse plasma when H89 / L118 or H553 / L118 is administered to mice at 2 mg / kg or 8 mg / kg (in a mixture with human IL-8).

[0361] [ Figure 25A ]

[0362] Figure 25A shows the change in relative values of antibody concentration-dependent chemiluminescence of antibody Hr9, H89 / L118 or H553 / L118 after storage in plasma for one week.

[0363] [ Figure 25B ]

[0364] Figure 25B shows the change in relative values of antibody concentration-dependent chemiluminescence of antibody Hr9, H89 / L118 or H553 / L118 after storage in plasma for two weeks.

[0365] [ Figure 25C ]

[0366] Figure 25C shows the change in relative values of antibody concentration-dependent chemiluminescence of antibody Hr9, H89 / L118 or H553 / L118 after storage in plasma for two weeks.

[0367] [ Figure 26 ]

[0368] Figure 26 shows the predicted frequency of ADA occurrence for each anti-IL-8 antibody (hWS4, Hr9, H89 / L118, H496 / L118 or H553 / L118) and the predicted frequency of ADA occurrence for other pre-existing therapeutic antibodies, predicted by EpiMatrix.

[0369] [ Figure 27 ]

[0370] Figure 27 shows the predicted frequency of ADA occurrence for each anti-IL-8 antibody (H496 / L118, H496vl / L118, H496v2 / L118, H496v3 / L118, H1004 / L118 or H1004 / L395) and the predicted frequency of ADA occurrence for other pre-existing therapeutic antibodies, predicted by EpiMatrix.

[0371] [ Figure 28A ]

[0372] Figure 28A Change in relative value of antibody concentration-dependent chemiluminescence for antibody Hr9, H89 / L118 or H1009 / L395-F1886s shown before storage in plasma.

[0373] [ Figure 28B ]

[0374] Figure 28B Change in relative value of antibody concentration-dependent chemiluminescence for antibody Hr9, H89 / L118 or H1009 / L395-F1886s shown after storage in plasma for one week.

[0375] [ Figure 28C ]

[0376] Figure 28C Change in relative value of antibody concentration-dependent chemiluminescence for antibody Hr9, H89 / L118 or H1009 / L395-F1886s shown after storage in plasma for two weeks.

[0377] [ Figure 29 ]

[0378] Figure 29 Change in human IL-8 concentration in mouse plasma shown when each of H1009 / L395, H553 / L118 and H998 / L63 (in a mixture with human IL-8) was administered to mice.

[0379] [ Figure 30 ]

[0380] Figure 30 Extracellular matrix binding shown when Hr9, H89 / L118 or H1009 / L395 were added to the extracellular matrix alone, and when they were added in a mixture with human IL-8.

[0381] [ Figure 31 ]

[0382] Figure 31 Change in mouse plasma antibody concentration shown when an antibody with the variable regions of H1009 / L395 and an Fc region that does not bind FcRn (F1942m) was administered to human FcRn transgenic mice, alone or in a mixture with human IL-8.

[0383] [ Figure 32 ]

[0384] Figure 32 Frequency of predicted occurrence of ADAs for H1009 / L395 and H1004 / L395 and frequency of predicted occurrence of ADAs for other pre-existing therapeutic antibodies predicted by EpiMatrix.

[0385] [ Figure 33 ]

[0386] Figure 33 Figure 8 shows the change in concentration of each anti-human IL-8 antibody in cynomolgus monkey plasma when administered as H89 / L118-IgGl, which has the variable regions of H89 / L118 and the Fc region of native human IgGl, and each antibody containing an Fc region variant with increased binding to FcRn (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m, and H89 / L118-F1927m).

[0387] [ Figure 34 ]

[0388] Figure 34 Figure 8 shows the change in concentration of each anti-human IL-8 antibody in cynomolgus monkey plasma when administered as H89 / L118-IgGl, which has the variable regions of H89 / L118 and the Fc region of native human IgGl, and each antibody containing an Fc region variant with increased binding to FcRn (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m, and H89 / L118-F1927m).

[0389] [ Figure 35 ]

[0390] Figure 35 Figure 8 shows the change in concentration of each anti-human IL-8 antibody in cynomolgus monkey plasma when administered as H89 / L118-IgGl, which has the variable regions of H89 / L118 and the Fc region of native human IgGl, and each antibody containing an Fc region variant with increased binding to FcRn (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m, and H89 / L118-F1927m).

[0391] [ Figure 36 ]

[0392] Figure 36 Figure 8 shows the change in concentration of each anti-human IL-8 antibody in cynomolgus monkey plasma when administered as H89 / L118-IgGl, which has the variable regions of H89 / L118 and the Fc region of native human IgGl, and each antibody containing an Fc region variant with increased binding to FcRn (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m, and H89 / L118-F1927m).

[0393] [ Figure 37 ]

[0394] Figure 37 Figure 8 shows the change in concentration of each anti-human IL-8 antibody in cynomolgus monkey plasma when administered as H89 / L118-IgGl, which has the variable regions of H89 / L118 and the Fc region of native human IgGl, and each antibody containing an Fc region variant with increased binding to FcRn (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m, and H89 / L118-F1927m).

[0395] [ Figure 38A ]

[0396] Figure 38 Figures 38A-38D Octet sensor maps of 25 selected pH-dependent and / or calcium-dependent antigen-binding clones are displayed.

[0397] [ Figure 38B ]

[0398] Figure 38B yes Figure 38A The continuation.

[0399] [ Figure 38C ]

[0400] Figure 38C yes Figure 38B The continuation.

[0401] [ Figure 38D ]

[0402] Figure 38D yes Figure 38C The continuation.

[0403] [ Figure 39 ]

[0404] Figure 39 The image shows the time-varying C5 plasma concentrations of some anti-C5 bispecific antibodies in C57BL6J mice, with regard to antibody variable region modification.

[0405] [ Figure 40 ]

[0406] Figure 40 The image shows the plasma IgE concentration-time curves of some anti-IgE antibodies in C57BL6J mice, with regard to antibody variable region modification. Detailed Implementation Plan

[0407] Detailed description

[0408] Non-limiting embodiments of disclosure A, B, or C are described below. All embodiments described in the examples below are intended to be correctly understood in the "Detailed Description" section and are not subject to any patent practices, regulations, guidelines, etc., that might attempt to narrowly interpret the embodiments in countries where this patent application is desired to be granted.

[0409] Disclosure A or Disclosure B

[0410] In some embodiments, the disclosure A relates to an antibody comprising an antigen binding domain whose antigen binding activity is altered depending on ion concentration conditions, wherein the isoelectric point (pi) is increased by a modification of at least one amino acid residue that can be exposed on the surface of the antibody (herein, also referred to as "ion concentration-dependent antibody with increased pi" within the scope of the disclosure A; and the antigen binding domain of the antibody is also referred to as "ion concentration-dependent antigen binding domain with increased pi"). The present invention is based, in part, on the inventors' surprising finding that the removal of an antigen from plasma can be facilitated by an ion concentration-dependent antibody whose isoelectric point (pi) is increased by a modification of at least one amino acid residue that can be exposed on the surface of the antibody (e.g., when the antibody is administered in vivo); and the binding of the antibody to the extracellular matrix can be increased with the ion concentration-dependent antibody having an increased (elevated) pi. The present invention is also based, in part, on the inventors' surprising finding that this beneficial effect comes from the combination of two completely different concepts: an ion concentration-dependent antigen binding domain or ion concentration-dependent antibody; and an antibody whose pi is increased by a modification of at least one amino acid residue that can be exposed on the surface (herein, also referred to as "antibody with increased pi" within the scope of the disclosure A; and an antibody whose pi is decreased (decreased) by a modification of at least one amino acid residue that can be exposed on the surface is also referred to as "antibody with decreased pi" within the scope of the disclosure A). The present invention thus falls into a category of pioneering research that can lead to a significant technological innovation in the field to which the disclosure A belongs (e.g., the medical field).

[0411] In general, for example, an antibody comprising an antigen binding domain and whose pi is increased by a modification of at least one amino acid residue that can be exposed on the surface of the antibody, which is further modified so that the antigen binding activity of the antigen binding domain is altered depending on ion concentration conditions, is also included within the scope of the disclosure A described herein (herein, the antibody is also referred to as "ion concentration-dependent antibody with increased pi" within the scope of the disclosure A).

[0412] In general, for example, an antibody comprising an ion concentration-dependent antigen binding domain, which at least one amino acid residue that can be exposed on the surface of the antibody has a different charge from at least one amino acid residue in the corresponding position in the antibody before the modification (native antibody (e.g., native Ig antibody, preferably native IgG antibody), or reference or parent antibody (e.g., antibody before the modification, or antibody before or during the construction of the library, etc.)), and whose net antibody pi is increased, is also included in the disclosure A described herein (the antibody is also referred to as "ion concentration-dependent antibody with increased pi" within the scope of the disclosure A described herein).

[0413] Generally, for example, antibodies containing ion-concentration-dependent antigen binding domains (wherein the pi of the antibody is increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody) are also included in the disclosure A described herein (which are also referred to as "ion-concentration-dependent antibodies with increased pi" within the scope of the disclosure A described herein).

[0414] Generally, for example, antibodies containing ion-concentration-dependent antigen binding domains (wherein the pi of the antibody is increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody) are also included in the disclosure A described herein (which are also referred to as "ion-concentration-dependent antibodies with increased pi" within the scope of the disclosure A described herein).

[0415] Within the scope of the disclosure A and B described herein, "amino acid" includes not only natural amino acids, but also unnatural amino acids. Within the scope of the disclosure A and B described herein, an amino acid or an amino acid residue can be represented by a single letter (e.g., A) or a three letter code (e.g., Ala), or both (e.g., Ala(A)).

[0416] "Modification of an amino acid", "modification of an amino acid residue", or a comparable term, when used in the context of the disclosure A and B, can be understood as not being limited to a modification of the amino acid sequence of an antibody by a molecular chemistry, but also as an 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 in the amino acid sequence of an antibody. The amino acid addition, deletion, substitution, or insertion can be performed on a nucleic acid encoding the amino acid sequence, e.g., by site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)) or overlap extension PCR; via affinity maturation of an antibody, or by using chain shuffling of an antibody heavy or light chain; or by antigen panning-based selection using a phage display library (Smith et al., Methods Enzymol. 217:228-257 (1993)); and these can be performed individually or in appropriate combinations. The amino acid modification is preferably performed by an amino acid addition, deletion, substitution, or insertion by replacing one or more amino acid residues in the amino acid sequence of an antibody with a different amino acid (respectively), and the amino acid sequence modification by humanization or chimerization can be performed by methods known in the art. The change or modification of an amino acid (residue), such as an amino acid addition, deletion, substitution, or insertion, can also be performed on an antibody variable region or an antibody constant region to be used for preparing a recombinant antibody for the antibody of the disclosure A or B.

[0417] In one embodiment in the context of the disclosure A and B described herein, a substitution of an amino acid (residue) refers to a replacement with a different amino acid (residue) and can be designed to modify, for example, matters such as each of (a) to (c): (a) polypeptide backbone structure in a region of folded or helical conformation; (b) charge or hydrophobicity of a target site; or (c) size of a side chain.

[0418] Amino acid residues are classified into groups based on the properties of side chains in the structure, for example, as follows: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, and He; (2) neutral, hydrophilic: Cys, Ser, Thr, Asn, and Gin; (3) acidic: Asp and Glu; (4) basic: His, Lys, and Arg; (5) residues that influence the orientation of the chain: Gly and Pro; and (6) aromatic: Trp, Tyr, and Phe.

[0419] Substitutions of amino acid residues within each group are referred to as conservative substitutions, while substitutions of amino acid residues between different groups are referred to as non-conservative substitutions. Substitutions of amino acid residues can be conservative substitutions, non-conservative substitutions, or a combination thereof. A variety of known suitable methods can be used to substitute amino acids with those other than the natural amino acids (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 containing tRNA can be used, in which a non-natural amino acid is linked to an amber suppressor tRNA complementary to the UAG codon (amber codon), which is a stop codon (Clover Direct (Protein Express)).

[0420] Within the scope of the disclosure A and B described herein, it is understood that the structure of an "antigen" is not limited to a specific structure, so long as the antigen comprises an epitope that binds to an antibody. The antigen can be an inorganic substance or an organic substance. The antigen can be any ligand, including various cytokines, e.g., interleukins, chemokines, and cell growth factors. Alternatively, often, a receptor that exists, e.g., in a soluble form or is modified to be in a soluble form in a biological fluid such as plasma can also be used as an antigen. Non-limiting examples of such soluble receptors include the soluble IL-6 receptor described in Mullberg et al., J. Immunol. 152(10):4958-4968 (1994). Further, the antigen can be monovalent (e.g., soluble IL-6 receptor) or multivalent (e.g., IgE).

[0421] In one embodiment, the antigen that can be bound by the antibody of the disclosure A and B is preferably a soluble antigen present in a biological fluid (e.g., a biological fluid described in WO2013 / 125667, preferably plasma, interstitial fluid, lymph fluid, ascites, or pleural fluid) of a subject (within the scope of the disclosure A and B described herein, the subject to which the antibody is administered (applied) can be virtually any animal, e.g., a human, a mouse, etc.); however, the antigen can also be a membrane antigen.

[0422] Within the scope of the disclosure A and B described herein, "prolonging the half-life of a target molecule in plasma" or "shortening the half-life of a target molecule in plasma" (the target molecule can be an antigen or an antibody), or equivalent terms thereof, can also be expressed more specifically using any other parameter other than the parameter of the half-life in plasma (t1 / 2), such as the mean residence time in plasma, the clearance in plasma (CL), and the area under the concentration curve (AUC) (Rikai no tame no kaihatsu to shori (Development and analysis for pharmacokinetics) Nanzando). These parameters can be specifically evaluated, for example, by noncompartmental analysis according to the protocol attached to the in vivo kinetics analysis software WinNonlin (Pharsight). It is known to those skilled in the art that these parameters are generally correlated with each other.

[0423] Within the scope of the disclosure A and B described herein, "epitope" refers to an antigenic determinant in an antigen and means a site on an antigen to which an antigen-binding domain of an antibody binds. Thus, an epitope can be defined, for example, based on its structure. Alternatively, an epitope can be defined by the antigen-binding activity of an antibody that recognizes the epitope. When an antigen is a peptide or a polypeptide, an epitope can be specified by the amino acid residues that constitute the epitope. Alternatively, when an epitope is a sugar chain, an epitope can be specified based on its specific sugar chain structure. The antigen-binding domain of the disclosure A and B can bind to a single epitope or different epitopes on an antigen.

[0424] A linear epitope can be a primary amino acid sequence. The linear epitope typically contains at least three and usually contains at least five, for example, 8 to 10 amino acids or 6 to 20 amino acids as a unique sequence.

[0425] In a conformational epitope, the amino acids that constitute the epitope are not usually present consecutively as a primary sequence. An antibody recognizes a conformational epitope in the three-dimensional structure of a peptide or a protein. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance (Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (Editor)).

[0426] Within the scope of the disclosure A and B described herein, "antibody" is not particularly limited and is used in the broadest sense of the term, as long as it can bind to a target antigen, non-limiting examples of antibodies broadly include known common antibodies (e.g., natural immunoglobulins (abbreviated as "Ig")), as well as molecules and variants derived therefrom, e.g., Fab, Fab', F(ab')2, diabodies, 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 (Orita et al., Blood 105:562-566 (2005)), scaffold proteins, one-armed antibodies (including all embodiments of one-armed antibodies described in WO 2005 / 063816), multispecific antibodies (e.g., bispecific antibodies: antibodies specific for two different epitopes, including antibodies recognizing different antigens and antibodies recognizing different epitopes on the same antigen). Within the scope of the disclosure A and B described herein, "bispecific antibodies" are not limited to but can be prepared, for example, as antibody molecules having common L chains described in WO 2005 / 035756, or by the method described in WO 2008 / 119353, in which two general types of antibodies having IgG4-like constant regions are mixed, causing a exchange reaction between the two types of the antibodies (known to those skilled in the art as the "Fab-arm exchange" method). In one alternative embodiment, they can be structural antibodies having a single chain in which a heavy chain variable region and a light chain variable region are linked together (e.g., sc(Fv)2). Alternatively, they can be antibody-like molecules resulting from linking a Fc region (a constant region lacking a CH1 domain) with a scFv (or sc(Fv)2) in which a heavy chain variable region (VH) is linked to a light chain variable region (VL) (e.g., scFv-Fc). A multispecific antibody consisting of scFv-Fc has a (scFv)2-Fc structure in which the first and second polypeptides are VH1-linker-VL1-Fc and VH2-linker-VL2-Fc, respectively. Alternatively, they can be 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) (US 2013 / 0171138), functional fragments thereof, substances equivalent in function thereto, and variants thereof modified in sugar chains.Herein, a native IgG (e.g., native IgGl) refers to a polypeptide containing the same amino acid sequence as a naturally occurring IgG (e.g., native IgGl) and belongs to the antibody type essentially encoded by immunoglobulin gamma genes. The native IgG can be a spontaneous mutant thereof, etc.

[0427] Generally, when the antibody has substantially the same or similar structure as the native IgG, a Y-shaped structure of four chains (two heavy chain polypeptides and two light chain polypeptides) can be the basic structure. Generally, the heavy chain and the light chain can be connected by a disulfide bond (SS bond) and form a heterodimer. The heterodimer can be connected together by a disulfide bond and form a Y-shaped heterotetramer. Two heavy chains or light chains can be the same as or different from each other.

[0428] For example, the IgG antibody can be cleaved into two Fab units (regions) and a single Fc unit (region) by papain cleavage of the hinge region (also referred to as "hinge" in the scope of the disclosure A and B described herein), in which the heavy chain Fab region is connected to the Fc region. Generally, the Fab region contains an antigen binding domain. Because phagocytes such as lymphocytes and macrophages have receptors (Fc receptors) capable of binding the Fc region, and can recognize the antigen-bound antibody via the Fc receptor and phagocytose the antigen (opsonization). Meanwhile, the Fc region is involved in the mediation of immune responses such as ADCC or CDC, and has an effector function of inducing a response after the antibody binds to the antigen. It is known that the antibody effector function changes depending on the type (isotype) of immunoglobulin. The Fc region of the IgG type will indicate, for example, a region spanning the cysteine at position 226 or the proline at position 230 (EU numbering) to the C-terminus; however, the Fc region is not limited thereto. The Fc region can be appropriately obtained by, for example, partially digesting a monoclonal IgGl, IgG2, IgG3, or IgG4 antibody, etc. with a protease such as pepsin, followed by eluting the adsorbed fraction from a protein A or protein G column.

[0429] In the scope of the disclosure A and B described herein, the position of the amino acid residue in the antibody variable region (one or more CDRs and / or one or more FRs) is shown according to Kabat, while the position of the amino acid residue in the constant region or Fc region is shown according to EU numbering based on the amino acid position according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991).

[0430] Within the scope of the disclosure A and B described herein, "library" can refer to a population of molecules, such as a plurality of antibodies having sequence variations, wherein their respective sequences can be identical or different from each other; a plurality of fusion polypeptides containing the antibodies; or nucleic acids or oligonucleotides encoding these amino acid sequences, as detailed in WO2013 / 125667 (e.g., paragraphs 0121-0125). The library can, for example, contain at least 10 4 antibody molecules, more preferably, at least 10 5 antibody molecules, even more preferably, at least 10 6 antibody molecules, particularly preferably, at least 10 7 antibody molecules or more. The library can be a phage library. The term "consisting essentially of means that the antibodies having different sequences in a number of independent clones in the library, which can have different antigen binding activities, occupy a certain portion. In one embodiment, an immune library based on antibody gene construction from lymphocytes of an animal immunized with a specific antigen, an infected patient, a human with elevated antibodies in the blood due to immunization, or a patient containing cancer or autoimmune disease can be suitably used as a randomized variable region library. In an alternative embodiment, a naive library containing naive sequences (antibody sequences without bias in the repertoire) from antibody gene construction from lymphocytes derived from a healthy human (Gejima et al., Human Antibodies 11 : 121-129 (2002)); Cardoso et al., Scand. J. Immunol. 51 : 337-344 (2000)) can also be suitably used as a randomized variable region library. Amino acid sequences containing naive sequences can refer to those obtained from the naive library. In an alternative embodiment, a synthetic library in which V genes from genomic DNA or CDR sequences of reconstructed functional V genes are replaced with a set of oligonucleotides containing sequences encoding codon sets of appropriate lengths can also be suitably used as a randomized variable region library. In this case, it is also possible to replace only the heavy chain CDR3 sequence because sequence changes are observed in the CDR3 gene. A standard approach to generate amino acid diversity in antibody variable regions can be to increase changes in amino acid residues at positions that can be exposed on the surface of the antibody.

[0431] In one embodiment, in the case of the antibodies of disclosure A or B, e.g., having substantially the same or similar structure as that of a native Ig antibody, they generally have 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: CH1 to CH3. Generally, the Fab region of a heavy chain contains VH and CH1, and generally the Fc region of a heavy chain contains CH2 and CH3. Generally, the hinge region is located between CH1 and CH2. Furthermore, the variable region generally has complementarity determining regions ("CDRs") and framework regions ("FRs"). Generally, the VH region and the VL region each have three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR4). Generally, the six CDRs in the variable regions of the heavy chain and the light chain interact with each other and form the antigen binding domain of the antibody. On the other hand, in the case of only one single CDR, it has the ability to recognize and bind to an antigen while known to have lower antigen binding affinity compared to the case where six CDRs are present.

[0432] Ig antibodies are classified into various types (isotypes) based on the structural differences of their constant regions. In many mammals, they are classified into five immunoglobulin types based on the structural differences of the constant regions: IgG, IgA, IgM, IgD, and IgE. Furthermore, in the case of humans, IgG has four types: IgG1, IgG2, IgG3, and IgG4; and IgA has two subtypes: IgA1 and IgA2. The heavy chains are classified into γ chain, μ chain, α chain, δ chain, and ε chain according to the differences in the constant regions, and based on these differences, there are five immunoglobulin types (isotypes): IgG, IgM, IgA, IgD, and IgE. On the other hand, there are two types of light chains: λ chain and κ chain, and all immunoglobulins have one of these two.

[0433] In one embodiment, the antibodies of disclosure A or B have a heavy chain, e.g., the heavy chain can be any one of, or can be derived from, γ chain, μ chain, α chain, δ chain, and ε chain, and wherein the antibodies of disclosure A or B have a light chain, e.g., the light chain can be κ chain or λ chain, or can be derived from any one of them. Furthermore, within the scope of disclosure A and B described herein, the antibodies can be any isotype (e.g., IgG, IgM, IgA, IgD, or IgE) and any subtype (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2; mouse IgG1, IgG2a, IgG2b, and IgG3), or can be derived from any one of them, but are not limited thereto.

[0434] Within the scope of the disclosure A and B described herein, "antigen binding domain" can have any structure as long as it binds to the antigen of interest. The domain can include, for example, variable regions of antibody heavy and light chains (e.g., 1 to 6 CDRs); a module of about 35 amino acids called A domain, which is included in Avimers (cell membrane proteins present in vivo) (WO 2004 / 044011 and WO 2005 / 040229); Adnectins containing 10Fn3 domains that bind to a protein in fibronectin expressed on a cell membrane (WO 2002 / 032925); Affibodies having a scaffold IgG-binding domain (a three-helix bundle of 58 amino acids that constitutes Protein A) (WO 1995 / 001937); Designed Ankyrin Repeat Proteins (DARPins), which are regions exposed on the molecular surface of ankyrin repeats (ARs) having a structure in which subunit repeats containing a 33-amino acid residue turn, two anti-parallel helices, and a loop are stacked (WO 2002 / 020565); Anticalins and the like, which are four-loop regions on one side of a central twisted barrel structure of eight anti-parallel strands highly conserved in molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO 2003 / 029462); and concave regions formed by parallel sheet structures inside a horseshoe-shaped structure formed by stacking of leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs) that do not have an immunoglobulin structure and are used in the system of acquired immunity in agnathans such as lampreys and hagfish (WO 2008 / 016854). Preferred antigen binding domains of the disclosure A or B can include those having IgG antibody heavy and light chain variable regions, and more specifically, ScFv, single chain antibody, Fv, scFv2 (single chain Fv2), Fab, and F(ab')2.

[0435] In one embodiment of the disclosure A, "ionic concentration" is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. Herein, "metal ion" can be any one of ions in Group I elements such as alkali metals and copper group elements, Group II elements such as alkaline earth metals and zinc group elements, Group III elements except boron, Group IV elements except carbon and silicon, Group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of Group V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. Metal atoms have a property of releasing valence electrons to become cations. This is called ionization tendency. Metals having a strong ionization tendency are considered to be chemically active.

[0436] In one embodiment of the disclosure A, the preferred metal ion can be a calcium ion, as detailed in WO2012 / 073992 and WO2013 / 125667.

[0437] In one embodiment of the disclosure A, the "one or more ion concentration conditions" can be conditions that focus on the difference in the biological behavior of the ion concentration-dependent antibody between low ion concentration and high ion concentration. In addition, "the antigen-binding activity is changed depending on the ion concentration conditions" can mean that the antigen-binding activity of the ion concentration-dependent antigen-binding domain or the ion concentration-dependent antibody of the disclosure A or B is changed between low ion concentration and high ion concentration. The cases include, for example, those having higher (stronger) or lower (weaker) antigen-binding activity at high ion concentration than at low ion concentration, but are not limited thereto.

[0438] In one embodiment of the disclosure A, the ion concentration can be hydrogen ion concentration (pH) or calcium ion concentration, in the case where 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 in the case where the ion concentration is calcium ion concentration, it can also be referred to as a "calcium ion concentration-dependent antigen-binding domain".

[0439] In one embodiment in the context of the disclosure A, the ion-concentration- dependent antigen binding domain, the ion-concentration-dependent antibody, the ion- concentration-dependent antigen binding domain having an increased pi, and the ion- concentration-dependent antibody having an increased pi can be obtained from a library consisting essentially of antibodies differing in sequence (having variability) and whose antigen binding domain contains at least one amino acid residue that causes the antigen binding activity of the antigen binding domain or antibody to change depending on the ion concentration conditions. The antigen binding domain can be preferably located within the light chain variable region (which can be modified) and / or the heavy chain variable region (which can be modified). Furthermore, for the construction of the library, the light chain or heavy chain variable region can be combined with a heavy chain or light chain variable region constructed as a library of random variable region sequences. In the case where the ion concentration is the concentration of hydrogen or calcium ions, non-limiting examples of the library include, for example, a library in which the heavy chain variable region constructed as a library of random variable region sequences is combined with a light chain variable region sequence in which one or more amino acid residues of the germline sequence such as SEQ ID NO: 1 (Vk1), SEQ ID NO: 2 (Vk2), SEQ ID NO: 3 (Vk3), or SEQ ID NO: 4 (Vk4) are replaced with at least one amino acid residue capable of changing the antigen binding activity depending on the ion concentration. Furthermore, in the case where the ion concentration is the concentration of calcium ions, the library includes, for example, those in which the heavy chain variable region sequence of SEQ ID NO: 5 (6RL#9-IgGl) or SEQ ID NO: 6 (6KC4-l#85-IgGl) is combined with a light chain variable region constructed as a library of random variable region sequences or a light chain variable region having a germline sequence.

[0440] In one embodiment, in the case where the ion concentration is the concentration of calcium ions, the high calcium ion concentration is not particularly limited to a specific value; however, the concentration can be selected between 100 μΜ and 10 mM, between 200 μΜ and 5 mM, between 400 μΜ and 3 mM, between 200 μΜ and 2 mM, or between 400 μΜ and 1 mM. A concentration selected between 500 μΜ and 2.5 mM (which is close to the in vivo plasma (blood) concentration of calcium ions) can also be preferred. The low calcium ion concentration is not particularly limited to a specific value; however, the concentration can be selected between 0.1 μΜ and 30 μΜ, between 0.2 μΜ and 20 μΜ, between 0.5 μΜ and 10 μΜ, or between 1 μΜ and 5 μΜ, or between 2 μΜ and 4 μΜ. A concentration selected between 1 μΜ and 5 μΜ (which is close to the in vivo calcium ion concentration in early endosomes) can also be preferred.

[0441] Whether or not the antigen-binding activity of the antigen-binding domain or the antibody containing the domain is changed depending on the metal ion concentration (e.g., calcium ion concentration) conditions can be easily determined by known methods, for example, by the methods described herein within the scope of Disclosure A, or the methods described in WO2012 / 073992. For example, the antigen-binding activity of the antigen-binding domain or the antibody containing the domain can be measured at low and high calcium ion concentrations and compared. In this case, the conditions other than the calcium ion concentration can preferably be the same. Furthermore, the conditions other than the calcium ion concentration in the determination of the antigen-binding activity can be appropriately selected by those skilled in the art. The antigen-binding activity can be determined, for example, at 37°C under the conditions of HEPES buffer, or using BIACORE (GE Healthcare) or the like.

[0442] In one embodiment in the case of Disclosure A, the ion-concentration-dependent antigen-binding domain, the ion-concentration-dependent antibody, the ion-concentration-dependent antigen-binding domain having an increased pi, or the ion-concentration-dependent antibody having an increased pi preferably has a higher antigen-binding activity under high calcium ion concentration conditions than under low calcium ion concentration conditions. In this case, the ratio between the antigen-binding activity under low calcium ion concentration conditions and the antigen-binding activity under high calcium ion concentration conditions is not limited; however, the ratio of the KD (dissociation constant) for the antigen under low calcium ion concentration conditions to the KD under high calcium ion concentration conditions, i.e., KD (3 μM Ca) / KD (2 mM Ca), can preferably be 2 or more, more preferably 10 or more, and still more preferably 40 or more. The upper limit of the KD (3 μM Ca) / KD (2 mM Ca) value is not limited, and can be any value such as 400, 1000, or 10000.

[0443] In the case where the antigen is a soluble antigen, the dissociation constant (KD) can be used as the value of the antigen-binding activity. Meanwhile, in the case where the antigen is a membrane antigen, the apparent dissociation constant (KD) can be used. The dissociation constant (KD) and the apparent dissociation constant (KD) can be determined by known methods, for example, by BIACORE (GE healthcare), Scatchard plot, or flow cytometry.

[0444] Alternatively, for example, the dissociation rate constant (kd) can also be used as another index for indicating the ratio of the binding activity. In the case of using the dissociation rate constant (kd) instead of the dissociation constant (KD) as an index for indicating the ratio of the antigen-binding activity, the ratio of the low-calcium-ion-concentration- condition dissociation rate constant (kd) to the high-calcium-ion-concentration-condition dissociation rate constant (kd), i.e., kd(low calcium ion concentration condition) / kd(high calcium ion concentration condition), can preferably be 2 or more, more preferably 5 or more, still more preferably 10 or more, and yet more preferably 30 or more. The upper limit of the value of kd(low calcium ion concentration condition) / kd(high calcium ion concentration condition) is not limited and can be any value such as 50, 100, or 200.

[0445] In the case where the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as a value of the antigen-binding activity. Meanwhile, in the case where the antigen is a membrane antigen, the apparent dissociation rate constant (kd) can be used. The dissociation rate constant (kd) and the apparent dissociation rate constant (kd) can be determined by a known method, for example, by BIACORE (GE healthcare) or flow cytometry.

[0446] In one embodiment, the method for producing or screening a calcium ion concentration-dependent antigen-binding domain or a calcium ion concentration-dependent antibody, or a library thereof, whose antigen-binding activity is higher in a high-calcium-ion-concentration condition than in a low-calcium-ion-concentration condition, is not limited. The method includes, for example, those described in WO2012 / 073992 (e.g., paragraphs 0200-0213).

[0447] The method can include, for example:

[0448] (a) determining the antigen-binding activity of the antigen-binding domain or the antibody in a low-calcium-ion-concentration condition;

[0449] (b) determining the antigen-binding activity of the antigen-binding domain or the antibody in a high-calcium-ion-concentration condition; and

[0450] (c) selecting the antigen-binding domain or the antibody whose antigen-binding activity in a low-calcium-ion-concentration condition is lower than the antigen-binding activity in a high-calcium-ion-concentration condition.

[0451] Alternatively, the method can include, for example:

[0452] (a) contacting the antigen with the antigen-binding domain or the antibody, or a library thereof, in a high-calcium-ion-concentration condition;

[0453] (b) incubating the antigen-binding domain or the antibody bound to the antigen in step (a) in a low-calcium-ion-concentration condition; and

[0454] (c) isolating the antigen binding domain or antibody that dissociates in step (b).

[0455] Alternatively, the method can comprise, for example:

[0456] (a) contacting the antigen binding domain or antibody, or a library thereof, with the antigen under low calcium ion concentration conditions;

[0457] (b) selecting the antigen binding domain or antibody that does not bind the antigen or has low antigen binding capacity in step (a);

[0458] (c) allowing the antigen binding domain or antibody selected in step (b) to bind to the antigen under high calcium ion concentration conditions; and

[0459] (d) isolating the antigen binding domain or antibody that binds to the antigen in step (c).

[0460] Alternatively, the method can comprise, for example:

[0461] (a) contacting the antigen binding domain or antibody, or a library thereof, with a column to which the antigen is immobilized under high calcium ion concentration conditions;

[0462] (b) eluting the antigen binding domain or antibody that binds to the column in step (a) from the column under low calcium ion concentration conditions; and

[0463] (c) isolating the antigen binding domain or antibody eluted in step (b).

[0464] Alternatively, the method can comprise, for example:

[0465] (a) passing the antigen binding domain or antibody, or a library thereof, through a column to which the antigen is immobilized under low calcium ion concentration conditions to collect the antigen binding domain or antibody that is eluted from the column without binding to the column;

[0466] (b) allowing the antigen binding domain or antibody collected in step (a) to bind to the antigen under high calcium ion concentration conditions; and

[0467] (c) isolating the antigen binding domain or antibody that binds to the antigen in step (b).

[0468] Alternatively, the method can comprise, for example:

[0469] (a) contacting the antigen binding domain or antibody, or a library thereof, with the antigen under high calcium ion concentration conditions;

[0470] (b) obtaining the antigen binding domain or antibody that binds to the antigen in step (a);

[0471] (c) incubating the antigen binding domain or antibody obtained in step (b) at low calcium ion concentration; and

[0472] (d) isolating the antigen binding domain or antibody whose antigen binding activity in step (c) is weaker than the standard selected in step (b).

[0473] Each step of these different screening methods can be repeated several times, or the steps can be appropriately combined to obtain the most suitable molecule. The above conditions can be appropriately selected for the low and high calcium ion concentration conditions. The desired calcium ion concentration-dependent antigen binding domain or calcium ion concentration-dependent antibody can be thus obtained.

[0474] In the case of the disclosure A, in one embodiment, the antigen binding domain or antibody as the starting material can be, for example, a modified antigen binding domain or antibody having an increased pi resulting from modification of the charge of at least one amino acid residue that can be exposed on the surface thereof. In an alternative embodiment, in the case where amino acids that change the binding activity of the ion concentration-dependent antigen binding domain are introduced into the sequence, they can be introduced together with modification of the charge of at least one amino acid residue that can be exposed on the surface of the antigen binding domain or antibody to increase the pi.

[0475] Alternatively, in the case of the present application A, for example, a pre-existing antigen binding domain or antibody, a pre-existing library (phage library, etc.); an antibody prepared from a hybridoma obtained by immunizing an animal or from B cells of an immunized animal, or a library thereof; or an antigen binding domain, an antibody, or a library obtained by introducing a natural or unnatural amino acid mutation capable of chelating calcium therein (described below) (for example, a library having an increased content of calcium-chelatable amino acids, or a library in which a calcium-chelatable amino acid is introduced at a specific site) can be used.

[0476] In one embodiment in the context of Disclosure A, in the case where the ion concentration is calcium ion concentration, there is no limitation on the type of amino acid that changes the ion concentration-dependent antigen binding domain or the binding activity of the ion concentration-dependent antigen binding domain having an elevated pi, as long as they are capable of forming a calcium-binding motif. For example, calcium-binding motifs are known to those skilled in the art (e.g., Springer et al. (Cells 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. (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)). Thus, in the case where the antigen binding domain has any calcium-binding motif such as that of C-type lectin, for example, ASGPR, CD23, MBR, or DC-SIGN, the antigen binding activity of the domain can change depending on the calcium ion concentration conditions. The calcium-binding motif can include, in addition to those described above, for example, the calcium-binding motif included in the antigen binding domain described in SEQ ID NO: 7 (which corresponds to "Vk5-2").

[0477] In one embodiment in the context of Disclosure A, in the case where the ion concentration is calcium ion concentration, an amino acid having metal-chelating activity can be used as the amino acid that changes the ion concentration-dependent antigen binding domain or the binding activity of the ion concentration-dependent antigen binding domain having an elevated pi. For example, any amino acid can be appropriately used as the amino acid having metal-chelating activity, as long as they are capable of forming a calcium-binding motif. Specifically, the amino acid includes those having electron-donating properties. The amino acid preferably includes, but is not limited to, Ser (S), Thr (T), Asn (N), Gin (Q), Asp (D), and Glu (E).

[0478] The position of the amino acid having metal-chelating activity in the antigen-binding domain is not limited to a particular position. In one embodiment, the amino acid can be located at any position in the heavy chain variable region and / or the light chain variable region that can form the antigen-binding domain. The at least one amino acid residue that causes a calcium ion concentration-dependent change in the antigen-binding activity of the antibody can be included in, for example, the CDRs (one or more of CDR1, CDR2, and CDR3) and / or the FRs (one or more of FR1, FR2, FR3, and FR4) of the heavy chain and / or the light chain. The one or more amino acid residues can be placed, for example, at one or more of positions 95, 96, 100a, and 101 in the heavy chain CDR3 according to the numbering of Kabat; one or more of positions 30, 31, and 32 in the light chain CDR1 according to the numbering of Kabat; position 50 in the light chain CDR2 according to the numbering of Kabat; and / or position 92 in the light chain CDR3 according to the numbering of Kabat. Those amino acid residues can be placed alone or in combination.

[0479] It is known that troponin C, calmodulin, parvalbumin, myosin light chain, etc. have multiple calcium-binding sites and are presumed to have originated from a common source in molecular evolution, and in one embodiment, one or more of the light chain CDR1, CDR2, and CDR3 can be designed to contain its binding motif. For the above purpose, for example, the cadherin domain; the EF hand contained in calmodulin; the C2 domain contained in protein kinase C; the Gla domain contained in blood clotting factor IX; the C-type lectin of asialoglycoprotein receptor or mannose-binding receptor; the A domain contained in LDL receptor; annexin; the third type of thrombin-sensitive protein domain; and the EGF-like domain can be appropriately used.

[0480] In one embodiment, in the case of ion concentration is hydrogen ion concentration (pH), the concentration condition of protons, i.e., the nucleus of hydrogen atoms, is used synonymously with the condition of hydrogen index (pH). In the case where the active amount of hydrogen ions in an aqueous solution is represented by aH + , pH is defined as -log10aH + . In the case where the ionic strength of an aqueous solution is low (for example, less than 10 -3 ), aH + is almost equal to the hydrogen ion strength. For example, the ion product of water at 25°C and 1 atmosphere is Kw = aH + *aOH = 10 -14 ; thus, for pure water, aH + = aOH = 10 -7In this case, pH = 7 is neutral, and an aqueous solution having a pH less than 7 is acidic, and an aqueous solution having a pH greater than 7 is basic. Thus, the hydrogen ion concentration condition can be a condition focusing on the difference in the biological behavior of the pH-dependent antibody at a high hydrogen ion concentration (acidic pH range) and at a low hydrogen ion concentration (neutral pH range) for the hydrogen ion concentration condition or the pH condition. For example, in the case of the disclosure A, "antigen binding activity under a condition of a high hydrogen ion concentration (acidic pH range) is lower than antigen binding activity under a condition of a low hydrogen ion concentration (neutral pH range)" can mean that the ion concentration-dependent antigen binding domain, the ion concentration-dependent antibody, the ion concentration-dependent antigen binding domain having an increased pi, or the ion concentration-dependent antibody having an increased pi has antigen binding activity that is weaker at a pH selected from the group consisting of 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 still more preferably pH 5.5 to pH 6.5, than at a pH selected from the group consisting of 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 still more preferably pH 7.0 to pH 8.0. Preferably, the above expression can mean that the antigen binding activity is weaker at the early endosomal intracellular pH than at the plasma pH in vivo; and specifically means that the antibody, for example, the antigen binding activity at pH 5.8 is weaker than the antigen binding activity at, for example, pH 7.4.

[0481] Whether the antigen binding activity of the antigen binding domain or the antibody containing the domain changes depending on the hydrogen ion concentration condition can be easily evaluated by a known method, for example, by the assay method described in the case of the disclosure A herein, or described in WO2009 / 125825. For example, the antigen binding activity of the antigen binding domain or the antibody containing the domain to the antigen of interest can be measured and compared at a low and a high hydrogen ion concentration. In this case, it is preferable that the conditions other than the hydrogen ion concentration are the same. In the case of determining the antigen binding activity, a person skilled in the art can appropriately select the conditions other than the hydrogen ion concentration, and, for example, the measurement can be performed at 37°C under the conditions of HEPES buffer, or using BIACORE (GE Healthcare) or the like.

[0482] Within the scope of the disclosure A described herein, unless otherwise specifically indicated in the context, the "neutral pH range" (also referred to as "low hydrogen ion concentration", "high pH", "neutral pH condition", or "neutral pH") is not particularly limited to a specific value; however, it can 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. The neutral pH range can be preferably pH 7.4, which is close to the in vivo pH in plasma (blood), but for the convenience of measurement, for example, pH 7.0 can be used.

[0483] Within the scope of the disclosure A described herein, unless otherwise specifically indicated in the context, the "acidic pH range" (also referred to as "high hydrogen ion concentration", "low pH", "acidic pH condition", or "acidic pH") is not particularly limited to a specific value; however, it can 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. The acidic pH range can be preferably pH 5.8, which is close to the in vivo hydrogen ion concentration of early endosomes, but for the convenience, for example, pH 6.0 can be used.

[0484] In one embodiment in the context of the disclosure A, in the case where the ion concentration is hydrogen ion concentration, it is preferable that the antigen-binding activity of the ion-concentration-dependent antigen-binding domain, the ion-concentration-dependent antibody, the ion-concentration-dependent antigen-binding domain having an increased pi, or the ion-concentration-dependent antibody having an increased pi is higher at the neutral pH condition than at the acidic pH condition. In this case, the ratio of the antigen-binding activity at the neutral pH condition to the antigen-binding activity at the acidic pH condition is not limited; however, the ratio of the dissociation constant (KD) of the antigen at the acidic pH condition to the KD at the neutral pH condition, i.e., KD(acidic pH range) / KD(neutral pH range), (e.g., KD(pH 5.8) / KD(pH 7.4)) can be 2 or more; 10 or more; or 40 or more. The upper limit of the KD(acidic pH range) / KD(neutral pH range) value is not limited, and can be any value such as 400, 1000, or 10000.

[0485] In an alternative embodiment, it is also possible to use, for example, the dissociation rate constant (kd) as an index to represent the above-mentioned ratio of binding activity. In the case where the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an index to represent the ratio of binding activity, the ratio of the dissociation rate constant (kd) of the antigen under high hydrogen ion concentration conditions to that under low hydrogen ion concentration conditions, i.e., kd(acidic pH range) / kd(neutral pH range) can 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 can be any value such as 50, 100, or 200.

[0486] In the case where the antigen is a soluble antigen, the value of the antigen binding activity can be represented by the dissociation rate constant (kd), while in the case where the antigen is a membrane antigen, the value can be represented by the apparent dissociation rate constant (apparent kd). The dissociation rate constant (kd) and the apparent dissociation rate constant (apparent kd) can be determined by known methods, for example, by using BIACORE (GE healthcare) or flow cytometry.

[0487] In one embodiment, the method for producing or screening a pH-dependent antigen binding domain or a pH-dependent antibody, or a library thereof, whose antigen binding activity is higher under neutral pH conditions than under acidic pH conditions, is not limited. The method includes, for example, those described in WO2009 / 125825 (e.g., paragraphs 0158-0190).

[0488] The method can include, for example:

[0489] (a) determining the antigen binding activity of the antigen binding domain or the antibody under acidic pH conditions;

[0490] (b) determining the antigen binding activity of the antigen binding domain or the antibody under neutral pH conditions; and

[0491] (c) selecting the antigen binding domain or the antibody whose antigen binding activity is lower under acidic pH conditions than under neutral pH conditions.

[0492] Alternatively, the method can include, for example:

[0493] (a) contacting the antigen with the antigen binding domain or the antibody, or a library thereof, under neutral pH conditions;

[0494] (b) incubating the antigen binding domain or the antibody bound to the antigen in step (a) under acidic pH conditions; and

[0495] (c) isolating the antigen binding domain or the antibody dissociated in step (b).

[0496] Alternatively, the method can comprise, for example:

[0497] (a) contacting the antigen with the antigen binding domain or antibody, or library thereof, under acidic pH conditions;

[0498] (b) selecting the antigen binding domain or antibody of step (a) that does not bind the antigen or has low antigen binding capacity;

[0499] (c) contacting the antigen binding domain or antibody selected in step (b) with the antigen under neutral pH conditions; and

[0500] (d) isolating the antigen binding domain or antibody that binds the antigen in step (c).

[0501] Alternatively, the method can comprise, for example:

[0502] (a) contacting the antigen binding domain or antibody, or library thereof, with a column to which the antigen is immobilized under neutral pH conditions;

[0503] (b) eluting the antigen binding domain or antibody bound to the column in step (a) from the column under acidic pH conditions; and

[0504] (c) isolating the antigen binding domain or antibody eluted in step (b).

[0505] Alternatively, the method can comprise, for example:

[0506] (a) passing the antigen binding domain or antibody, or library thereof, through a column to which the antigen is immobilized under acidic pH conditions to collect eluted antigen binding domains or antibodies that do not bind the column;

[0507] (b) contacting the antigen binding domain or antibody collected in step (a) with the antigen under neutral pH conditions; and

[0508] (c) isolating the antigen binding domain or antibody that binds the antigen in step (b).

[0509] Alternatively, the method can comprise, for example:

[0510] (a) contacting the antigen with the antigen binding domain or antibody, or library thereof, under neutral pH conditions;

[0511] (b) obtaining the antigen binding domain or antibody that binds the antigen in step (a);

[0512] (c) incubating the antigen binding domain or antibody obtained in step (b) under acidic pH conditions; and

[0513] (d) isolating an antigen binding domain or antibody whose antigen binding activity in step (c) is weaker than the standard selected in step (b).

[0514] Each step of these different screening methods can be repeated several times, or the steps can be combined. For acidic and neutral pH conditions, the above conditions can be appropriately selected. A desired pH-dependent antigen binding domain or pH-dependent antibody can be thus obtained.

[0515] In the case of the disclosure A, in one embodiment, the antigen binding domain or antibody as a starting material can be, for example, a modified antigen binding domain or antibody having an increased pi due to modification of the charge of at least one amino acid residue capable of being exposed on the surface thereof. In an alternative embodiment, in the case where amino acids that change the ion concentration-dependent binding activity of the antigen binding domain are introduced into the sequence, they can be introduced together with modification of the charge of at least one amino acid residue capable of being exposed on the surface of the antigen binding domain or antibody to increase the pi.

[0516] Alternatively, in the case of the present application A, for example, a pre-existing antigen binding domain or antibody, a pre-existing library (phage library, etc.); an antibody prepared from a hybridoma obtained by immunizing an animal or from B cells of an immunized animal, or a library thereof; or an antigen binding domain, an antibody, or a library obtained by introducing a natural or unnatural amino acid mutation having a side chain pKa of 4.0-8.0 (described below) thereto (for example, a library having an increased natural or unnatural amino acid mutation having a side chain pKa of 4.0-8.0, or a library in which a natural or unnatural amino acid mutation having a side chain pKa of 4.0-8.0 is introduced at a specific site) can be used. The preferred antigen binding domain can have, for example, an amino acid sequence in which at least one amino acid residue is substituted with an amino acid having a side chain pKa of 4.0-8.0 and / or is inserted with an amino acid having a side chain of 4.0-8.0, as described in WO2009 / 125825.

[0517] In one embodiment in the context of the disclosure A, the site of introduction of an amino acid mutation having a side chain pKa of 4.0-8.0 is not limited, and the mutation can be introduced to any site as long as the antigen binding activity is weaker in the acidic pH range than in the neutral pH range (KD(acidic pH range) / KD(neutral pH range) value increases or kd(acidic pH range) / kd(neutral pH range) value increases) compared to before the substitution or insertion. In the case of an antibody having a variable region or one or more CDRs, the site can be within the variable region or one or more CDRs. The number of amino acids substituted or inserted can be appropriately determined by one skilled in the art; and the number can be one or more. Furthermore, other amino acids can be deleted, added, inserted, and / or substituted, or modified (in addition to the above-mentioned substitution or insertion). Substitution or insertion of an amino acid having a side chain pKa of 4.0-8.0 with an amino acid having a side chain pKa of 4.0-8.0 can be performed in a random manner by a scanning method such as a histidine scan, in which histidine is used instead of alanine in an alanine scan known to one skilled in the art, and / or an antibody whose KD(acidic pH range) / KD(neutral pH range) value or kd(acidic pH range) / kd(neutral pH range) value increases compared to before the mutation can be selected from among antigen binding domains or antibodies, or libraries thereof, obtained by mutating these amino acids with random substitution or random insertion of these amino acids.

[0518] Furthermore, the antigen binding domain or antibody can preferably be those whose antigen binding activity is not significantly decreased, not substantially decreased, substantially the same, or increased in the neutral pH range before and after these mutations; and in other words, the activity can be maintained at at least 10% or more, preferably 50% or more, still more preferably 80% or more, and yet more preferably 90% or more, or even higher. In the case where the binding activity of the antigen binding domain or antibody is decreased due to substitution or insertion of an amino acid having a pKa of 4.0-8.0 with an amino acid having a pKa of 4.0-8.0, the binding activity can be restored or increased by, for example, substituting, deleting, adding, or inserting one or more amino acids at sites other than the above-mentioned substitution or insertion site.

[0519] In an alternative embodiment, amino acids having a side chain pKa of 4.0-8.0 can be placed at any position within the variable region of the heavy chain and / or light chain that can form an antigen binding domain. At least one amino acid residue having a side chain pKa of 4.0-8.0 can be located, for example, 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 residues include, but are not limited to, one or more of the amino acid residues at positions 24, 27, 28, 31, 32 and 34 in CDR1 of the light chain variable region; one or more of the amino acid residues at positions 50, 51, 52, 53, 54, 55 and 56 in CDR2 of the light chain variable region; and / or one or more of the amino acid residues at positions 89, 90, 91, 92, 93, 94 and 95A in CDR3 of the light chain variable region according to the Kabat numbering. Those amino acid residues can be included individually or in combination, as long as the antigen binding activity of the antibody changes depending on the hydrogen ion concentration.

[0520] In one embodiment within the scope of the disclosure A, any amino acid residue can be appropriately used as an amino acid residue whose antigen binding activity of the antigen binding domain or antibody changes depending on the hydrogen ion concentration. Specifically, the amino acid residues can include those having a side chain pKa of 4.0-8.0. The amino acids having electron-donating properties can include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (US 2009 / 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)). The amino acid residues can preferably include, for example, amino acids having a side chain pKa of 6.0-7.0, particularly His (H).

[0521] Within the scope of the disclosure A described herein, unless otherwise specified and unless there is a contextually inconsistent, it is to be understood that the isoelectric point (pi) can be a theoretically or experimentally determined isoelectric point, and it is also referred to as "pi".

[0522] The pi value can be determined experimentally, for example, by isoelectric focusing electrophoresis. Meanwhile, the theoretical pi value can be calculated using gene and amino acid sequence analysis software (Genetyx, etc.).

[0523] In one embodiment, whether the pi of an antibody having an increased pi or an antibody of the disclosure A is increased compared to the antibody prior to modification (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody) or a reference antibody (e.g., an antibody prior to modification of the antibody, or prior to or during construction of a library)) can be determined by performing antibody pharmacokinetic testing in combination with methods such as BIACORE, cell proliferation assays, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), or fluorescence immunoassay using plasma (e.g., from a mouse, rat, rabbit, dog, monkey, or human) in addition to or instead of the methods described above.

[0524] Within the context of the disclosure A described herein, "amino acid residues capable of being exposed on the surface" can generally refer to amino acid residues located on the surface of the polypeptide that makes up the antibody. "Amino acid residues located on the surface of the polypeptide" can refer to amino acid residues whose side chains can be in contact with solvent molecules, which can generally be predominantly water molecules. However, the side chains do not necessarily have to be completely in contact with the solvent molecules, and even when a part of the side chain is in contact with the solvent molecules, the amino acid residues are defined as "amino acids located on the surface." Amino acid residues located on the surface of the polypeptide can also include amino acid residues adjacent to the surface of the antibody and can thereby have a common charge influence from other one or more amino acid residues whose side chains, even partially, are in contact with the solvent molecules. One of ordinary skill in the art can prepare a homology model of the polypeptide or antibody by, for example, using commercially available software. Alternatively, methods such as X-ray crystallography can be used. Amino acid residues that can be exposed on the surface can be determined, for example, using a three-dimensional model of the antibody, for example, using computer software such as the Insight II 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)). Sites that can be exposed on the surface can be determined using software suitable for protein modeling and three-dimensional structural information obtained from the antibody. Software available for this purpose includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires user input of size parameters, the "size" of the probe used in the calculation can be set to a radius of about 1.4 angstroms (A) or less. In addition, a method for determining surface-exposed regions and areas using software for personal computers is described by Pacios (Pacios, Comput. Chem 18(4):377-386 (1994); J. Mol. Model. 1:46-53 (1995)). Based on the information described above, appropriate amino acid residues located on the surface of the polypeptide that makes up the antibody can be selected.

[0525] Methods of increasing the pi of a protein are, for example, decreasing the number of amino acids having a negatively charged side chain (e.g., aspartic acid and glutamic acid) under neutral pH conditions and / or increasing the number of amino acids having a positively charged side chain (e.g., arginine, lysine, and histidine). Amino acid residues having a negatively charged side chain have a negative charge of -1 under pH conditions sufficiently above their side chain pKa, which is a well known tenet to those skilled in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the side chain has a negative charge of -1 under neutral pH conditions (e.g., in a solution at pH 7.0). Conversely, amino acid residues having a positively charged side chain have a positive charge of +1 under pH conditions sufficiently below their side chain pKa. For example, the theoretical pKa of the side chain of arginine is 12.5, and the side chain has a positive charge of +1 under neutral pH conditions (e.g., in a solution at pH 7.0). Amino acid residues whose side chains are not charged under neutral pH conditions (e.g., in a solution at pH 7.0) are known to include 15 types of natural amino acids, namely, alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. In general, it is to be understood that the amino acids used to alter the pi can be non-natural amino acids.

[0526] In summary, as a method for increasing the pi of a protein under neutral pH conditions (e.g., in a solution at pH 7.0), for example, for an aspartic acid (residue) or glutamic acid (residue) in the amino acid sequence of a protein whose side chain has a negative charge of -1, a +1 charge change can be imparted to the protein of interest by substituting the amino acid (residue) with a non-charged side chain. Further, for example, for an amino acid (residue) whose side chain is not charged, a +1 charge change can be imparted to the protein by substituting arginine or lysine whose side chain has a positive charge of +1. Further, for aspartic acid or glutamic acid whose side chain has a negative charge of -1, a +2 charge change can be imparted to the protein each time by substituting arginine or lysine whose side chain has a positive charge of +1. Alternatively, to increase the pi of a protein, amino acids having a non-charged side chain and / or amino acids having a positively charged side chain can be added or inserted into the amino acid sequence of the protein, or amino acids whose side chain is not charged and / or amino acids whose side chain is negatively charged present in the amino acid sequence of the protein can be deleted. It is to be understood that, for example, in addition to the charge derived from the side chain, the N-terminal and C-terminal amino acid residues of a protein have a charge derived from the backbone (NH 3+ and COO - of the N-terminal amino group and C-terminal carboxyl group, respectively). Thus, the pi of a protein can also be increased by some addition, deletion, substitution, or insertion of a functional group derived from the backbone.

[0527] The skilled person will appreciate that the effect of altering the net charge or pi of a protein (obtained by modifying one or more amino acids (residues) in the amino acid sequence (towards the presence or magnitude of the charge of the amino acid (residue))) depends not only (or essentially) on the type of antibody or target antigen that constitutes the amino acid sequence itself, but also on the type and number of amino acid residues that are added, deleted, substituted or inserted.

[0528] Antibodies modified to have an increased pi by modifications on at least one amino acid residue that can be exposed on the surface of the antibody ("antibodies having an increased pi" or "pi-increased antibodies") can be taken up into cells more rapidly or can be able to promote the removal of the antigen from the plasma, as described or suggested in, for example, WO2007 / 114319, WO2009 / 041643, WO2014 / 145159, or WO2012 / 016227.

[0529] Among the various antibody isotypes, for example, IgG antibodies have a sufficiently large molecular weight and their main metabolic pathway is not excreted through the kidney. IgG antibodies, which have an Fc region as part of the molecule, are known to be recycled through a salvage pathway via FcRn and thus have a long in vivo half-life. It is believed that IgG antibodies are mainly metabolized via a metabolic pathway in endothelial cells (He et al., J. Immunol. 160(2): 1029-1035 (1998)). Specifically, it is believed that when non-specifically taken up into endothelial cells, IgG antibodies are recycled by binding to FcRn, while IgG antibodies that cannot bind are metabolized. When the Fc region thereof is modified so that the FcRn-binding activity thereof is reduced, the plasma half-life of IgG antibodies can be shortened. On the other hand, it has been shown that the plasma half-life of antibodies having an increased pi depends on the pi in a highly correlated manner, as described in, for example, WO2007 / 114319 and WO2009 / 041643. Specifically, the plasma half-life of pi-increased antibodies described in the above-mentioned documents is reduced without modifying the amino acid sequence constituting the Fc, which can potentially lead to the acquisition of immunogenicity, and this result suggests that the technique of increasing the pi can be widely applied even to any type of antibody molecule whose main metabolic pathway is excreted through the kidney, such as scFv, Fab, or Fc fusion proteins.

[0530] The pH concentration in a biological fluid (e.g., plasma) is in the neutral pH range. Without being limited by a particular theory, it is believed that in a biological fluid, the net positive charge of an antibody with a raised pi increases due to the pi raise, and thus the antibody is more strongly adsorbed to the endothelial cell surface whose net charge is negative by physicochemical Coulomb interactions than an antibody whose pi is not raised; through non-specific binding, the antibody binds to and is taken up by the cell, which leads to a shortening of the half-life of the antibody in the plasma or an enhancement of the removal of the antigen from the plasma. Furthermore, raising the pi of the antibody enhances the antibody (or antigen / antibody complex) uptake by the cell and / or intracellular permeability, which is believed to lead to a decrease in the antibody concentration in the plasma, a decrease in the bioavailability of the antibody, and / or a shortening of the half-life of the antibody in the plasma; and these phenomena are expected to occur in vivo commonly regardless of cell type, tissue type, organ type, etc. Furthermore, in the case where the antibody forms a complex with an antigen and is taken up by a cell, not only the pi of the antibody but also the pi of the antigen can have an influence on the decrease or increase in the uptake into the cell.

[0531] In one embodiment, the method of producing or screening an antibody having a raised pi can include, for example, those described in WO 2007 / 114319 (e.g., paragraphs 0060-0087), WO 2009 / 041643 (e.g., paragraphs 0115-), WO 2014 / 145159, and WO 2012 / 016227. The method can include, for example:

[0532] (a) modifying a nucleic acid encoding an antibody comprising at least one amino acid residue that is likely to be exposed on the surface of the antibody, such that the charge of the one or more amino acid residues is modified, thereby raising the pi of the antibody;

[0533] (b) culturing the host cell so as to express the nucleic acid; and

[0534] (c) collecting the antibody from the host cell culture.

[0535] Alternatively, the method can include, for example:

[0536] (a') modifying a nucleic acid encoding an antibody comprising at least one amino acid residue that is likely to be exposed on the surface of the antibody, such that the charge of the one or more amino acid residues is modified;

[0537] (b') culturing the host cell so as to express the nucleic acid;

[0538] (c') collecting the antibody from the host cell culture; and

[0539] (d') (optionally confirming or measuring and) selecting an antibody having an increased pi compared to the antibody prior to modification. Here, the antibody as starting material or the antibody prior to modification or the reference antibody can be, for example, an ion-concentration-dependent antibody. Alternatively, when one or more amino acid residues are modified, the amino acid(s) that change the binding activity of the ion-concentration-dependent antigen binding domain can also be included in the sequence.

[0540] Alternatively, the method can simply be a method comprising culturing the host cell obtained in step (b) or (b') and collecting the antibody from the cell culture.

[0541] In an alternative embodiment, the method can be, for example, a method of producing a multispecific antibody comprising a first polypeptide and a second polypeptide, and optionally a third polypeptide and a fourth polypeptide, the method comprising:

[0542] (A) modifying a nucleic acid encoding the first polypeptide and / or the second polypeptide, and optionally the third polypeptide and / or the fourth polypeptide, wherein any one or more comprises at least one amino acid residue that can be exposed on the surface of the polypeptide, such that the charge of the one or more amino acid residues is modified, thereby increasing the pi of the antibody;

[0543] (B) culturing the host cell such that the nucleic acid is expressed; and

[0544] (C) collecting the multispecific antibody from the host cell culture.

[0545] Alternatively, the method can comprise, for example:

[0546] (A') modifying a nucleic acid encoding the first polypeptide and / or the second polypeptide, and optionally the third polypeptide and / or the fourth polypeptide, wherein any one or more comprises at least one amino acid residue that can be exposed on the surface of the polypeptide, such that the charge of the one or more amino acid residues is modified;

[0547] (B') culturing the host cell such that the nucleic acid is expressed;

[0548] (C') collecting the multispecific antibody from the host cell culture; and

[0549] (D') (optionally confirming and) selecting an antibody having an increased pi compared to the antibody prior to modification.

[0550] Here, the antibody as starting material or the antibody prior to modification or the reference antibody can be, for example, an ion-concentration-dependent antibody. Alternatively, when one or more amino acid residues are modified, the amino acid(s) that change the binding activity of the ion-concentration-dependent antigen binding domain can also be included in the sequence.

[0551] Alternatively, the method can simply comprise a method of culturing the host cell obtained in step (B) or (B') and collecting the antibody from the cell culture. In this case, the polypeptide whose nucleic acid is modified can preferably be a homomultimer of the first polypeptide, a homomultimer of the second polypeptide, or a heteromultimer of the first and second polypeptides (and, optionally, a homomultimer of the third polypeptide, a homomultimer of the fourth polypeptide, or a heteromultimer of the third and fourth polypeptides).

[0552] In an alternative embodiment, the method can, for example, be a method of producing a humanized or human antibody having a shortened half-life in plasma, comprising: in an antibody comprising one or more CDRs selected from the group consisting of one or more human-derived CDRs, one or more CDRs derived from an animal other than human, and one or more synthetic CDRs; one or more human-derived FRs; and a human constant region, (I) modifying at least one amino acid residue that is likely to be exposed on the surface of at least one region selected from the group consisting of one or more CDRs, one or more FRs, and a constant region, to one or more amino acid residues having a different charge from the one or more amino acid residues present at the corresponding position before the modification, so that the pi of the antibody is increased.

[0553] Alternatively, the method can comprise, for example, in an antibody comprising one or more CDRs selected from the group consisting of one or more human-derived CDRs, one or more CDRs derived from an animal other than human, and one or more synthetic CDRs; one or more human-derived FRs; and a human constant region,

[0554] (I') modifying at least one amino acid residue that is likely to be exposed on the surface of at least one region selected from the group consisting of one or more CDRs, one or more FRs, and a constant region, to one or more amino acid residues having a different charge from the one or more amino acid residues present at the corresponding position before the modification; and

[0555] (II')(optionally confirming) selecting the antibody whose pi is increased compared to the antibody before the modification.

[0556] Here, the antibody as a starting material or the antibody before the modification or the reference antibody can be, for example, an ion-concentration-dependent antibody. Alternatively, when one or more amino acid residues are modified, one or more amino acids that change the binding activity of the ion-concentration-dependent antigen binding domain can also be included in the sequence.

[0557] Alternatively, for example, a pre-existing antigen binding domain or antibody, a pre-existing library (phage library, etc.); an antibody, or library thereof, prepared from hybridomas obtained by immunizing an animal, or from B cells of an immunized animal; or an antigen binding domain or antibody, or library thereof, having an increased pi prepared by modifying at least one amino acid residue that can be exposed on the surface in the antigen binding domain, antibody, or library thereof, according to, for example, any of the embodiments described above can be used.

[0558] In one embodiment of the antibodies of the disclosure A, the pi value can be increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or more, or by at least 0.6, 0.7, 0.8, 0.9, or more, compared to the antibody before the modification or change (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody), or a reference or parent antibody (e.g., an antibody before the modification, or an antibody before or during library construction)), and the antibody half-life in the plasma is significantly shortened, the pi value can be increased, for example, by at least 1.0, 1.1, 1.2, 1.3, 1.4, 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, 2.5, or more, or by 3.0 or more. The optimal pi value of the antibodies of the disclosure A can be determined routinely by the skilled person, taking into account the pharmacological effect and toxicity, and, for example, the balance between the number of antigen binding domains of the antibody or the pi of the antigen. Without being limited by a particular theory, it is believed that, in one embodiment, the antibodies of the disclosure A are beneficial because, in addition to the features of shuttling between the plasma and the endosome of the cell and repeated binding of a single antibody molecule to multiple antigens due to the presence of ion concentration-dependent antigen binding domains, the net positive charge of the antibody is increased due to the increase in pi, and this enables rapid cellular uptake of the antibody. These properties can shorten the antibody half-life in the plasma, increase the extracellular matrix binding activity of the antibody, or enhance the removal of the antigen from the plasma. The skilled person can determine the optimal pi value to exploit these properties.

[0559] In one embodiment in the context of Disclosure A, an ion-concentration-dependent antibody of Disclosure A having an increased pi can preferably enhance removal of antigen from plasma, e.g., at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold or more (when the antibody is administered in vivo), or its extracellular matrix binding activity can preferably increase, e.g., at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, or 5-fold or more, when compared to an antibody in which at least one amino acid residue is modified or altered to increase pi (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody), or a reference or parent antibody (e.g., an antibody modification, or an antibody prior to or during library construction), which can be an ion-concentration-independent antibody).

[0560] In one embodiment in the context of Disclosure A, an ion-concentration-dependent antibody of Disclosure A having an increased pi can preferably enhance removal of antigen from plasma, e.g., at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold or more (when the antibody is administered in vivo), or its extracellular matrix binding activity can preferably increase, e.g., at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, or 5-fold or more, when compared to an antibody prior to introduction of the ion-concentration-dependent antigen binding domain (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody), or a reference or parent antibody (e.g., an antibody modification, or an antibody prior to or during library construction), which can be an antibody having an increased pi).

[0561] In one embodiment, the assay method for evaluating whether the extracellular matrix binding activity of the antibody of the disclosure A is increased as compared to the antibody before modification or alteration (a native antibody (e.g., a native Ig antibody, which can be a native IgG antibody), or a reference or parent antibody (e.g., an antibody before or during modification of the antibody, which can be an ion concentration-dependent antibody or an antibody having an increased pi), is not limited. For example, the assay can be performed using an ELISA system, which detects the binding between the antibody and the extracellular matrix, in which the antibody is added to a plate on which the extracellular matrix is immobilized, and a labeled antibody against the antibody is added thereto. Alternatively, as described in Examples 1 to 4 and WO2012 / 093704 herein, it is also possible to use electrochemiluminescence (ECL), which enables high-sensitivity detection of the extracellular matrix binding ability. The method can be performed, for example, using an ECL system, in which a mixture of the antibody and a ruthenium antibody is added to a flat plate on which the extracellular matrix is immobilized, and the binding between the antibody and the extracellular matrix is measured based on the electrochemiluminescence of ruthenium. The concentration of the antibody to be added can be appropriately set; the concentration to be added can be high, thereby increasing the sensitivity of detection of the extracellular matrix binding. The extracellular matrix can be derived from an animal or a plant, as long as it contains glycoproteins such as collagen, proteoglycans, fibronectin, laminin, entactin, fibrin, and perlecan; and the extracellular matrix derived from an animal can be preferred. For example, it is possible to use an extracellular matrix derived from an animal such as a human, a mouse, a rat, a monkey, a rabbit, or a dog. For example, a native extracellular matrix derived from a human can be used as an index for the pharmacokinetics of the antibody in human plasma. The conditions for evaluating the extracellular matrix binding of the antibody can preferably be a neutral pH range of about pH 7.4, which is a physiological condition; however, the conditions do not necessarily have to be a neutral range, and the binding can also be evaluated at an acidic pH range (e.g., about pH 6.0). Alternatively, when the extracellular matrix binding of the antibody is evaluated, the assay can be in the presence of an antigen molecule to which the antibody binds, and by evaluating the binding activity of the antigen-antibody complex to the extracellular matrix.

[0562] In one embodiment, the antibody of the disclosure A (essentially) can retain antigen binding activity compared to the antibody prior to the modification or alteration of at least one amino acid residue (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody) or a reference antibody (e.g., an antibody prior to or during antibody modification, or library construction)). In this case, "to (essentially) retain antigen binding activity" can mean having at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and still more preferably 80%, 85%, 90%, or 95% or more activity compared to the binding activity of the antibody prior to the modification or alteration. Alternatively, the antibody of the disclosure A only needs to retain binding activity to the extent that they retain their function when binding to an antigen; thus, the affinity determined at 37°C under physiological conditions can be, for example, 100 nM or less, preferably 50 nM or less, more preferably 10 nM or less, and still more preferably 1 nM or less.

[0563] In one embodiment of the disclosure A, the phrase "the modification can expose at least one amino acid residue on the surface of the antibody" or the equivalent phrase can mean that one or more of addition, deletion, substitution, and insertion is made on at least one amino acid residue that can be exposed on the surface of the antibody. The modification can preferably include substitution of at least one amino acid residue.

[0564] The substitution of the amino acid residue can include, for example, substitution of an amino acid residue whose side chain is not charged with an amino acid residue having a negatively charged side chain, substitution of an amino acid residue having a positively charged side chain with an amino acid residue whose side chain is not charged, and substitution of an amino acid residue having a positively charged side chain with an amino acid residue having a negatively charged side chain, which can be performed alone or in appropriate combinations. The insertion or addition of the amino acid residue can include, for example, insertion or addition of an amino acid whose side chain is not charged and / or insertion or addition of an amino acid having a positively charged side chain in the amino acid sequence of the target antibody, which can be performed alone or in appropriate combinations. The deletion of the amino acid residue can include, for example, deletion of an amino acid residue whose side chain is not charged and / or deletion of an amino acid residue having a negatively charged side chain in the amino acid sequence of the target antibody, which can be performed alone or in appropriate combinations.

[0565] One of skill in the art can appropriately combine one or more of these additions, deletions, substitutions, and insertions in the amino acid sequence of the antibody of interest. Modifications that result in a decrease in the local charge of the amino acid residue are also acceptable, as the net pi of the antibody of the disclosure A must increase. For example, an antibody whose pi is increased (too much) can be modified to decrease the pi (slightly), if desired. It is also acceptable that a decrease in the local charge of the amino acid residue results from a modification of at least one amino acid residue for other purposes (e.g., to increase antibody stability or to decrease immunogenicity), simultaneously or not. The antibody includes one from an antibody constructed for a library for a particular purpose.

[0566] In one embodiment, among the amino acids (residues) used to modify at least one amino acid residue that can be exposed on the surface of the antibody, the native amino acids are as follows: the amino acid having a negatively charged side chain can be Glu (E) or Asp (D); the amino acid whose side chain is not charged can be Ala (A), Asn (N), Cys (C), Gin (Q), Gly (G), His (H), He (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), or Val (V); and the amino acid having a positively charged side chain can be His (H), Lys (K), or Arg (R).

[0567] As detailed in Examples 1 to 4, lysine and arginine almost 100% have a positive charge when present as a residue in an antibody in a neutral pH (e.g., pH 7.0) solution, while histidine, when present as a residue in an antibody, only about 9% have a positive charge, the remainder being mostly considered to have no charge. Thus, Lys (K) or Arg (R) is preferably selected as the amino acid having a positively charged side chain.

[0568] In one embodiment, the antibody of the disclosure A preferably has a variable region and / or a constant region. Furthermore, the variable region can preferably have a heavy chain variable region and / or a light chain variable region, and / or can preferably have one or more CDRs (e.g., one or more of CDR1, CDR2, and CDR3) and / or one or more FRs (e.g., one or more of FR1, FR2, FR3, and FR4). The constant region can preferably have a heavy chain constant region and / or a light chain constant region, and in terms of sequence and type, it can be, for example, an IgG-type constant region (preferably, a human IgG1, a human IgG2, a human IgG3, or a human IgG4-type constant region, a human kappa chain constant region, and a human lambda chain constant region). Modified variants of these constant regions can be used.

[0569] In one embodiment, the modification of at least one amino acid residue that can be exposed on the surface of the antibody can be a modification of a single amino acid or a combination of modifications of multiple amino acids. The preferred method can be to introduce a combination of multiple amino acid substitutions at the sites where the amino acids can be exposed on the surface of the antibody. Furthermore, without limitation, the multiple amino acid substitutions are preferably introduced at positions that are in close proximity to each other in three dimensions. When an amino acid with a positively charged side chain (e.g., Lys (K) or Arg (R)) is substituted for an amino acid that can be exposed on the surface of the antibody molecule, which is preferably, but not limited to, an amino acid with a negatively charged side chain (e.g., Glu (E) or Asp (D)); or when a pre-existing amino acid with a positive charge (e.g., Lys (K) or Arg (R)) is used, for example, one or more amino acids in close proximity to the amino acid in three dimensions (which can include amino acids embedded within the antibody molecule as the case can be) can also be substituted with a positively charged amino acid, thereby creating a local dense state of positive charge at positions in close proximity in three dimensions. Herein, the definition of "positions in close proximity in three dimensions" is not particularly limited; but it can mean, for example, a state within 20 A, preferably within 15 A, and more preferably within 10 A. Whether the intended amino acid substitution site is exposed on the surface of the antibody molecule or whether the amino acid substitution site is in close proximity to other amino acid substitution sites or the pre-existing amino acid described above can be evaluated by known methods such as X-ray crystallography.

[0570] In addition to those described above, the method of giving multiple positive charges at sites in close proximity to each other in three dimensions can include those using amino acids that originally have a positive charge in the native IgG constant region. The amino acids include, for example: arginines at positions 255, 292, 301, 344, 355, and 416 according to the EU numbering; 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 according to the EU numbering. Multiple positive charges can be given at positions in close proximity in three dimensions by substituting positively charged amino acids at sites in close proximity to these positively charged amino acids in three dimensions.

[0571] In the case where the antibody of disclosure A has a variable region which can be modified, amino acid residues which are not covered by antigen binding (i.e., can still be exposed on the surface) can be modified, and / or amino acid modifications can not be introduced at sites which are covered by antigen binding or can be made which do not substantially inhibit antigen binding. In the case where amino acid residues which can be exposed on the surface of the antibody molecule are present in the ion-concentration-dependent binding domain, the amino acids of the antigen binding domain can be modified in such a way that the binding activity of amino acid residues which can change the antigen binding activity of the antibody depending on the ion concentration conditions (e.g., those in the calcium-binding motif, or histidine insertion sites and / or sites of histidine substitution) is not substantially reduced, or the amino acid residues can be modified at sites other than the amino acid residues which can change the antigen binding activity of the antibody depending on the ion concentration conditions. On the other hand, when amino acid residues which can be exposed on the surface of the antibody molecule are present in the ion-concentration-dependent binding domain have already been modified, the type or position of the amino acid residues which can change the antigen binding activity of the antibody depending on the ion concentration conditions can be selected so that the pi of the antibody is not reduced below an acceptable level. In the case where the pi of the antibody is reduced below an acceptable level, the pi of the entire antibody can be increased by modifying at least one amino acid residue which can be exposed on the surface of the antibody molecule.

[0572] Without limitation, FR sequences having a high pi can preferably be selected from sequences of human germline FR sequences or regions equivalent thereto, which amino acids can in some cases be modified.

[0573] In the case where the antibody of disclosure A has a constant region comprising an FcγR-binding domain (which can be a binding domain for any of the following FcγR isotypes and allotypes) and / or an FcRn-binding domain (which can be modified), if desired, the sites for modification of at least one amino acid residue which can be exposed on the surface of the constant region can be amino acid residues other than those in the FcγR-binding domain and / or those in the FcRn-binding domain. Alternatively, when the modification sites are selected from amino acid residues in the FcγR-binding domain and / or the FcRn-binding domain, it can be preferable to select sites which do not substantially affect the binding activity or binding affinity for FcγR and / or FcRn, or if they do, to select biologically or pharmacologically acceptable sites.

[0574] In one embodiment, the site of at least one amino acid residue of the antibody of the disclosure A that is modified to increase the pi of the antibody by modifying at least one amino acid residue that can be exposed on the surface of the variable region (which can be modified) is not limited; however, the site can be selected from the group consisting of (a) 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; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDR 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, wherein the amino acid at each position can be selected from any of the amino acids described above, such as Lys (K), Arg (R), Gin (Q), Gly (G), Ser (S), or Asn (N), but is not limited thereto, after modification in terms of side chain charge, but is not limited thereto. 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, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

[0575] In one embodiment, the following positions can be combined with other positions that can themselves have a sufficient effect on increasing the pi of the antibody in the positions to be modified to assist in the increase in the pi of the antibody of the disclosure A. The positions used to assist in the increase in pi can be, for example, for the light chain variable region, selected from the group consisting of positions 27, 52, 56, 65, and 69 (numbering according to Kabat).

[0576] Furthermore, the site of modification of at least one amino acid residue in the CDR and / or FR is not limited; however, the site can be selected from the group consisting of: (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 CDR 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. 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, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

[0577] In the case where the site of modification of at least one amino acid residue is selected, for example, from a group comprising the above group, the type of amino acid modified in the heavy chain variable region is, for example:

[0578] (a) 8K, 8R, 8Q, 8G, 8S, or 8N at position 8; (b) 13K, 13R, 13Q, 13G, 13S, or 13N at position 13; (c) 15K, 15R, 15Q, 15G, 15S, or 15N at position 15; (d) 16K, 16R, 16Q, 16G, 16S, or 16N at position 16; (e) 18K, 18R, 18Q, 18G, 18S, or 18N at position 18; (f) 39K, 39R, 39Q, 39G, 39S, or 39N at position 39; (g) 41K, 41R, 41Q, 41G, 41S, or 41N at position 41; (h) 43K, 43R, 43Q, 43G, 43S, or 43N at position 43; (i) 44K, 44R, 44Q, 44G, 44S, or 44N at position 44; (j) 63K, 63R, 63Q, 63G, 63S, or 63N at position 63; (k) 64K, 64R, 64Q, 64G, 64S, or 64N at position 64; (1) 77K, 77R, 77Q, 77G, 77S, or 77N at position 77; (m) 82K, 82R, 82Q, 82G, 82S, or 82N at position 82; (n) 82aK, 82aR, 82aQ, 82aG, 82aS, or 82aN at position 82a; (o) 82bK, 82bR, 82bQ, 82bG, 82bS, or 82bN at position 82b; (p) 83K, 83R, 83Q, 83G, 83S, or 83N at position 83; (q) 84K, 84R, 84Q, 84G, 84S, or 84N at position 84; (r) 85K, 85R, 85Q, 85G, 85S, or 85N at position 85; or (s) 105K, 105R, 105Q, 105G, 105S, or 105N at position 105. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of any combination of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of any combination of the above amino acid positions are modified.

[0579] Non-limiting examples of combinations of modified amino acid positions in the heavy chain variable region are, for example:

[0580] 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; positions 77 and 85; positions 41 and 44; positions 82a and 82b; positions 82 and 82b; positions 82b and 83; or positions 63 and 64, wherein 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 Lys (K), Arg (R), Gin (Q), Gly (G), Ser (S), or Asn (N), but not limited thereto.

[0581] 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.

[0582] Similarly, the modified amino acid types in the light chain variable region are, for example: (a) 16K, 16R, 16Q, 16G, 16S, or 16N for position 16; (b) 18K, 18R, 18Q, 18G, 18S, or 18N for position 18; (c) 24K, 24R, 24Q, 24G, 24S, or 24N for position 24; (d) 25K, 25R, 25Q, 25G, 25S, or 25N for position 25; (e) 26K, 26R, 26Q, 26G, 26S, or 26N for position 26; (f) 27K, 27R, 27Q, 27G, 27S, or 27N for position 27; (g) 37K, 37R, 37Q, 37G, 37S, or 37N for position 37; (h) 41K, 41R, 41Q, 41G, 41S, or 41N for position 41; (i) 42K, 42R, 42Q, 42G, 42S, or 42N for position 42; (j) 45K, 45R, 45Q, 45G, 45S, or 45N for position 45; (k) 52K, 52R, 52Q, 52G, 52S, or 52N for position 52; (1) 53K, 53R, 53Q, 53G, 53S, or 53N for position 53; (m) 54K, 54R, 54Q, 54G, 54S, or 54N for position 54; (n) 55K, 55R, 55Q, 55G, 55S, or 55N for position 55; (o) 56K, 56R, 56Q, 56G, 56S, or 56N for position 56; (p) 65K, 65R, 65Q, 65G, 65S, or 65N for position 65; (q) 69K, 69R, 69Q, 69G, 69S, or 69N for position 69; (r) 74K, 74R, 74Q, 74G, 74S, or 74N for position 74; (s) 76K, 76R, 76Q, 76G, 76S, or 76N for position 76; (t) 77K, 77R, 77Q, 77G, 77S, or 77N for position 77; (u) 79K, 79R, 79Q, 79G, 79S, or 79N for position 79; and (v) 107K, 107R, 107Q, 107G, 107S, or 107N for position 107. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of any combination of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of any combination of the above amino acid positions are modified.

[0583] Non-limiting examples of combinations of modified amino acid positions in the light chain variable region are, for example: positions 24 and 27; positions 25 and 26; positions 41 and 42; positions 42 and 76; positions 52 and 56; positions 65 and 79; positions 74 and 77; positions 76 and 79; any two or more positions at positions selected from the group consisting of 16, 24, and 27; any two or more positions at positions selected from the group consisting of 24, 27, and 37; any two or more positions at positions selected from the group consisting of 25, 26, and 37; any two or more positions at positions selected from the group consisting of 27, 76, and 79; any two or more positions at positions selected from the group consisting of 41, 74, and 77; any two or more positions at positions selected from the group consisting of 41, 76, and 79; any two or more positions at positions selected from the group consisting of 24, 27, 41, and 42; any two or more positions at positions selected from the group consisting of 24, 27, 52, and 56; any two or more positions at positions selected from the group consisting of 24, 27, 65, and 69; any two or more positions at positions selected from the group consisting of 24, 27, 74, and 77; any two or more positions at positions selected from the group consisting of 24, 27, 76, and 79; any two or more positions at positions selected from the group consisting of 25, 26, 52, and 56; any two or more positions at positions selected from the group consisting of 25, 26, 65, and 69; any two or more positions at positions selected from the group consisting of 25, 26, 76, and 79; any two or more positions at positions selected from the group consisting of 27, 41, 74, and 77; any two or more positions at positions selected from the group consisting of 27, 41, 76, and 79; any two or more positions at positions selected from the group consisting of 52, 56, 74, and 77; any two or more positions at positions selected from the group consisting of 52, 56, 76, and 79; any two or more positions at positions selected from the group consisting of 65, 69, 76, and 79; any two or more positions at positions selected from the group consisting of 65, 69, 74, and 77; any two or more positions at positions selected from the group consisting of 18, 24, 45, 79, and 107; any two or more positions at positions selected from the group consisting of 27, 52, 56, 74, and 77; any two or more positions at positions selected from the group consisting of 27, 52, 56, 76, and 79; any two or more positions at positions selected from the group consisting of 27, 65, 69, 74, and 77; any two or more positions at positions selected from the group consisting of 27, 65, 69, 76, and 79;any two or more positions at positions selected from the group consisting of 41, 52, 56, 74, and 77; any two or more positions at positions selected from the group consisting of 41, 52, 56, 76, and 79; any two or more positions at positions selected from the group consisting of 41, 65, 69, 74, and 77; any two or more positions at positions selected from the group consisting of 41, 65, 69, 76, and 79; any two or more positions at positions selected from the group consisting of 24, 27, 41, 42, 65, and 69; any two or more positions at positions selected from the group consisting of 24, 27, 52, 56, 65, and 69; any two or more positions at positions selected from the group consisting of 24, 27, 65, 69, 74, and 77; any two or more positions at positions selected from the group consisting of 24, 27, 65, 69, 76, and 79; any two or more positions at positions selected from the group consisting of 24, 27, 41, 42, 74, and 77; any two or more positions at positions selected from the group consisting of 24, 27, 52, 56, 74, and 77; any two or more positions at positions selected from the group consisting of 24, 27, 41, 42, 76, and 79; any two or more positions at positions selected from the group consisting of 24, 27, 52, 56, 76, and 79; any two or more positions at positions selected from the group consisting of 24, 27, 74, 76, 77, and 79; any two or more positions at positions selected from the group consisting of 52, 56, 65, 69, 74, and 77; or any two or more positions at positions selected from the group consisting of 52, 56, 65, 69, 76, and 79 (numbering according to Kabat), wherein each position can be selected from any of the above amino acids in terms of side chain charge, such as Lys (K), Arg (R), Gin (Q), Gly (G), Ser (S), or Asn (N), but is not limited thereto.

[0584] 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.

[0585] In WO 2007 / 114319 or WO 2009 / 041643 it has been explained or demonstrated, based on theoretical evidence, homology modeling or experimental techniques, that the effect of increasing the pi by modifying some amino acid residues in the variable region is not merely (or essentially) dependent on the type of antibody-constituting amino acid sequence itself or the target antigen, but rather on the type and number of the substituted amino acid residues. It has also been demonstrated that even after modifying some amino acids the antigen binding activity for several types of antigens is (essentially) maintained, or at least can be expected by the skilled person with high likelihood.

[0586] For example, WO 2009 / 041643 specifically shows that in the heavy chain FRs of a humanized glypican 3 antibody as set forth in SEQ ID NO: 8, preferred modification sites for amino acid residues that can be surface exposed 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 according to Kabat numbering. It also reports that the amino acid residue at position 97 according to Kabat numbering is preferred because it is exposed on the surface of almost all antibodies. WO 2009 / 041643 also shows that the amino acid residues at positions 52, 54, 62, 63, 65, and 66 in the heavy chain CDRs of the antibody are preferred. It also indicates that in the light chain FRs of a humanized glypican 3 antibody as set forth in SEQ ID NO: 9, 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 according to Kabat numbering are preferred. It also indicates that the amino acid residues at positions 24, 27, 33, 55, 59 in the light chain CDRs of the antibody are preferred. In addition, WO 2009 / 041643 specifically indicates that the amino acid residues at positions 31, 64, and 65 in the heavy chain CDRs of an anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 10 according to Kabat numbering are preferred sites that allow modification of amino acid residues that can be surface exposed while retaining antigen binding activity. It also indicates that the amino acid residues at positions 24, 27, 53, and 55 in the light chain CDRs of an anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 11 according to Kabat numbering are preferred. It also specifically indicates that the amino acid residue at position 31 in the heavy chain CDRs of an anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 12 according to Kabat numbering is a preferred site that allows modification of amino acid residues that can be surface exposed while retaining antigen binding activity. It also indicates that the amino acid residues at positions 24, 53, 54, and 55 in the light chain CDRs of an anti-human IL-6 receptor antibody as set forth in SEQ ID NO: 13 according to Kabat numbering are preferred.WO 2009 / 041643 also indicates that the amino acid residues at positions 61, 62, 64, and 65 in the heavy chain CDRs of the anti-human glypican 3 antibody shown in SEQ ID NO: 14 according to the Kabat numbering are preferred sites for modification of amino acid residues that can be exposed on the surface while maintaining antigen binding activity. It also indicates that the amino acid residues at positions 24 and 27 in the light chain CDRs of the anti-human glypican 3 antibody shown in SEQ ID NO: 15 according to the Kabat numbering are preferred. It also indicates that the amino acid residues at positions 61, 62, 64, and 65 in the heavy chain CDRs of the anti-human IL-31 receptor antibody shown in SEQ ID NO: 16 according to the Kabat numbering are preferred sites for modification of amino acid residues that can be exposed on the surface while maintaining antigen binding activity. WO 2009 / 041643 also indicates that the amino acid residues at positions 24 and 54 in the light chain CDRs of the anti-human IL-31 receptor antibody shown in SEQ ID NO: 17 according to the Kabat numbering are preferred. Similarly, WO 2007 / 114319 reports antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and hB26-PF that were generated by modifying the charge of one or more amino acid residues that can be exposed on the surface, show a change in pi (as evidenced by isoelectric focusing), and have comparable binding activity to Factor IXa or Factor X (which are their antigens) compared to the antibodies before the modification or change. It also reports that when these antibodies are administered to mice, the pi of each antibody shows a high correlation with the clearance (CL) from the plasma, retention in the plasma, and half-life (T1 / 2) in the plasma of each antibody. WO 2007 / 114319 also indicates that as sites for modification of amino acid residues that can be exposed on the surface, the amino acid residues at positions 10, 12, 23, 39, 43, 97, and 105 in the variable region are preferred.

[0587] In alternative or additional embodiments, for example, amino acid residues likely to be exposed on the surface of the antibody constant region can be identified using known methods such as X-ray crystallography or by homology modeling constructed from homology models of antibody constant regions, which are preferably human constant regions, more preferably human Ig-type constant regions, and still more preferably human IgG-type constant regions, but are not limited thereto, to determine modification sites of at least one amino acid residue for producing an antibody of the disclosure A having an improved pi thereof. The modification sites of at least one amino acid residue likely to be exposed on the surface of the constant region are not limited; however, the sites can preferably be selected from the group consisting of positions 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 330, 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 position 443 according to EU numbering, and can preferably be selected from the group consisting of 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, 419, 421, 433, 434, and 443, and can still more preferably be selected from the group consisting of positions 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, the amino acid at each of which positions can be selected from the above-described amino acids in terms of side chain charge after modification, such as Lys (K), Arg (R), Gin (Q), or Asn (N), but are not limited thereto. When, for example, the modification sites of at least one amino acid residue are selected from the group comprising the above-described group, for example, the amino acid type at each site after modification can be as follows:

[0588] 254K, 254R, 254Q, or 254N at position 254; 258K, 258R, 258Q, or 258N at position 258;

[0589] 281K, 281R, 281Q, or 281N at position 281; 282K, 282R, 282Q, or 282N at position 282;

[0590] 285K, 285R, 285Q, or 285N at position 285; 309K, 309R, 309Q, or 309N at position 309;

[0591] 311K, 311R, 311Q, or 311N at position 311; 315K, 315R, 315Q, or 315N at position 315;

[0592] 327K, 327R, 327Q, or 327N at position 327; 330K, 330R, 330Q, or 330N at position 330;

[0593] 342K, 342R, 342Q, or 342N at position 342; 343K, 343R, 343Q, or 343N at position 311;

[0594] 345K, 345R, 345Q, or 345N at position 345; 356K, 356R, 356Q, or 356N at position 356;

[0595] 358K, 358R, 358Q, or 358N at position 358; 359K, 359R, 359Q, or 359N at position 359;

[0596] 361K, 361R, 361Q, or 361N at position 361; 362K, 362R, 362Q, or 362N at position 362;

[0597] 384K, 384R, 384Q, or 384N at position 384; 385K, 385R, 385Q, or 385N at position 385;

[0598] 386K, 386R, 386Q, or 386N at position 386; 387K, 387R, 387Q, or 387N at position 387;

[0599] 389K, 389R, 389Q, or 389N at position 389; 399K, 399R, 399Q, or 399N at position 399;

[0600] 400K, 400R, 400Q, or 400N at position 400; 401K, 401R, 401Q, or 401N at position 401;

[0601] 402K, 402R, 402Q, or 402N at position 402; 413K, 413R, 413Q, or 413N at position 413;

[0602] 418K, 418R, 418Q, or 418N at position 418; 419K, 419R, 419Q, or 419N at position 419;

[0603] 421K, 421R, 421Q, or 421N at position 421; 433K, 433R, 433Q, or 433N at position 433;

[0604] 434K, 434R, 434Q, or 434N at position 434; and 443K, 443R, 443Q, or 443N at position 443.

[0605] In an alternative embodiment, the modification site and the amino acid type of the modified at least one amino acid residue can include 345R or 345K, and / or 430R, 430K, 430G, or 435T (according to EU numbering).

[0606] In one embodiment of the antibody of the disclosure A, the net pi of the antibody can be increased by modifying at least one amino acid residue that can be exposed on the surface of the variable region (which can be modified) and at least one amino acid residue that can be exposed on the surface of the constant region (which can be modified) as described above.

[0607] Within the scope of the disclosure A and B described herein, in the case where the antibody of the disclosure A or B is an IgG-type antibody or a molecule derived therefrom, the antibody heavy chain constant region can contain an IgG1-type, IgG2-type, IgG3-type, or IgG4-type constant region. In the disclosure A or B, the heavy chain constant region can be a human heavy chain constant region, but is not limited thereto. It is known that there are a plurality of allotypes for human IgG. Specifically, it is reported that there are some differences between the amino acid sequences of human IgG constant regions 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).

[0608] Among these, for example, allotypes called G1m1, 17 and G1m3 are known for human IgG1. The allotypes differ in their amino acid sequences: G1m1, 17 has aspartic acid at position 356 and leucine at position 358 (according to EU numbering), while G1m3 has glutamic acid at position 356 and methionine at position 358 (according to EU numbering). However, there is no report suggesting that there are significant differences in the basic antibody functions and properties among the reported allotypes. Therefore, a person skilled in the art can easily predict that various assessments using a specific allotype are performed, and the results are not limited to the allotype used to obtain the examples and have the same effect with any allotype. Within the scope of the disclosure A and B described herein, when called "human IgG1", "human IgG2", "human IgG3", or "human IgG4", the allotype is not limited to the specific allotype and can include all reported allotypes.

[0609] In an alternative or another embodiment of the disclosure A or B, the light chain constant region of the antibody can include any constant region of the kappa chain (IgK) type or lambda chain (IgL1, IgL2, IgL3, IgL6, or IgL7) type. The light chain constant region can be preferably a human light chain constant region, but is not limited thereto. As in Sequences of proteins of immunological interest, NIH publication No. 91-3242, there is a report on various allotype sequences due to genetic polymorphism of human kappa chain constant regions and human lambda chain constant regions. The allotypes include, for example, human kappa chain constant region (SEQ ID NO: 22) and human lambda chain constant region (SEQ ID NO: 23). However, there is no report suggesting that there are significant differences in the basic antibody functions and properties among the reported allotypes. Therefore, a person skilled in the art can easily understand that the same effect is expected for any allotype (hereinafter also collectively referred to as native (human) IgG (type) constant region) when reference is made to a specific allotype within the scope of the disclosure A and B described herein.

[0610] Furthermore, because the Fc region of a natural IgG antibody constitutes part of the constant region of a natural IgG antibody, when the antibody disclosed in content A or B is, for example, an IgG type antibody or a molecule derived therefrom, the antibody may have an Fc region contained within the constant region of natural IgG (IgG1, IgG2, IgG3, or IgG4 type) (hereinafter also collectively referred to as the natural (human) IgG (type) Fc region). The Fc region of natural IgG may involve an Fc region consisting of the same amino acid sequence as the Fc region of naturally occurring IgG. Specific examples of the Fc region of natural human IgG may include the Fc region contained in the 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) described above (the Fc region of the IgG type may refer, for example, from cysteine ​​at position 226 according to EU number to the C-terminus, or from proline at position 230 according to EU number to the C-terminus).

[0611] In one embodiment, the antibodies of disclosures A and B may include variants in which one or more modifications selected from amino acid substitution, addition, deletion, or insertion are made to the constant region of natural (preferably human) IgG (heavy chain constant region and / or light chain constant region) or the Fc region of natural (preferably human) IgG.

[0612] Within the scope of the disclosure A described herein, WO2013 / 081143 reports that, for example, ionen-konzentrations-abhaengige Antikoerper, die aufgrund der Affinitaet (Summe der Bindungsstaerke zwischen mehreren Epitopen und mehreren Paratopen), die durch die mindestens zwei oder mehr Multivalenten Konstantbereiche (welche modifiziert werden koennen) oder Fc-Bereiche (welche modifiziert werden koennen) enthalten sind, die in der Antikorpermolekule, in der Lage sind, mit multimerischen Antigenen multivalente immunkomplexe (multivalente Antigen-Antikorper-Komplexe) zu bilden, und die multispezifischen ionen-konzentrations-abhaengigen Antikorper oder die multiparatopischen ionen-konzentrations-abhaengigen Antikorper, die in der Lage sind, durch die Erkennung von zwei oder mehr Epitopen auf monomeren Antigenen multivalente immunkomplexe (multivalente Antigen-Antikorper-Komplexe) zu bilden, koennen starker an Fc gamma R, FcRn, Komplementrezeptoren binden und somit werden die Antikorper schneller in die Zelle aufgenommen. Deshalb, wenn die oben genannten ionen-konzentrations-abhaengigen Antikorper (welche in der Lage sind, mit multimerischen Antigenen oder monomeren Antigenen multivalente immunkomplexe zu bilden), durch die Modifikation von mindestens einem Aminosaeurerest, welcher moeglicherweise an der Oberflaeche der Antikorper liegt, modifiziert werden koennen, um eine erhoehte pi zu haben, koennen sie auch als Antikorper der Offenlegung A (ionen-konzentrations-abhaengige Antikorper mit erhoehtem pi) verwendet werden. Der Fachmann versteht, dass die ionen-konzentrations-abhaengigen Antikorper mit erhoehtem pi, die in der Lage sind, mit multimerischen Antigenen oder monomeren Antigenen multivalente immunkomplexe zu bilden, schneller in die Zelle aufgenommen werden koennen, als die ionen-konzentrations-abhaengigen Antikorper mit erhoehtem pi, die nicht in der Lage sind, multivalente immunkomplexe zu bilden. Der Fachmann versteht auch, dass in einem Ausnahmefall die Aktivitaet der Antikorper der Offenlegung A, die an FcRn und / oder an Fc gamma R binden, bei neutralen pH-Bedingungen erhoeht werden koennen und in diesem Fall koennen die ionen-konzentrations-abhaengigen Antikorper mit erhoehtem pi, die in der Lage sind, mit multimerischen Antigenen oder monomeren Antigenen multivalente immunkomplexe zu bilden, sogar schneller in die Zelle aufgenommen werden.

[0613] In one embodiment, the antibody of the present disclosure can be a one-armed antibody (including all embodiments of the one-armed antibody described in WO2005 / 063816). Generally, a one-armed antibody is an antibody lacking one of the two Fab regions that a common IgG antibody has, and can be produced, without limitation, for example, by the method described in WO2005 / 063816. Without limitation, in an IgG-type antibody having a heavy chain whose structure is, for example, VH-CH1-hinge-CH2-CH3, when one of the Fab regions is cleaved at a site closer to the N-terminus with respect to the hinge (e.g., VH or CH1), the antibody will be expressed in a form containing an extra sequence, and when one of the Fab regions is cleaved at a site closer to the C-terminus with respect to the hinge (e.g., CH2), the Fc region will have an incomplete form. Thus, without limitation, from the viewpoint of the stability of the antibody molecule, it is preferable that the one-armed antibody be produced by cleavage in the hinge region (hinge) of one of the two Fab regions of an IgG antibody. More preferably, the heavy chain is linked to the un-cleaved heavy chain by an intramolecular disulfide bond after cleavage. WO2005 / 063816 reports that such a one-armed antibody has increased stability compared to a Fab molecule. Antibodies having an increased or decreased pi can also be produced by preparing such a one-armed antibody. Furthermore, when an ion-concentration-dependent antigen-binding domain is introduced into an antibody having an increased pi that is a one-armed antibody, the half-life of the antibody in the plasma can be further shortened, the cellular uptake of the antibody can be further enhanced, the removal of the antigen from the plasma can be further enhanced, or the affinity of the antibody to the extracellular matrix can be further increased compared to the antibody having an increased pi without the ion-concentration-dependent antigen-binding domain.

[0614] Without being limited by a particular theory, it is conceivable that the embodiment in the case where the cell-uptake-accelerating effect of the one-armed antibody is expected is, but not limited to, a case where the pi of the soluble antigen is lower than that of the antibody. The net pi of the complex consisting of the antibody and the antigen can be calculated by a known method, taking into account that the complex is 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. When a common IgG antibody molecule (having two Fabs) binds to a single low-pi soluble antigen compared to binding to two low-pi soluble antigens, the net pi of the complex in the latter case is lower. When such a common antibody is converted into a one-armed antibody, only one antigen can bind to the single molecule of the antibody; the decrease in the complex pi due to the binding of the second antigen can thereby be suppressed. In other words, it is believed that when the pi of the soluble antigen is lower than that of the antibody, conversion into a one-armed antibody increases the pi of the complex compared to the common antibody, and accelerates the intake into the cell.

[0615] Furthermore, without being limited, when the Fab of a common IgG-type antibody molecule (having two Fabs) has a lower pi than the Fc, the conversion to a single-arm antibody increases the net pi of the complex consisting of the single-arm antibody and the antigen. Furthermore, when such conversion to a single-arm antibody is made, it is preferable from the viewpoint of stability of the single-arm antibody that one of the Fabs is cleaved in the hinge region at the junction between the Fab and the Fc. In this case, it is expected that the pi can be effectively increased by selecting a site that will increase the pi of the single-arm antibody to the desired degree.

[0616] Thus, as can be understood by those skilled in the art, the pi of an antibody can be increased and the cellular uptake of an accompanying antigen can be accelerated by converting the antibody to a single-arm antibody, not only (or substantially) depending on the antibody amino acid sequence itself and the type of soluble antigen, by calculating the theoretical pi of the antibody (the theoretical pi of the Fc and the theoretical pi of the Fab) and the theoretical pi of the soluble antigen and predicting the relationship on the difference of their theoretical pi values.

[0617] In one embodiment, the antibody of the disclosure A or B can be a multispecific antibody, which can be, but is not limited to, a bispecific antibody. The multispecific antibody can be a multispecific antibody containing a first polypeptide and a second polypeptide. Here, the "multispecific antibody containing a first polypeptide and a second polypeptide" means an antibody that binds at least two or more types of different antigens or at least two or more types of epitopes in the same antigen. The first polypeptide and the second polypeptide can preferably contain a heavy chain variable region, and more preferably the variable region contains one or more CDRs and / or one or more FRs. In another embodiment, the first polypeptide and the second polypeptide can each preferably contain a heavy chain constant region. In another embodiment, the multispecific antibody can contain a third polypeptide and a fourth polypeptide, each of which contains a light chain variable region and preferably also a light chain constant region. In this case, the first to fourth polypeptides can be assembled together to form a multispecific antibody.

[0618] In one embodiment, in the case where the antibody of Disclosure A is a multispecific antibody and the multispecific antibody contains a heavy chain constant region, in order to reduce the pi thereof, for example, the following sequences can be used: an IgG2 or IgG4 sequence at position 137; an IgGl, IgG2, or IgG4 sequence at position 196; an IgG2 or IgG4 sequence at position 203; an IgG2 sequence at position 214; an IgGl, IgG3, or IgG4 sequence at position 217; an IgGl, IgG3, or IgG4 sequence at position 233; an IgG4 sequence at position 268; an IgG2, IgG3, or IgG4 sequence at position 274; an IgGl, 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 IgGl, IgG2, or IgG4 sequence at position 435. Meanwhile, in order to increase the pi thereof, for example, the following sequences can be used: an IgGl or IgG3 sequence at position 137; an IgG3 sequence at position 196; an IgGl or IgG3 sequence at position 203; an IgGl, IgG3, or IgG4 sequence at position 214; an IgG2 sequence at position 217; an IgG2 sequence at position 233; an IgGl, IgG2, or IgG3 sequence at position 268; an IgGl sequence at position 274; an IgG3 sequence at position 276; an IgGl, IgG2, or IgG3 sequence at position 355; an IgGl, IgG2, or IgG4 sequence at position 392; an IgGl, IgG2, or IgG3 sequence at position 419; or an IgG3 sequence at position 435.

[0619] In one embodiment, in the case where the antibody of Disclosure A has two heavy chain constant regions, the pis of the two heavy chain constant regions can be the same as or different from each other. The heavy chain constant regions can be IgGl, IgG2, IgG3, and IgG4 heavy chain constant regions originally having different pis. Alternatively, a pi difference can be introduced between the two heavy chain constant regions. Modification sites of at least one amino acid residue for introducing such a pi difference in the constant region can be one or more positions described above or one or more positions selected from, for example, the group consisting of position 137, position 196, position 203, position 214, position 217, position 233, position 268, position 274, position 276, position 297, position 355, position 392, position 419, and position 435 in the heavy chain constant region described in WO 2009 / 041643 (according to EU numbering). Alternatively, the amino acid residue of position 297, which is a glycosylation site, can be modified to remove a sugar chain, because removal of a sugar chain from the heavy chain constant region results in a pi difference.

[0620] In one embodiment, the antibody of disclosure A or B can be a polyclonal antibody or a monoclonal antibody, and a monoclonal antibody of mammalian origin is preferred. Monoclonal antibodies include those produced by hybridomas or those produced by host cells transformed with expression vectors that carry antibody genes by way of genetic engineering techniques. The antibody of disclosure A or B can be, for example, an antibody such as a chimeric antibody, a humanized antibody, or an antibody produced by affinity maturation, or a molecule derived therefrom.

[0621] In one embodiment, the antibody of disclosure A or B can be derived from, without limitation, any animal species (e.g., human; or non-human animals such as mouse, rat, hamster, rabbit, monkey, cynomolgus monkey, rhesus monkey, hamadryas baboon, chimpanzee, goat, sheep, dog, pig, or camel), or any bird; and the antibody is preferably derived from human, monkey, or mouse.

[0622] In one embodiment, the antibody of disclosure A or B can be an Ig-type antibody, and can preferably be an IgG-type antibody.

[0623] Within the scope of disclosure A and B described herein, Fc receptor (also referred to as "FcR") means a receptor protein that can bind to the Fc region of an immunoglobulin (antibody) or a molecule derived therefrom, or a variant of the Fc region. For example, within the scope of disclosure A described herein, Fc receptors for IgG, IgA, IgE, and IgM are known as FcyR, FcaR, FcsR, and FcμR, respectively. Within the scope of disclosure A and B described herein, the Fc receptor can also be, for example, FcRn (also referred to as "neonatal Fc receptor").

[0624] Within the scope of the disclosure A described herein, "FcyR" can refer to a receptor protein that can bind an Fc region of an IgGl, IgG2, IgG3, or IgG4 antibody or a molecule derived therefrom, or an Fc region variant, and can include any one or more or all of the members of a family of proteins substantially encoded by FcyR genes. In humans, the family includes, but is not limited to, FcyRI (CD64) (including isoforms FcyRIa, FcyRIb, and FcyRIc); FcyRII (CD32) (including isoforms FcyRIIa (including allotypes H131 (type H) and R131 (type R)), FcyRIIb (including FcyRIIb-l and FcyRIIb-2), and FcyRIIc); and FcyRIII (CD16) (including isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NA1 and FcyRIIIb-NA2)), as well as all unidentified human FcyRs and FcyR isoforms and allotypes. In addition, FcyRIIbl and FcyRIIb2 have been reported as splice variants of human FcyRIIb (hFcyRIIb). There is also a report of a splice variant termed FcyRIIb3 (Brooks et al., J. Exp. Med, 170: 1369-1385 (1989)). hFcyRIIb includes all splice variants in addition to those described above, such as those recorded in NCBI under NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. hFcyRIIb also includes all genetic polymorphisms that have been reported, e.g., FcyRIIb (Li et al., 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)), as well as all genetic polymorphisms that will be reported in the future.

[0625] FcγRs can be derived from any organism, and can include those derived from humans, mice, rats, rabbits, or monkeys, but are not limited thereto. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), as well as all unidentified mouse FcγRs, and FcγR isoforms and allotypes. The preferred FcγRs include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), or FcγRIIIB (CD16). Because FcγRs exist in membrane form in vivo, they can be used in experimental systems after being artificially converted into an appropriate soluble form.

[0626] For example, as shown in WO2014 / 163101, the oligonucleotide sequence and amino acid sequence of FcγRI can be the sequences shown in NM_000566.3 and NP_000557.1, respectively; the oligonucleotide sequence and amino acid sequence of FcγRIIA can be the sequences shown in BC020823.1 and AAH20823.1, respectively. The oligonucleotide sequence and amino acid sequence of FcγRIIB can be the sequences shown in BC146678.1 and AAI46679.1, respectively; the oligonucleotide sequence and amino acid sequence of FcγRIIIA can be the sequences shown in BC033678.1 and AAH33678.1, respectively; the oligonucleotide sequence and amino acid sequence of FcγRIIIB can be the sequences shown in BC128562.1 and AAI28563.1, respectively (showing RefSeq accession numbers).

[0627] FcγRIIa has two genetic polymorphisms in which the amino acid at position 131 of FcγRIIa is replaced by histidine (type H) or arginine (type R) (J. Exp. Med. 172: 19-25, 1990).

[0628] Among FcyRI (CD64) (which includes FcyRIa, FcyRIb, and FcyRIc), and FcyRIII (CD16) (which includes FcyRIIIa (including allotypes V158 and F158)), the a chain that binds the Fc region of IgG associates with a common gamma chain that has an ITAM that transmits an activating signal inside the cell. FcyRIIIb (including allotypes FcyRIIIb-NA1 and FcyRIIIb-NA2) is a GPI-anchored protein. Meanwhile, the cytoplasmic domain of FcyRII (CD32) (which includes FcyRIIa (including allotypes H131 and R131) and FcyRIIc isoforms) contains an ITAM. These receptors are expressed on many immune cells such as macrophages, mast cells, and antigen-presenting cells. The activating signals transduced upon binding of these receptors to the Fc region of IgG promote phagocytosis by macrophages, production of inflammatory cytokines, degranulation of mast cells, and increased function of antigen-presenting cells. FcyRs with the ability to transduce activating signals as described above are also referred to as activating FcyRs in the context of Disclosure A and B described herein.

[0629] Meanwhile, the cytoplasmic domain of FcyRIIb (including FcyRIIb-1 and FcyRIIb-2) contains an ITIM that transmits an inhibitory signal. In B cells, cross-linking between FcyRIIb and the B cell receptor (BCR) inhibits the activating signal from the BCR, which leads to inhibition of antibody production by the BCR. In macrophages, cross-linking of FcyRIII and FcyRIIb inhibits phagocytosis and the ability to produce inflammatory cytokines. FcyRs with the ability to transduce inhibitory signals as described above are also referred to as inhibitory Fcy receptors in the context of Disclosure A and B described herein.

[0630] Within the scope of the disclosure A described herein, the binding activity of the antibody or Fc region (variant) to various FcyRs is increased, or (substantially) maintained, or decreased compared to the antibody or Fc region (variant) before modification can be evaluated by methods known to those skilled in the art. The method is not particularly limited and those described in the present example can be used, and BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010) based on surface plasmon resonance (SPR) phenomenon, for example, can be used. Alternatively, ELISA and fluorescence-activated cell sorting (FACS), and ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay), for example. In these assays, the extracellular domain of human FcyR can be used as a soluble antigen (e.g., WO2013 / 047752).

[0631] For the pH condition for measuring the binding activity between the FcyR-binding domain contained in the antibody or Fc region (variant) and the FcyR, acidic or neutral pH conditions can be appropriately used. For the temperature used in the measurement condition, the binding activity (binding affinity) between the FcyR-binding domain and the FcyR can be evaluated, for example, at any of between 10°C to 50°C. The preferred temperature for determining the binding activity (binding affinity) of the human FcyR-binding domain to the FcyR is, for example, 15°C to 40°C. More preferably, in order to determine the binding activity (binding affinity) between the FcyR-binding domain and the FcyR, any temperature of 20°C to 35°C can be used, for example, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C. A non-limiting example of the temperature is 25°C.

[0632] In one embodiment, in the case where the antibody of the disclosure A or B has a constant region (which can be modified), the constant region can have an Fc region or an Fc region variant (preferably, a human Fc region or a human Fc region variant), and preferably has an FcyR-binding domain within the scope of the disclosure A and an FcRn-binding domain within the scope of the disclosure A and B described herein.

[0633] In one embodiment, in the case where the antibody of the disclosure A has FcyR-binding activity, it can have an FcyR-binding domain, preferably a human FcyR-binding domain. The FcyR-binding domain is not particularly limited as long as the antibody has binding activity or affinity to the FcyR at acidic pH and / or neutral pH, and it can be a domain having direct or indirect binding activity to the FcyR.

[0634] In one embodiment, in the case where the antibody of Disclosure A has FcγR-binding activity, it is preferred that the FcγR-binding activity of the antibody is increased under neutral pH conditions, as compared with a reference antibody containing a native IgG constant region. From the viewpoint of comparing the FcγR-binding activity of both, it is preferred that, without limitation, the antibody of Disclosure A and the reference antibody containing a native IgG constant region have the same amino acid sequence in a region other than the constant region of the antibody of Disclosure A, which is preferably modified at one or more amino acid residues (e.g., the variable region).

[0635] In one embodiment, in the case where the antibody of Disclosure A has FcγR-binding activity or increased FcγR-binding activity under neutral pH conditions (e.g., pH 7.4), without being bound by theory, it is considered that the antibody has a combination of the properties of shuttling between the plasma and endosomes and repeatedly binding multiple antigens as a single antibody molecule with an ion-concentration-dependent antigen-binding domain; the property of being rapidly taken into cells by having an increased pi and increased positive charge in the whole antibody; and the property of being rapidly taken into cells by having increased FcγR-binding activity under neutral pH conditions. Thereby, the half-life of the antibody in the plasma can be further shortened, or the binding activity of the antibody to the extracellular matrix can be further increased, or the removal of the antigen from the plasma can be further promoted; thus the antibody of Disclosure A is beneficial. One skilled in the art can routinely determine the optimal pi value for the antibody to utilize these properties.

[0636] In one embodiment, an Fc gamma R-binding domain with higher Fc gamma R-binding activity than the Fc region or constant region of native human IgG in which the sugar chain attached at position 297 according to the EU numbering is a fucose-containing sugar chain can be produced by modifying the amino acid residues in the Fc region or constant region of native human IgG (see WO2013 / 047752). Furthermore, the domain of any structure that binds to Fc gamma R can be used as the Fc gamma R-binding domain. In this case, the Fc gamma R-binding domain can be produced without the need to introduce amino acid modifications, and alternatively, its affinity to Fc gamma R can be increased by introducing additional modifications. The Fc gamma R-binding domain can include the Fab fragment antibody, the single-domain antibody of camelid origin, and the single-chain Fv antibody that bind to Fc gamma RIIIa 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), and the loop peptide that binds to Fc gamma Rl described in Bonetto et al., FASEB J. 23(2): 575-585 (2008). Whether the Fc gamma R-binding activity of the Fc gamma R-binding domain is higher than the Fc region or constant region of native human IgG in which the sugar chain attached at position 297 according to the EU numbering is a fucose-containing sugar chain can be appropriately evaluated using the methods described above.

[0637] In one embodiment of the disclosure A, the starting FcγR-binding domain preferably includes, for example, a (human) IgG Fc region or a (human) IgG constant region. Any Fc region or constant region can be used as the starting Fc region or the starting constant region as long as a variant of the starting Fc region or the starting constant region is capable of binding to a human FcγR in a neutral pH range. An Fc region or a constant region further obtained by further modifying one or more amino acid residues of the starting Fc region or the starting constant region which has been modified from the Fc region or the constant region can also be appropriately used as the Fc region or the constant region of the disclosure A. The starting Fc region or the starting constant region can refer to a polypeptide itself, a composition containing the starting Fc region or the starting constant region, or an amino acid sequence encoding the starting Fc region or the starting constant region. The starting Fc region or the starting constant region can include a known Fc region or a known constant region produced by a recombinant technique. The origin of the starting Fc region or the starting constant region is not limited, and it can be obtained from any organism of a non-human animal or a human. In addition, the starting FcγR-binding domain can be obtained from a cynomolgus monkey, a marmoset, a macaque, a chimpanzee, or a human. The starting Fc region or the starting constant region can be preferably obtained from a human IgG1; however, it is not limited to a specific IgG type. This means that an Fc region of a human IgG1, IgG2, IgG3, or IgG4 can be used as an appropriate starting FcγR-binding domain, and it also means that, within the scope of the disclosure A described herein, an Fc region or a constant region of an IgG type or subclass derived from any organism can be preferably used as the starting Fc region or the starting constant region. Examples of native IgG variants or modified forms are described in well-known documents such as 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.

[0638] In one embodiment, the amino acid residues of the starting FcγR-binding domain, the starting Fc region, or the starting constant region can contain, for example, one or more mutations: for example, substitutions with amino acid residues different from those in the starting Fc region or the starting constant region; insertions of one or more amino acid residues into the amino acid residues in the starting Fc region or the starting constant region; or deletions of one or more amino acid residues from those in the starting Fc region or the starting constant region. The amino acid sequence of the modified Fc region or constant region is preferably an amino acid sequence containing at least a portion of an Fc region or a constant region that can not be naturally occurring. The variant necessarily has less than 100% sequence identity or similarity to the starting Fc region or the starting constant region. For example, the variant has about 75% to less than 100%, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, still more preferably about 90% to less than 100%, and also more preferably about 95% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting Fc region or the starting constant region. In a non-limiting example, there are at least one amino acid difference between the modified Fc region or constant region of Disclosure A and the starting Fc region or the starting constant region.

[0639] In one embodiment, the Fc region or constant region having FcγR-binding activity at acidic pH range and / or at neutral pH range (which can be included in the antibody of Disclosure A) can be obtained by any method. In particular, the Fc region or constant region variant having FcγR-binding activity at neutral pH range can be obtained by modifying the amino acid of the human IgG antibody which can be used as the starting Fc region or the starting constant region. The IgG antibody Fc region or IgG antibody constant region suitable for modification can include, for example, the Fc region or constant region of human IgG (IgG1, IgG2, IgG3, or IgG4, or a variant thereof), and mutants spontaneously generated therefrom. For the Fc region or constant region of human IgG1, human IgG2, human IgG3, or human IgG4 antibody, many allotype sequences due to genetic polymorphism are described in "Sequences of proteins of immunological interest", NIH publication No. 91-3242, and any of them can be used in Disclosure A. In particular, for the human IgG1 sequence, the amino acid sequence according to EU numbering positions 356 to 358 can be DEL or EEM.

[0640] In another embodiment within the scope of the disclosure, modifications of other amino acids are not limited as long as the variant has FcyR-binding activity in the neutral pH range. The modified one or more amino acid positions are reported in, for example, WO 2007 / 024249, WO 2007 / 021841, WO 2006 / 031370, WO 2000 / 042072, WO 2004 / 029207, WO 2004 / 099249, WO 2006 / 105338, WO 2007 / 041635, WO 2008 / 092117, WO 2005 / 070963, WO 2006 / 020114, WO 2006 / 116260, WO 2006 / 023403, WO 2013 / 047752, WO 2006 / 019447, WO 2012 / 115241, WO 2013 / 125667, WO 2014 / 030728, WO 2014 / 163101, WO 2013 / 118858, and WO 2014 / 030750.

[0641] The site of the amino acid modification in the constant region or Fc region to increase FcyR-binding activity in the neutral pH range can include, for example, one or more positions selected from the group consisting of positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 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 (according to EU numbering) as shown in WO2013 / 047752. The modification of the amino acid residues can increase the binding of the Fc region or constant region of the IgG antibody to FcyR under neutral pH conditions. WO2013 / 047752 describes that, as a preferred modification in the IgG-type constant region or Fc region, for example, the modification of one or more amino acid residues is selected from the group consisting of: an amino acid modification of position 221 to Lys or Tyr; an amino acid modification of position 222 to any one of Phe, Trp, Glu, and Tyr; an amino acid modification of position 223 to any one of Phe, Trp, Glu, and Lys; an amino acid modification of position 224 to any one of Phe, Trp, Glu, and Tyr; an amino acid modification of position 225 to any one of Glu, Lys, and Trp; an amino acid modification of position 227 to any one of Glu, Gly, Lys, and Tyr; an amino acid modification of position 228 to any one of Glu, Gly, Lys, and Tyr; an amino acid modification of position 230 to any one of Ala, Glu, Gly, and Tyr; an amino acid modification of position 231 to any one of Glu, Gly, Lys, Pro, and Tyr; an amino acid modification of position 232 to any one of Glu, Gly, Lys, and Tyr;an amino acid modification at position 233 that is any one of Ala, Asp, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 234 that is any one of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 235 that is any one of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 236 that is any one of Ala, Asp, Glu, Phe, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 237 that is any one of Asp, Glu, Phe, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 238 that is any one of Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 239 that is any one of Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 240 that is any one of Ala, lie, Met, and Thr; an amino acid modification at position 241 that is any one of Asp, Glu, Leu, Arg, Trp, and Tyr; an amino acid modification at position 243 that is any one of Glu, Leu, Gin, Arg, Trp, and Tyr; an amino acid modification at position 244 that is His; an amino acid modification at position 245 that is Ala; an amino acid modification at position 246 that is any one of Asp, Glu, His, and Tyr; an amino acid modification at position 247 that is any one of Ala, Phe, Gly, His, lie, Leu, Met, Thr, Val, and Tyr; an amino acid modification at position 249 that is any one of Glu, His, Gin, and Tyr; an amino acid modification at position 250 that is Glu or Gin; an amino acid modification at position 251 that is Phe; an amino acid modification at position 254 that is any one of Phe, Met, and Tyr; an amino acid modification at position 255 that is any one of Glu, Leu, and Tyr;an amino acid modification at position 256 that is any of Ala, Met, and Pro; an amino acid modification at position 258 that is any of Asp, Glu, His, Ser, and Tyr; an amino acid modification at position 260 that is any of Asp, Glu, His, and Tyr; an amino acid modification at position 262 that is any of Ala, Glu, Phe, lie, and Thr; an amino acid modification at position 263 that is any of Ala, lie, Met, and Thr; an amino acid modification at position 264 that is any of Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Trp, and Tyr; an amino acid modification at position 265 that is any of Ala, Leu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 266 that is any of Ala, lie, Met, and Thr; an amino acid modification at position 267 that is any of Asp, Glu, Phe, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 268 that is any of Asp, Glu, Phe, Gly, lie, Lys, Leu, Met, Pro, Gin, Arg, Thr, Val, and Trp; an amino acid modification at position 269 that is any of Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 270 that is any of Glu, Phe, Gly, His, lie, Leu, Met, Pro, Gin, Arg, Ser, Thr, Trp, and Tyr; an amino acid modification at position 271 that is any of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 272 that is any of Asp, Phe, Gly, His, lie, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 273 that is Phe or lie; an amino acid modification at position 274 that is any of Asp, Glu, Phe, Gly, His, lie, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 275 that is Leu or Trp;the amino acid modification at position 276 is any one of Asp, Glu, Phe, Gly, His, lie, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 278 is any one of Asp, Glu, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, and Trp; the amino acid modification at position 279 is Ala; the amino acid modification at position 280 is any one of Ala, Gly, His, Lys, Leu, Pro, Gin, Trp, and Tyr; the amino acid modification at position 281 is any one of Asp, Lys, Pro, and Tyr; the amino acid modification at position 282 is any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid modification at position 283 is any one of Ala, Gly, His, lie, Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid modification at position 284 is any one of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid modification at position 285 is any one of Asp, Glu, Lys, Gin, Trp, and Tyr; the amino acid modification at position 286 is any one of Glu, Gly, Pro, and Tyr; the amino acid modification at position 288 is any one of Asn, Asp, Glu, and Tyr; the amino acid modification at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid modification at position 291 is any one of Asp, Glu, Gly, His, lie, Gin, and Thr; the amino acid modification at position 292 is any one of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid modification at position 293 is any one of Phe, Gly, His, lie, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 294 is any one of Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 295 is any one of Asp, Glu, Phe, Gly, His, lie, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 296 is any one of Ala, Asp, Glu, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, and Val;an amino acid modification at position 297 that is any one of Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 298 that is any one of Ala, Asp, Glu, Phe, His, lie, Lys, Met, Asn, Gin, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 299 that is any one of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Val, Trp, and Tyr; an amino acid modification at position 300 that is any one of Ala, Asp, Glu, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, and Trp; an amino acid modification at position 301 that is any one of Asp, Glu, His, and Tyr; an amino acid modification at position 302 that is lie; an amino acid modification at position 303 that is any one of Asp, Gly, and Tyr; an amino acid modification at position 304 that is any one of Asp, His, Leu, Asn, and Thr; an amino acid modification at position 305 that is any one of Glu, lie, Thr, and Tyr; an amino acid modification at position 311 that is any one of Ala, Asp, Asn, Thr, Val, and Tyr; an amino acid modification at position 313 that is Phe; an amino acid modification at position 315 that is Leu; an amino acid modification at position 317 that is Glu or Gin; an amino acid modification at position 318 that is any one of His, Leu, Asn, Pro, Gin, Arg, Thr, Val, and Tyr; an amino acid modification at position 320 that is any one of Asp, Phe, Gly, His, lie, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 322 that is any one of Ala, Asp, Phe, Gly, His, lie, Pro, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 323 that is lie; an amino acid modification at position 324 that is any one of Asp, Phe, Gly, His, lie, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 325 that is any one of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr;an amino acid modification at position 326 to any one of Ala, Asp, Glu, Gly, lie, Leu, Met, Asn, Pro, Gin, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 327 to any one of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 328 to any one of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 329 to any one of Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 330 to any one of Cys, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 331 to any one of Asp, Phe, His, lie, Leu, Met, Gin, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 332 to any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 333 to any one of Ala, Asp, Glu, Phe, Gly, His, lie, Leu, Met, Pro, Ser, Thr, Val, and Tyr; an amino acid modification at position 334 to any one of Ala, Glu, Phe, lie, Leu, Pro, and Thr; an amino acid modification at position 335 to any one of Asp, Phe, Gly, His, lie, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; an amino acid modification at position 336 to any one of Glu, Lys, and Tyr; an amino acid modification at position 337 to any one of Glu, His, and Asn; an amino acid modification at position 339 to any one of Asp, Phe, Gly, lie, Lys, Met, Asn, Gin, Arg, Ser, and Thr; an amino acid modification at position 376 to Ala or Val; an amino acid modification at position 377 to Gly or Lys; an amino acid modification at position 378 to Asp; an amino acid modification at position 379 to Asn; an amino acid modification at position 380 to any one of Ala, Asn, and Ser;amino acid modification at position 421 to Lys; amino acid modification at position 427 to Asn; amino acid modification at position 428 to Phe or Leu; amino acid modification at position 429 to Met; amino acid modification at position 434 to Trp; amino acid modification at position 436 to lie; and amino acid modification at position 440 to any one of Gly, His, lie, Leu, and Tyr (according to the EU numbering). The number of amino acids to be modified is not particularly limited, and only one position of amino acid or two or more positions of amino acid can be modified. Combinations of amino acid modifications at two or more positions are shown in Table 5 of WO2013 / 047752. Modification of these amino acid residues can also be appropriately introduced into the antibody of Disclosure A.

[0642] In one embodiment, the binding activity of the FcγR-binding domain of the antibody of Disclosure A to (human) FcγR, such as any one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb, can be higher than that of a natural IgG or a reference antibody containing a starting Fc region or a starting constant region (of the Fc region or the constant region). For example, the FcγR-binding activity of the FcγR-binding domain of the antibody of Disclosure A can be 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, 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 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, 5-fold or more, 7.5-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, or 100-fold or more than the FcγR-binding activity of the reference antibody.

[0643] In another embodiment, the level of increase in the binding activity to the inhibitory FcγR (FcγRIIb-1 and / or FcγRIIb-2) (in the neutral pH range) can be greater than the level of increase in the binding activity to the 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)).

[0644] In one embodiment, the antibody of the disclosure A can have the binding activity to FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

[0645] In one embodiment, the preferred FcγR-binding domain of the disclosure A further includes, for example, a FcγR-binding domain having selective FcγR-binding activity, whose binding activity to a specific FcγR is greater than that to other FcγRs. In the case of using an antibody (or an Fc region as a FcγR-binding domain), a single antibody molecule can bind only to a single FcγR molecule. Therefore, a single antibody molecule in the state of binding to an inhibitory FcγR cannot bind to other activating FcγRs, and a single antibody molecule in the state of binding to an activating FcγR cannot bind to other activating FcγRs or inhibitory FcγRs.

[0646] As described above, the activating FcγR preferably includes, for example, FcγRI (CD64) such as FcγRIa, FcγRIb, or FcγRIc; and FcγRIII (CD16) such as FcγRIIIa (such as allotype V158 or F158) or FcγRIIIb (such as allotype FcγRIIIb-NA1 or FcγRIIIb-NA2). Meanwhile, the inhibitory FcγR preferably includes, for example, FcγRIIb (such as FcγRIIb-1 or FcγRIIb-2).

[0647] In one embodiment, the FcγR-binding domain having higher binding activity to an inhibitory FcγR than to an activating FcγR can be used as the selective FcγR-binding domain included in the antibody of the disclosure A. The selective FcγR-binding domain can include, for example, the FcγR-binding domain having higher binding activity to FcγRIIb (such as FcγRIIb-1 and / or FcγRIIb-2) than to any one or more of the activating FcγRs selected from the group consisting of FcγRI (CD64) such as FcγRIa, FcγRIb, or FcγRIc; FcγRIII (CD16) such as FcγRIIIa (such as allotype V158 or F158) or FcγRIIIb (such as FcγRIIIb-NA1 or FcγRIIIb-NA2); FcγRII (CD32) such as FcγRIIa (including allotype H131 or R131); and FcγRIIc.

[0648] Further, whether the FcγR-binding domain has selective binding activity can be evaluated by comparing the binding activity to each FcγR determined by the above-described methods, for example, by comparing the value (ratio) obtained by dividing the KD value to the activating FcγR by the KD value to the inhibitory FcγR, more specifically by comparing the FcγR selectivity index shown in Equation 1 below:

[0649] [Equation 1] FcγR selectivity index = KD value to activating FcγR / KD value to inhibitory FcγR

[0650] In Equation 1, the KD value to the activating FcγR refers to the KD value to one or more of the following: FcγRIa; FcγRIb; FcγRIc; FcγRIIIa (including allotype V158 and / or F158); FcγRIIIb (including FcγRIIIb-NA1 and / or FcγRIIIb-NA2); FcγRIIa (including allotype H131 and / or R131); and FcγRIIc; and the KD value to the inhibitory FcγR refers to the KD value to FcγRIIb-1 and / or FcγRIIb-2. The activating FcγR and the inhibitory FcγR used for determining the KD value can be selected in any combination. For example, the value (ratio) determined by dividing the KD value to FcγRIIa (including allotype H131) by the KD value to FcγRIIb-1 and / or FcγRIIb-2 can be used, without limitation.

[0651] The FcyR selectivity index can be, 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, 100 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, 920 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 100000 or more; without being limited thereto.

[0652] In one embodiment, human IgG (IgGl, IgG2, IgG3, or IgG4) Fc region variants or constant region variants (antibodies containing the same) in which the amino acid at position 238 or 328 according to EU numbering is Asp or Glu, respectively, can be preferred for use as the antibodies of disclosure A containing the Fc region variants or constant region variants, as they have higher binding activity to FcyRIIb-1 and / or FcyRIIb-2 than to FcyRIa, FcyRIb, FcyRIc, FcyRIIIa (including allotype V158), FcyRIIIa (including allotype F158), FcyRIIIb (including allotype FcyRIIIb-NA1), FcyRIIIb (including allotype FcyRIIIb-NA2), FcyRIIa (including allotype H131), FcyRIIa (including allotype R131), and / or FcyRIIc, as specifically described in WO2013 / 125667, WO2012 / 115241, and WO2013 / 047752. In this embodiment, the antibodies of disclosure A have binding activity to all activating FcyRs (herein, which is selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb, FcyRIIa) and FcyRIIb, and their FcyRIIb-binding activity is maintained or increased compared to a reference antibody containing a native IgG constant region or a native IgG Fc region, and / or their binding activity to all activating FcyRs is decreased.

[0653] In one embodiment, for the antibodies of disclosure A containing the Fc region variants or constant region variants, their binding activity to FcyRIIb can be maintained or increased compared to a reference antibody having a constant region or Fc region of native IgG, and their binding activity to FcyRIIa (type H) and FcyRIIa (type R) can be decreased. The antibodies can have increased binding selectivity to FcyRIIb over FcyRIIa.

[0654] Within the scope of the disclosure A described herein, the extent of "reduced binding activity to all activating FcyRs" can be, 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, 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.

[0655] Within the context of the disclosure A described herein, the "extent to which FcyRIIb-binding activity is maintained or increased," the "extent to which binding activity to FcyRIIb is maintained or increased," or the "extent of maintained or increased binding activity to FcyRIIb" can be, but is not limited to, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101% or greater, 102% or greater, 103% or greater, 104% or greater, 105% or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 112% or greater, 114% or greater, 116% or greater, 118% or greater, 120% or greater, 122% or greater, 124% or greater, 126% or greater, 128% or greater, 130% or greater, 132% or greater, 134% or greater, 136% or greater, 138% or greater, 140% or greater, 142% or greater, 144% or greater, 146% or greater, 148% or greater, 150% or greater, 155% or greater, 160% or greater, 165% or greater, 170% or greater, 175% or greater, 180% or greater, 185% or greater, 190% or greater, 195% or greater, 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 7-fold or greater, 8-fold or greater, 9-fold or greater, 10-fold or greater, 20-fold or greater, 30-fold or greater, 40-fold or greater, 50-fold or greater, 60-fold or greater, 70-fold or greater, 80-fold or greater, 90-fold or greater, 100-fold or greater, 200-fold or greater, 300-fold or greater, 400-fold or greater, 500-fold or greater, 600-fold or greater, 700-fold or greater, 800-fold or greater, 900-fold or greater, 1000-fold or greater, 10000-fold or greater, or 100000-fold or greater.

[0656] Within the scope of the disclosure A described herein, the degree of "reduced binding activity for FcyRIIa (type H) and FcyRIIa (type R)" or "reduced binding activity for FcyRIIa (type H) and FcyRIIa (type R)" can be, 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, 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.

[0657] Within the scope of the disclosure A described herein, modifications that increase the binding selectivity for FcyRIIb over FcyRIIa (type R) can be preferred, and modifications that increase the binding selectivity for FcyRIIb over FcyRIIa (type H) can be more preferred, and preferred amino acid substitutions for such modifications can include, for example, according to EU numbering: (a) a modification by substitution of Gly at position 237 with Trp; (b) a modification by substitution of Gly at position 237 with Phe; (c) a modification by substitution of Pro at position 238 with Phe; (d) a modification by substitution of Asn at position 325 with Met; (e) a modification by substitution of Ser at position 267 with lie; (f) a modification by substitution of Leu at position 328 with Asp; (g) a modification by substitution of Ser at position 267 with Val; (h) a modification by substitution of Leu at position 328 with Trp; (i) a modification by substitution of Ser at position 267 with Gin; (j) a modification by substitution of Ser at position 267 with Met; (k) a modification by substitution of Gly at position 236 with Asp; (1) a modification by substitution of Ala at position 327 with Asn; (m) a modification by substitution of Asn at position 325 with Ser; (n) a modification by substitution of Leu at position 235 with Tyr; (o) a modification by substitution of Val at position 266 with Met; (p) a modification by substitution of Leu at position 328 with Tyr; (q) a modification by substitution of Leu at position 235 with Trp; (r) a modification by substitution of Leu at position 235 with Phe; (s) a modification by substitution of Ser at position 239 with Gly; (t) a modification by substitution of Ala at position 327 with Glu; (u) a modification by substitution of Ala at position 327 with Gly; (v) a modification by substitution of Pro at position 238 with Leu; (w) a modification by substitution of Ser at position 239 with Leu; (x) a modification by substitution of Leu at position 328 with Thr; (y) a modification by substitution of Leu at position 328 with Ser; (z) a modification by substitution of Leu at position 328 with Met; (aa) a modification by substitution of Pro at position 331 with Trp; (ab) a modification by substitution of Pro at position 331 with Tyr; (ac) a modification by substitution of Pro at position 331 with Phe; (ad) a modification by substitution of Ala at position 327 with Asp; (ae) a modification by substitution of Leu at position 328 with Phe; (af) a modification by substitution of Pro at position 271 with Leu;(ag) a modification at position 267 by substitution of Ser with Glu; (ah) a modification at position 328 by substitution of Leu with Ala; (ai) a modification at position 328 by substitution of Leu with lie; (aj) a modification at position 328 by substitution of Leu with Gin; (ak) a modification at position 328 by substitution of Leu with Val; (al) a modification at position 326 by substitution of Lys with Trp; (am) a modification at position 334 by substitution of Lys with Arg; (an) a modification at position 268 by substitution of His with Gly; (ao) a modification at position 268 by substitution of His with Asn; (ap) a modification at position 324 by substitution of Ser with Val; (aq) a modification at position 266 by substitution of Val with Leu; (ar) a modification at position 271 by substitution of Pro with Gly; (as) a modification at position 332 by substitution of lie with Phe; (at) a modification at position 324 by substitution of Ser with lie; (au) a modification at position 333 by substitution of Glu with Pro; (av) a modification at position 300 by substitution of Tyr with Asp; (aw) a modification at position 337 by substitution of Ser with Asp; (ax) a modification at position 300 by substitution of Tyr with Gin; (ay) a modification at position 335 by substitution of Thr with Asp; (az) a modification at position 239 by substitution of Ser with Asn; (ba) a modification at position 326 by substitution of Lys with Leu; (bb) a modification at position 326 by substitution of Lys with lie; (bc) a modification at position 239 by substitution of Ser with Glu; (bd) a modification at position 326 by substitution of Lys with Phe; (be) a modification at position 326 by substitution of Lys with Val; (bf) a modification at position 326 by substitution of Lys with Tyr; (bg) a modification at position 267 by substitution of Ser with Asp; (bh) a modification at position 326 by substitution of Lys with Pro; (bi) a modification at position 326 by substitution of Lys with His; (bj) a modification at position 334 by substitution of Lys with Ala; (bk) a modification at position 334 by substitution of Lys with Trp; (bl) a modification at position 268 by substitution of His with Gin; (bm) a modification at position 326 by substitution of Lys with Gin; (bn) a modification at position 326 by substitution of Lys with Glu; (bo) a modification at position 326 by substitution of Lys with Met; (bp) a modification at position 266 by substitution of Val with lie; (bq) a modification at position 334 by substitution of Lys with Glu;(br) a modification made by substituting Tyr at position 300 with Glu; (bs) a modification made by substituting Lys at position 334 with Met; (bt) a modification made by substituting Lys at position 334 with Val; (bu) a modification made by substituting Lys at position 334 with Thr; (bv) a modification made by substituting Lys at position 334 with Ser; (bw) a modification made by substituting Lys at position 334 with His; (bx) a modification made by substituting Lys at position 334 with Phe; (by) a modification made by substituting Lys at position 334 with Gin; (bz) a modification made by substituting Lys at position 334 with Pro; (ca) a modification made by substituting Lys at position 334 with Tyr; (cb) a modification made by substituting Lys at position 334 with He; (cc) a modification made by substituting Gin at position 295 with Leu; (cd) a modification made by substituting Lys at position 334 with Leu; (ce) a modification made by substituting Lys at position 334 with Asn; (cf) a modification made by substituting His at position 268 with Ala; (cg) a modification made by substituting Ser at position 239 with Asp; (ch) a modification made by substituting Ser at position 267 with Ala; (ci) a modification made by substituting Leu at position 234 with Trp; (cj) a modification made by substituting Leu at position 234 with Tyr; (ck) a modification made by substituting Gly at position 237 with Ala; (cl) a modification made by substituting Gly at position 237 with Asp; (cm) a modification made by substituting Gly at position 237 with Glu; (cn) a modification made by substituting Gly at position 237 with Leu; (co) a modification made by substituting Gly at position 237 with Met; (cp) a modification made by substituting Gly at position 237 with Tyr; (cq) a modification made by substituting Ala at position 330 with Lys; (cr) a modification made by substituting Ala at position 330 with Arg; (cs) a modification made by substituting Gin at position 233 with Asp; (ct) a modification made by substituting His at position 268 with Asp; (cu) a modification made by substituting His at position 268 with Glu; (cv) a modification made by substituting Lys at position 326 with Asp; (cw) a modification made by substituting Lys at position 326 with Ser; (cx) a modification made by substituting Lys at position 326 with Thr; (cy) a modification made by substituting Val at position 323 with He; (cz) a modification made by substituting Val at position 323 with Leu; (da) a modification made by substituting Val at position 323 with Met; (db) a modification made by substituting Tyr at position 296 with Asp;(dc) a modification by substitution of Lys at position 326 with Ala; (dd) a modification by substitution of Lys at position 326 with Asn; and (de) a modification by substitution of Ala at position 330 with Met.

[0658] The above modifications can be individually at a single position or in combination at two or more positions. Alternatively, the preferred modifications can include, for example, those shown in Tables 14-15, 17-24, and 26-28 of WO 2013 / 047752, e.g., variants of human constant regions or human Fc regions in which the amino acid at position 238 according to EU numbering is Asp and the amino acid at position 271 according to EU numbering is Gly in human IgG (IgGl, IgG2, IgG3, or IgG4); 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 according to EU numbering can be substituted. In this case, the variants can include, but are not limited to, variants of human constant regions or human Fc regions containing one or more of the following:

[0659] Asp at position 233, Tyr at position 234, Asp at position 237, lie at position 264, Glu at position 265, any one of Phe, Met, and Leu at position 266, any one of Ala, Glu, Gly, and Gin at position 267, Asp or Glu at position 268, Asp at position 269, any one of Asp, Phe, lie, Met, Asn, and Gin at position 272, Asp at position 296, Ala or Asp at position 326, Gly at position 327, Lys or Arg at position 330, Ser at position 331, Thr at position 332, any one of Thr, Lys, and Arg at position 333, and any one of Asp, Glu, Phe, lie, Lys, Leu, Met, Gin, Arg, and Tyr at position 396 (according to EU numbering).

[0660] In an alternative embodiment, the antibody of the disclosure A containing a Fc region variant or constant region variant can have a binding activity to FcyRIIb that is maintained or increased and a binding activity to FcyRIIa (type H) and FcyRIIa (type R) that is reduced compared to a reference antibody containing a native IgG constant region or Fc region. Preferred sites of amino acid substitution of such a variant can be as reported in WO2014 / 030728, for example, at least one amino acid according to EU numbering at amino acid position 238 and a position selected from the group consisting of: position 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 (according to EU numbering).

[0661] More preferably, the variant can have an Asp at position 238 according to EU numbering and at least one amino acid of the group of amino acids selected from: Asp at position 233, Tyr at position 234, Phe at position 235, Asp at position 237, lie at position 264, Glu at position 265, Phe, Leu, or Met at position 266, Ala, Glu, Gly, or Gin at position 267, Asp, Gin, or Glu at position 268, Asp at position 269, Gly at position 271, Asp, Phe, lie, Met, Asn, Pro, or Gin at position 272, Gin at position 274, Asp or Phe at position 296, Ala or Asp at position 326, 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 Gin at position 355, Glu at position 356, Met at position 358, Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, or Tyr at position 396, Arg at position 409, and Glu at position 419 (according to EU numbering).

[0662] In an alternative embodiment, the antibody of the disclosure A containing a Fc region variant or a constant region variant can have a maintained binding activity to FcyRIIb and a reduced binding activity to all activating FcyRs, FcyRIIa (type R) compared to a reference antibody containing a native IgG constant region or Fc region. The preferred sites for amino acid substitution of such a variant can be as reported in WO2014 / 163101, for example, at least one amino acid selected from the group consisting of positions 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 according to EU numbering, in addition to the amino acid at position 238 according to EU numbering). More preferably, the variant can have an Asp at position 238 according to EU numbering, and at least one amino acid from the group consisting of Phe at position 235; Gin 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; lie at position 323; Asn or His at position 324; and His or Tyr at position 330 according to EU numbering.

[0663] Within the scope of the disclosure A described herein, the level of "maintained binding activity to FcyRIIb" can be, 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 2-fold or more.

[0664] Within the scope of the disclosure A described herein, the aforementioned "reduced binding activity to all activating FcyRs, especially FcyRIIa (type R)" can be, but is not limited to, 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.

[0665] WO2014 / 030750 also reports variants of mouse constant regions and Fc regions. In one embodiment, the antibody of disclosure A or B can comprise such a variant.

[0666] Within the scope of the disclosure A and B described herein, unlike FcyRs, which belong to the immunoglobulin superfamily, "FcRn", especially human FcRn, is structurally similar to the polypeptides of the major histocompatibility complex (MHC) class I and presents 22% to 29% sequence identity with MHC class I molecules (Ghetie et al., Immunol. Today 18(12), 592-598 (1997)). FcRn is expressed as a heterodimer, consisting of a soluble β or light chain (β2 microglobulin) complexed with a transmembrane α or heavy chain. Like MHC, the α chain of FcRn contains three extracellular domains (α1, α2, and α3), and its short cytoplasmic domain connects the protein to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain of the Fc region of antibodies (Raghavan et al., Immunity 1 :303-315 (1994)).

[0667] FcRn is expressed in the maternal placenta and yolk sac of mammals, and is involved in IgG transfer from mother to fetus. In addition, in the small intestine of neonatal rodents where FcRn is expressed, FcRn is involved in the transfer of mature IgG across the brush border epithelium from ingested colostrum or milk. FcRn is expressed in various other tissues and endothelial cell systems of various species. FcRn is also expressed in the vascular endothelium of adults, the muscle vasculature, and the liver sinusoidal capillaries. FcRn is thought to play a role in the maintenance of plasma IgG concentration by binding to IgG and recycling IgG to the serum. In general, the binding of FcRn to IgG molecules is strictly pH-dependent. Optimal binding is observed in the acidic pH range below 7.0.

[0668] The oligonucleotide and amino acid sequences of human FcRn can be derived from, for example, the precursor (containing a signal sequence) shown in NM_004107.4 and NP_004098.1, respectively (RefSeq accession numbers shown in parentheses).

[0669] The precursor forms a complex with human β2-microglobulin in vivo. Therefore, by using known recombinant expression techniques, a soluble human FcRn capable of forming a complex with human β2-microglobulin can be prepared, and is suitably used in various experimental systems. The soluble human FcRn can be used to evaluate the FcRn-binding activity of an antibody or Fc region variant. In the disclosure A or B, FcRn is not particularly limited as long as it is in a form capable of binding to the FcRn-binding domain; however, a preferred FcRn can be human FcRn.

[0670] Within the scope of the disclosure A and B described herein, an antibody or Fc region variant can have an "FcRn-binding domain", preferably a human FcRn-binding domain, in case it has FcRn-binding activity. The FcRn-binding domain is not particularly limited as long as the antibody has binding activity or affinity to FcRn at acidic pH and / or at neutral pH; or it can be a domain having activity to bind FcRn directly or indirectly. The domain includes, but is not limited to, the Fc region of IgG-type immunoglobulin, albumin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides, and anti-FcRn scaffold molecules (which have activity to bind FcRn directly), and molecules binding IgG or albumin (which have activity to bind FcRn indirectly. In the disclosure A or B, it is also possible to use a domain having FcRn-binding activity at the acidic pH range and / or at the neutral pH range. If the domain originally has FcRn-binding activity at the acidic pH range and / or at the neutral pH range, they can be used without further modification. If the domain has only weak or no FcRn-binding activity at the acidic pH range and / or at the neutral pH range, amino acid residues in the FcRn-binding domain of the antibody or Fc region variant can be modified to have FcRn-binding activity at the acidic pH range and / or at the neutral pH range. Alternatively, a domain originally having FcRn-binding activity at the acidic pH range and / or at the neutral pH range can be modified for amino acids to further increase its FcRn-binding activity. The FcRn-binding activity at the acidic pH range and / or at the neutral pH range can be compared before and after the amino acid modification to find the amino acid modification of interest for the FcRn-binding domain.

[0671] The FcRn-binding domain can preferably be a region that binds FcRn directly. The preferred FcRn-binding domain includes, for example, the constant region and Fc region of an antibody. However, a region that is capable of binding a polypeptide having FcRn-binding activity, such as albumin and IgG, can bind FcRn indirectly via albumin, IgG. Thus, the FcRn-binding region can be a region that binds a polypeptide having binding activity for albumin or IgG. Without limitation, in order to facilitate removal of an antigen from plasma, the FcRn-binding activity thereof is higher at neutral pH, while in order to improve retention of an antibody in plasma, the FcRn-binding activity thereof is higher at acidic pH. For example, an FcRn-binding domain whose FcRn-binding activity is higher at neutral pH or acidic pH can be selected. Alternatively, the amino acids of an antibody or Fc region variant can be modified to impart FcRn-binding activity at neutral pH or acidic pH. Alternatively, pre-existing FcRn-binding activity at neutral pH or acidic pH can be increased.

[0672] Within the scope of the disclosure A and B described herein, the FcRn-binding activity of an antibody or Fc region (variant) can be increased, (substantially) maintained, or decreased compared to the antibody or Fc region (variant) before modification can be assessed by known methods, such as those described in the Examples herein, and, for example, BIACORE, Scatchard plot, and flow cytometry (see WO2013 / 046722). The extracellular domain of human FcRn can be used as a soluble antigen in these assays. The conditions (in addition to pH) can be appropriately selected by one skilled in the art in the measurement of the FcRn-binding activity of an antibody or Fc region (variant). The assay can be performed, for example, under the conditions of MES buffer and 37°C, as described in WO2009 / 125825. The FcRn-binding activity of an antibody or Fc region (variant) can be assessed, for example, by loading FcRn as an analyte on a chip on which an antibody is immobilized.

[0673] The FcRn-binding activity of an antibody or Fc region (variant) can be assessed based on the dissociation constant (KD), the apparent dissociation constant (apparent KD), the dissociation rate (kd), the apparent dissociation (apparent kd).

[0674] For the pH condition for measuring the binding activity between FcRn and the FcRn-binding domain contained in the antibody or Fc region (variant), either acidic pH condition or neutral pH condition can be appropriately used. For the temperature condition for measuring the binding activity (binding affinity) between FcRn and the FcRn-binding domain, any temperature between 10°C to 50°C can be used. In order to determine the binding activity (binding affinity) between FcRn and the human FcRn-binding domain, it can be preferable to use a temperature between 15°C to 40°C. More preferably, any temperature between 20°C to 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 be used. A non-limiting example of the temperature can be 25°C.

[0675] In one embodiment, in the case where the antibody of the disclosure A or B has FcRn-binding activity, it can 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 pH and / or neutral pH, and it can be a domain having direct or indirect binding activity for FcRn. In one specific embodiment, it can be preferable that the antibody of the disclosure A or B has, for example, increased FcRn-binding activity under neutral pH conditions compared to a reference antibody containing a constant region of a native IgG (see WO2013 / 046722). From the perspective of comparing the FcRn-binding activity of both, it can be preferable that the antibody of the disclosure A or B and the reference antibody containing a constant region of a native IgG have the same amino acid sequence in the region (e.g., variable region) other than the constant region of the antibody of the disclosure A or B, which is preferably modified at one or more amino acid residues.

[0676] In one embodiment, within the scope of the disclosure A described herein, in the case where the antibody of the disclosure A has increased FcRn-binding activity under neutral pH conditions, without being limited by a particular theory, the antibody of the disclosure A can have a combination of any two or more of the following properties: the property of shuttling between plasma and endosomes and repeatedly binding multiple antigens as a single antibody molecule with an ion concentration-dependent antigen-binding domain; the property of being rapidly taken up into cells by having an increased pi and increased positive charge throughout the antibody; and the property of being rapidly taken up into cells by having increased FcRn-binding activity under neutral pH conditions. Thereby, the antibody half-life in plasma can be further shortened, or the binding activity of the antibody to the extracellular matrix can be further increased, or the removal of antigen from plasma can be further promoted. One skilled in the art can determine the optimal pi value of the antibody of the disclosure A to utilize these properties.

[0677] Within the scope of the disclosure A and B described herein, according to Yeung et al. (J. Immunol. 182:7663-7671 (2009)), the activity of native human IgGl binding to human FcRn is KD 1.7 μΜ in the acidic pH range (pH 6.0), while the activity is hardly detectable in the neutral pH range. Therefore, in order to increase the FcRn-binding activity in the neutral pH range, as an antibody of the disclosure A or B, it can be preferable to use: an antibody or constant region variant or Fc region variant whose human FcRn-binding activity in the acidic pH range is KD 20 μΜ or stronger and whose human FcRn-binding activity in the neutral pH range is comparable to or stronger than that of native human IgGl; preferably an antibody or constant region variant or Fc region variant whose human FcRn-binding activity in the acidic pH range is KD 2.0 μΜ or stronger and whose human FcRn-binding activity in the neutral pH range is KD 40 μΜ or stronger; and more preferably an antibody or constant region variant or Fc region variant whose human FcRn-binding activity in the acidic pH range is KD 0.5 μΜ or stronger and whose human FcRn-binding activity in the neutral pH range is KD 15 μΜ or stronger. The 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 loading human FcRn as an analyte).

[0678] Within the scope of the disclosure A and B described herein, the domain of any structure binding to FcRn can be used as the FcRn-binding domain. In this case, the FcRn-binding domain can be produced without the need to introduce an amino acid modification, or the affinity to FcRn can be increased by introducing an additional modification.

[0679] Within the scope of the disclosure A and B described herein, the starting FcRn-binding domain can include, for example, an Fc region or a constant region of (human) IgG. Any Fc region or constant region can be used as the starting Fc region or the starting constant region as long as the variant of the starting Fc region or the starting constant region is capable of binding to FcRn in the acidic pH range and / or in the neutral pH range. Or, an Fc region or a constant region obtained by further modifying the starting Fc region or the starting constant region, the amino acid residues of which have been modified from the Fc region or the constant region, can also be appropriately used as the Fc region or the constant region. The starting Fc region or the starting constant region can include an Fc region known to be produced by recombination. The starting Fc region or the starting constant region can refer to the polypeptide itself, a composition containing the starting Fc region or the starting constant region, or an amino acid sequence encoding the starting Fc region or the starting constant region, depending on the context. The origin of the starting Fc region or the starting constant region is not limited, and it can be obtained from any organism of a non-human animal or a human. In addition, the starting FcRn-binding domain can be obtained from cynomolgus monkey, a marmoset, a rhesus monkey, a chimpanzee, and a human. The starting Fc region or the starting constant region can be obtained from human IgG1, but is not limited to any particular IgG type. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as an appropriate starting FcRn-binding domain, and the Fc region or the constant region of an IgG type or a subclass derived from any organism can be used as the starting Fc region or as the starting constant region. Examples of native IgG variants or modified forms are described in, for example, 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).

[0680] Within the scope of the disclosure A and B described herein, the amino acid residues of the starting FcRn-binding domain, the starting Fc region, or the starting constant region can contain, for example, one or more mutations: for example, substitution mutations with amino acid residues different from the amino acid residues in the starting Fc region or the starting constant region; insertion of one or more amino acid residues into the amino acid residues in the starting Fc region or the starting constant region; or deletion of one or more amino acid residues from the amino acid residues of the starting Fc region or the starting constant region. The amino acid sequence of the modified Fc region or constant region can preferably be an amino acid sequence containing at least a portion of the Fc region or the constant region that does not occur naturally. The variant must have less than 100% sequence identity or similarity to the starting Fc region or the starting constant region. For example, the variant has about 75% to less than 100%, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, still more preferably about 90% to less than 100%, and yet more preferably about 95% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting Fc region or the starting constant region. In a non-limiting example, at least one amino acid is different between the modified Fc region or constant region of the disclosure A or B and the starting Fc region or the starting constant region.

[0681] Within the scope of the disclosure A and B described herein, the Fc region or the constant region having FcRn-binding activity in the acidic pH range and / or in the neutral pH range can be obtained by any method. Specifically, the variant of the Fc region or the constant region having FcRn-binding activity in the acidic pH range and / or in the neutral pH range can be obtained by modifying the amino acid of the human IgG-type antibody that can be used as the starting Fc region or the starting constant region. The IgG-type antibody Fc region or constant region suitable for modification includes, for example, the Fc region or the constant region of human IgG (IgG1, IgG2, IgG3, and IgG4, and variants thereof), and mutants spontaneously generated therefrom are also included in the IgG Fc region or the constant region. For the Fc region or the constant region of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, many allotype sequences due to genetic polymorphism are described in "Sequences of proteins of immunological interest", NIH publication No. 91-3242, and any of them can be used for the disclosure A or B. Especially, for the human IgG1 sequence, the amino acid sequence according to the EU numbering positions 356 to 358 can be DEL or EEM.

[0682] In one embodiment of the disclosure A or B, the modification to other amino acids is not particularly limited as long as the resulting variant has FcRn-binding activity in the acidic pH range and / or in the neutral pH range, and preferably in the neutral pH range. Sites of the modified amino acids to increase the FcRn-binding activity under neutral pH conditions are described, for example, in WO2013 / 046722. The modification sites include, for example, one or more positions selected from the group consisting of positions 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, 291 to 312, 315 to 320, 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 in the Fc region or constant region of a human IgG antibody (according to EU numbering), as described in WO2013 / 046722. WO2013 / 046722 also describes, as part of the preferred modifications in the Fc region or constant region, for example, modification of one or more amino acids selected from the group consisting of amino acid modification of position 256 to Pro, amino acid modification of position 280 to Lys, amino acid modification of position 339 to Thr, amino acid modification of position 385 to His, amino acid modification of position 428 to Leu, and amino acid modification of position 434 to Trp, Tyr, Phe, Ala, or His (according to EU numbering). The number of amino acids to be modified is not particularly limited, and the modification can be made singly at a single position or at two or more positions. The modification of these amino acid residues can enhance the FcRn binding of the Fc region or constant region of an IgG-type antibody under neutral pH conditions. The modification of these amino acid residues can also be appropriately introduced into the antibody of the disclosure A or B.

[0683] In further or alternative embodiments, it is also possible to use appropriate amino acid modification sites for increasing FcRn-binding activity at acidic pH conditions. In such modification sites, one or more modification sites that allow for an increase in FcRn binding can also be appropriate for the disclosure A or B at neutral pH ranges. Such modification sites include, for example, those reported in WO2011 / 122011, WO2013 / 046722, WO2013 / 046704, and WO2013 / 046722. The amino acid sites and modified amino acid types of such modifications of the constant region or Fc region of a human IgG-type antibody are reported in Table 1 of WO2013 / 046722. WO2013 / 046722 further describes, as particularly preferred, modification sites in the constant region or Fc region, for example, sites at one or more amino acid positions selected from the group consisting of: positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 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 (according to EU numbering). Modification of these amino acid residue positions can also enhance human FcRn binding of the FcRn-binding domain at neutral pH ranges. WO2013 / 046722 further describes, as part of the preferred modifications in the IgG-type constant region or Fc region, for example, modifications of one or more amino acid residues selected from the group consisting of: (a) an amino acid modification at position 237 to Met; (b) an amino acid modification at position 238 to Ala; (c) an amino acid modification at position 239 to Lys; (d) an amino acid modification at position 248 to lie; (e) an amino acid modification at position 250 to any one of Ala, Phe, lie, Met, Gin, Ser, Val, Trp, and Tyr; (f) an amino acid modification at position 252 to any one of Phe, Trp, and Tyr; (g) an amino acid modification at position 254 to Thr; (h) an amino acid modification at position 255 to Glu; (i) an amino acid modification at position 256 to any one of Asp, Glu, and Gin; (j) an amino acid modification at position 257 to any one of Ala, Gly, lie, Leu, Met, Asn, Ser, Thr, and Val; (k) an amino acid modification at position 258 to His; (1) an amino acid modification at position 265 to Ala; (m) an amino acid modification at position 270 to Phe; (n) an amino acid modification at position 286 to Ala or Glu;(o) an amino acid modification at position 289 to His; (p) an amino acid modification at position 297 to Ala; (q) an amino acid modification at position 298 to Gly; (r) an amino acid modification at position 303 to Ala; (s) an amino acid modification at position 305 to Ala; (t) an amino acid modification at position 307 to any one of Ala, Asp, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Val, Trp, and Tyr; (u) an amino acid modification at position 308 to any one of Ala, Phe, lie, Leu, Met, Pro, Gin, and Thr; (v) an amino acid modification at position 309 to any one of Ala, Asp, Glu, Pro, and Arg; (w) an amino acid modification at position 311 to any one of Ala, His, and lie; (x) an amino acid modification at position 312 to Ala or His; (y) an amino acid modification at position 314 to Lys or Arg; (z) an amino acid modification at position 315 to Ala or His; (aa) an amino acid modification at position 317 to Ala; (ab) an amino acid modification at position 325 to Gly; (ac) an amino acid modification at position 332 to Val; (ad) an amino acid modification at position 334 to Leu; (ae) an amino acid modification at position 360 to His; (af) an amino acid modification at position 376 to Ala; (ag) an amino acid modification at position 380 to Ala; (ah) an amino acid modification at position 382 to Ala; (ai) an amino acid modification at position 384 to Ala; (aj) an amino acid modification at position 385 to Asp or His; (ak) an amino acid modification at position 386 to Pro; (al) an amino acid modification at position 387 to Glu; (am) an amino acid modification at position 389 to Ala or Ser; (an) an amino acid modification at position 424 to Ala; (ao) an amino acid modification at position 428 to any one of Ala, Asp, Phe, Gly, His, lie, Lys, Leu, Asn, Pro, Gin, Ser, Thr, Val, Trp, and Tyr; (ap) an amino acid modification at position 433 to Lys; (aq) an amino acid modification at position 434 to Ala, Phe, His, Ser, Trp, and Tyr; and (ar) an amino acid modification at position 436 to His (according to the EU numbering). The number of amino acids to be modified is not particularly limited, and the modification can be made singly at a single position or at two or more positions. Combinations of amino acid modifications at two or more positions include, for example, those shown in Table 2 of WO2013 / 046722. The modifications of these amino acid residues can also be appropriately introduced into the antibodies of Disclosure A and B.

[0684] In one embodiment, the FcRn-binding activity of the FcRn-binding domain of the antibody of disclosure A or B is increased when compared to a reference antibody comprising a native IgG Fc region or constant region or a reference antibody comprising a starting Fc region or starting constant region. That is, the FcRn-binding activity of the Fc region variant or constant region variant of disclosure A or B, or an antibody comprising said variant, is greater than the binding activity of the reference antibody. This can mean that the FcRn-binding activity of the antibody of disclosure A or B can 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, when compared to the FcRn-binding activity of the reference antibody.

[0685] In one embodiment, the amino acid sequence to be modified in the antibody of disclosure A or B can preferably contain a human sequence (a sequence present in a naturally human-derived antibody), so as not to increase the immunogenicity of the antibody when the antibody is administered in vivo (preferably, into a human body). Alternatively, after the modification, mutations can be introduced into positions other than the amino acid modification site in such a way that one or more of the FRs (FR1, FR2, FR3, and FR4) are replaced with a human sequence. Methods for replacing one or more FRs with a human sequence 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). Methods for humanization are known in the art and include, but are not limited to, those reported in Methods 36(1):43-60 (2005).

[0686] In one embodiment, the framework region sequence (also referred to as "FR sequence") of the heavy chain and / or light chain variable region of the antibody of disclosure A or B can contain a human germline framework region sequence. When the framework region sequence is entirely a human germline sequence, the antibody is expected to cause little or no immunogenic reaction when administered to a human (e.g., to treat or prevent a certain disease).

[0687] FR sequences can preferably include, for example, fully human FR sequences such as those shown in V-Base (vbase.mrc-cpe.cam.ac.uk / ). These FR sequences can be adapted for use in either disclosure A or B. Germline sequences can be categorized 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)). Preferred germline sequences can be suitably selected from: V kappa, which is categorized into seven subgroups; V lambda, which is categorized into ten subgroups; and VH, which is categorized into seven subgroups.

[0688] Fully human VH sequences can preferably include, for example, the following VH sequences: subgroup VH1 (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, and VH1-69); subgroup VH2 (e.g., VH2-5, VH2-26, and VH2-70); subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, and VH3-74); subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, and VH4-61); subgroup VH5 (VH5-51); subgroup VH6 (VH6-1); or subgroup VH7 (VH7-4 and VH7-81). These are also described in, for example, Matsuda et al. (J. Exp. Med. 188:1973-1975 (1998)), and one of skill in the art can design based on the information of these sequences as appropriate. Other fully human FR sequences or sequences of regions comparable thereto can also be preferably used.

[0689] Fully human Vκ sequences can preferably include, for example: A20, A30, LI, L4, L5, L8, L9, LI 1, L12, L14, L15, L18, L19, L22, L23, L24, 02, 04, 08, 012, 014, or 018, which are classified as subgroup VK1; Al, A2, A3, A5, A7, A17, A18, A19, A23, Ol, and Oll, which are classified as subgroup VK2; Al 1, A27, L2, L6, L10, L16, L20, and L25, which are classified as subgroup VK3; B3, which is classified as subgroup VK4; B2 (also known as "Vk5-2"), which is classified as subgroup VK5; or AlO, A14, and A26, which are classified as subgroup VK6 (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)). ...

Claims

1. An antibody whose ability to remove an antigen from plasma is promoted, comprising an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions, wherein the antibody comprises (i) a modified light chain consisting of an amino acid sequence comprising amino acid substitutions in the amino acid sequence of SEQ ID NO: 32, wherein the amino acid substitutions consist of Ser18Arg, Gln24Arg, Glu45Lys, Glu79Gln, and Glu107Lys according to Kabat numbering, and wherein the isoelectric point (pi) of the modified light chain is higher than the pi of a light chain consisting of the amino acid sequence of SEQ ID NO: 32; and (ii) a heavy chain modified from any one of (a) to (d): (a) a modified heavy chain consisting of an amino acid sequence containing amino acid substitutions in the amino acid sequence of SEQ ID NO: 24, wherein the amino acid substitutions consist of Glu 16 Gin, Glu 43 Arg, Qn 64 Lys, and Glu 105 Gin according to the numbering of Kabat, and wherein the isoelectric point (pi) of the modified heavy chain is higher than the pi of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 24; (b) a modified heavy chain consisting of an amino acid sequence containing amino acid substitutions in the amino acid sequence of SEQ ID NO: 26, wherein the amino acid substitutions consist of Glu 16 Gin, Glu 43 Arg, Qn 64 Lys, and Glu 105 Gin according to the numbering of Kabat, and wherein the isoelectric point (pi) of the modified heavy chain is higher than the pi of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 26; (c) a modified heavy chain consisting of an amino acid sequence containing amino acid substitutions in the amino acid sequence of SEQ ID NO: 28, wherein the amino acid substitutions consist of Glu 16 Gin, Glu 43 Arg, Qn 64 Lys, and Glu 105 Gin according to the numbering of Kabat, and wherein the isoelectric point (pi) of the modified heavy chain is higher than the pi of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 28; and (d) a modified heavy chain consisting of an amino acid sequence containing amino acid substitutions in the amino acid sequence of SEQ ID NO: 30, wherein the amino acid substitutions consist of Glu 16 Gin, Glu 43 Arg, Qn 64 Lys, and Glu 105 Gin according to the numbering of Kabat, wherein the isoelectric point (pi) of the modified heavy chain is higher than the pi of a heavy chain consisting of the amino acid sequence of SEQ ID NO:

30.

2. The antibody of claim 1, wherein the antibody binds to a soluble antigen.

3. The antibody of claim 1 or 2, wherein the antigen-binding domain is a domain whose antigen-binding activity under high ion concentration conditions is higher than that under low ion concentration conditions.

4. The antibody of claim 1 or 2, wherein the ion concentration is hydrogen ion concentration or calcium ion concentration.

5. The antibody of claim 3, wherein the ion concentration is hydrogen ion concentration or calcium ion concentration.

6. The antibody of claim 4, wherein its KD ratio for an antigen in an acidic pH range to that in a neutral pH range is 2 or more.

7. The antibody of claim 5, wherein its KD ratio for an antigen in an acidic pH range to that in a neutral pH range is 2 or more.

8. The antibody of claim 1 or 2, wherein the extracellular matrix binding activity of the antibody is enhanced compared to the antibody prior to the substitution.

9. The antibody of claim 3, wherein the extracellular matrix binding activity of the antibody is enhanced compared to the antibody prior to the substitution.

10. The antibody of claim 4, wherein the extracellular matrix binding activity of the antibody is enhanced compared to the antibody prior to the substitution.

11. The antibody of any one of claims 5 to 7, wherein the extracellular matrix binding activity of the antibody is enhanced compared to the antibody prior to the substitution.

12. The antibody of claim 1 or 2, wherein the antibody is an IgG antibody.

13. The antibody of claim 3, wherein the antibody is an IgG antibody.

14. The antibody of claim 4, wherein the antibody is an IgG antibody.

15. The antibody of any one of claims 5 to 7, wherein the antibody is an IgG antibody.

16. The antibody of claim 8, wherein the antibody is an IgG antibody.

17. The antibody of claim 9 or 10, wherein the antibody is an IgG antibody.

18. The antibody of claim 11, wherein the antibody is an IgG antibody.

19. A pharmaceutical composition comprising the antibody of any one of claims 1 to 18.

20. The pharmaceutical composition of claim 19 for use in facilitating the removal of an antigen from plasma or for use in enhancing binding to extracellular matrix.

21. A nucleic acid encoding the antibody of any one of claims 1 to 18.

22. A vector comprising the nucleic acid of claim 21.

23. A host cell comprising the vector of claim 22.

24. A method for producing an antibody comprising an antigen binding domain whose antigen binding activity is altered depending on ionic concentration conditions, wherein the method comprises culturing the host cell of claim 23 and collecting the antibody from the cell culture.

25. A method for producing an antibody whose ability to remove an antigen from plasma is facilitated, the antibody comprising an antigen binding domain whose antigen binding activity is altered depending on ionic concentration conditions, wherein the method comprises introducing an amino acid substitution to increase the isoelectric point (pi), wherein the amino acid substitution consists of (i) Ser 18 Arg, Gln 24 Arg, Glu 45 Lys, Glu 79 Gin, and Glu 107 Lys according to Kabat numbering in a light chain consisting of the amino acid sequence of SEQ ID NO: 32 and (ii) Glu 16 Gin, Glu 43 Arg, Gln 64 Lys, and Glu 105 Gin according to Kabat numbering in a heavy chain consisting of the amino acid sequence of SEQ ID NO: 24, 26, 28, or 30.

26. A method for producing a modified antibody comprising an antigen binding domain that facilitates the removal of an antigen from plasma compared to the antibody prior to the modification, and the method comprises: (i) introducing an amino acid substitution to increase the isoelectric point (pi), wherein the amino acid substitution consists of (i) Ser 18 Arg, Gln 24 Arg, Glu 45 Lys, Glu 79 Gin, and Glu 107 Lys according to Kabat numbering in a light chain consisting of the amino acid sequence of SEQ ID NO: 32 and (ii) Glu 16 Gin, Glu 43 Arg, Gln 64 Lys, and Glu 105 Gin according to Kabat numbering in a heavy chain consisting of the amino acid sequence of SEQ ID NO: 24, 26, 28, or 30. (a) introducing amino acid substitutions, wherein the amino acid substitutions consist of (i) Ser 18 Arg, GIn 24 Arg, GIu 45 Lys, GIu 79 GIn, and GIu 107 Lys according to Kabat numbering in a light chain consisting of the amino acid sequence of SEQ ID NO: 32 and (ii) GIu 16 GIn, GIu 43 Arg, GIn 64 Lys, and GIu 105 GIn according to Kabat numbering in a heavy chain consisting of the amino acid sequence of SEQ ID NO: 24, 26, 28, or 30; (b) modifying the antigen binding domain in such a way that the resulting antigen binding activity changes depending on ionic concentration conditions, wherein the (a) and (b) can be performed simultaneously or sequentially; (c) culturing host cells to express nucleic acids encoding the modified antibody; and (d) collecting the modified antibody from the host cell culture.

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