Antibodies comprising an antigen-binding domain with ion concentration dependence, Fc region variants, IL-8-binding antibodies and their uses

Abstract

A non-exclusive aspect provides molecules further improved from antibodies capable of binding antigen in an ion concentration-dependent manner. Alternative non-exclusive aspects provide safe and more favorable Fc region variants with reduced binding to pre-existing ADA. Alternative non-exclusive aspects provide novel IL-8 antibodies that are excellent as drugs.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680005280.2, with an application date of February 4, 2016 and an invention title of "Antibodies Comprising Antigen-Binding Domains with Ion Concentration-Dependent Antigen-Binding Activity, Fc Region Variants, IL-8-Binding Antibodies, and Their Applications". Technical Field

[0002] Cross - reference to related applications

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

[0004] In one non-exclusive aspect, the present disclosure relates to antibodies comprising antigen-binding domains whose antigen-binding activity changes according to ionic concentration conditions, and pharmaceutical compositions containing such antibodies. Nucleic acids encoding these antibodies and host cells containing such nucleic acids are also provided, as well as uses and production methods of the antibodies and pharmaceutical compositions. In another non-exclusive aspect, the present disclosure provides Fc region variants, antibodies containing such variants, and pharmaceutical compositions containing such Fc region variants and antibodies. Nucleic acids encoding the Fc region variants and antibodies, and host cells containing such nucleic acids are also provided, as well as applications and production methods of the Fc region variants and antibodies and pharmaceutical compositions. In a third non-exclusive aspect, the present disclosure provides anti-IL-8 antibodies, pharmaceutical compositions containing such antibodies, nucleic acids encoding such antibodies, and host cells containing such nucleic acids. Production methods of the IL-8 antibodies and pharmaceutical compositions and their applications in treating, for example, IL-8-related disorders are also provided. Background Art

[0005] Antibodies are attracting attention as drugs because they are highly stable in plasma and have few side effects. Many IgG-type therapeutic antibodies are on the market, and even now many therapeutic antibodies are in development (Reichert et al., Nat. Biotechnol. 23: 1073-1078 (2005) (NPL1); Pavlou et al., Eur. J. Pharm. Biopharm. 59(3): 389-396 (2005) (NPL2)). At the same time, various technologies are being developed for second-generation therapeutic antibodies; 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) (NPL3)). The doses of therapeutic antibodies are usually very high, and thus the development of therapeutic antibodies encounters problems such as difficulty in producing subcutaneous formulations and high production costs. Methods for improving the pharmacokinetics, pharmacodynamics and antigen-binding properties of therapeutic antibodies provide a way to reduce the doses and production costs associated with therapeutic antibodies.

[0006] Substitution of amino acid residues in the constant region provides a method for improving antibody pharmacokinetics (Hinton et al., J. Immunol. 176(1): 346-356 (2006) (NPL4); Ghetie et al., Nat. Biotechnol. 15(7): 637-640 (1997)) (NPL5). Affinity maturation techniques provide methods for enhancing the antigen-neutralizing ability of antibodies (Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24): 8466-8471 (2005) (NPL6); Wu et al., J. Mol. Biol. 368: 652 (2007) (NPL7)), and one or more mutations can be introduced into one or more amino acid residues in the CDRs and / or framework regions of the antibody variable domain to increase antigen-binding activity. Improving the antigen-binding properties of antibodies can improve the in vitro biological activity of antibodies or reduce the dose, and can further improve the in vivo (in the body) efficacy (Wu et al., J. Mol. Biol. 368: 652-665 (2007) (NPL8)).

[0007] The amount of antigen that can be neutralized by one antibody molecule depends on the affinity of the antibody for the antigen; and thus, it may be possible to neutralize the antigen with a small amount of antibody by increasing the affinity. The affinity of an antibody for an antigen can be increased using various known methods (see, e.g., Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466 - 8471(2005)(NPL6)). Additionally, if it can bind covalently to the antigen such that the affinity is infinite, it would be theoretically possible to neutralize one antigen molecule with one antibody molecule (2 antigen molecules when the antibody is bivalent). Nevertheless, a limitation in therapeutic antibody development to date is that one antibody molecule typically binds and neutralizes only one antigen molecule (2 antigen molecules when the antibody is bivalent). Recently, it has been reported that using an antibody that binds to the antigen in a pH - dependent manner (also referred to hereinafter as a "pH - dependent antibody" or "pH - dependent - binding antibody") enables one antibody molecule to bind and neutralize multiple antigen molecules (see, e.g., WO2009 / 125825(PTL1); Igawa et al., Nat. Biotechnol. 28:1203 - 1207(2010)(NPL9)). The pH - dependent antibody binds strongly to the antigen under neutral pH conditions in plasma and dissociates from the antigen under acidic pH conditions within the endosome of the cell. After dissociating from the antigen, the antibody is recycled to the plasma via FcRn and subsequently freely binds and neutralizes another antigen molecule; and thus one pH - dependent antibody can repeatedly bind and neutralize multiple antigen molecules.

[0008] Recently, it has been reported that antibody recycling properties can be achieved by focusing on the difference in calcium (Ca) ion concentration between plasma and endosomes and using an antibody showing calcium - dependent antigen - antibody interaction (also referred to hereinafter as a "calcium ion concentration - dependent antibody") (WO2012 / 073992(PTL2)). (Hereinafter, pH - dependent antibodies and "calcium ion concentration - dependent antibodies" are collectively referred to as "pH / Ca concentration - dependent antibodies".)

[0009] By binding to FcRn, IgG antibodies have a long retention time in plasma. The binding between IgG antibodies and FcRn is strong under acidic pH conditions (e.g., pH 5.8), but there is little binding under neutral pH conditions (e.g., pH 7.4). IgG antibodies are non - specifically internalized into cells and return to the cell surface under acidic pH conditions in endosomes by binding to FcRn in the endosomes. IgG then dissociates from FcRn under neutral pH conditions in plasma.

[0010] It has been reported that pH-dependent antibodies modified to increase their FcRn binding at neutral pH have the ability to repeatedly bind and remove antigen molecules from plasma; and thus the use of such antibodies allows for the removal of antigens from plasma (WO2011 / 122011 (PTL3)). According to this report, compared to pH-dependent antibodies containing the Fc region of natural IgG antibodies, pH-dependent antibodies modified to increase their FcRn binding at neutral pH (e.g., pH 7.4) can further accelerate the removal of antigens (WO2011 / 122011 (PTL3)).

[0011] Meanwhile, when mutations are introduced into the Fc region of an IgG antibody to eliminate its binding to FcRn at acidic pH, it may no longer recycle from endosomes into plasma, which significantly weakens the retention of the antibody in plasma. Subsequently, methods for increasing FcRn binding at acidic pH have been reported as methods for improving the plasma retention of IgG antibodies. Introducing amino acid modifications into the Fc region of an IgG antibody to increase its FcRn binding at acidic pH can enhance the efficacy of recycling from endosomes into plasma, which thereby results in improved plasma retention. For example, it has been reported that modifying M252Y / S254T / T256E (YTE; Dall'Acqua et al., J. Biol. Chem. 281: 23514-235249 (2006) (NPL10)), M428L / N434S (LS; Zalevsky et al., Nat. Biotechnol. 28: 157-159 (2010)) (NPL11), and N434H (Zheng et al., Clin. Pharm. & Ther. 89(2): 283-290 (2011) (NPL12)) results in an increased antibody half-life relative to native IgG1.

[0012] However, in addition to considering that immunogenicity or the incidence of aggregates may be worse in antibodies containing such Fc region variants (whose FcRn binding is increased at neutral pH or acidic pH), it has also been further reported that there is increased binding to anti-drug antibodies (hereinafter also referred to as "pre-existing ADA") (e.g., rheumatoid factor) present in patients prior to the administration of therapeutic antibodies (WO2013 / 046722 (PTL4), WO2013 / 046704 (PTL5)).

[0013] WO2013 / 046704 (PTL5) reported that compared with unmodified native Fc, an Fc region variant containing specific mutations (represented by modification of two residues of Q438R / S440E according to EU numbering) increased the binding to FcRn under acidic pH conditions and also showed a significant reduction in the binding to rheumatoid factor. However, WO2013 / 046704 (PTL5) did not specifically demonstrate that the Fc region variant has better plasma retention than antibodies with native Fc regions.

[0014] Therefore, there is a need for a safe and more favorable Fc region variant with further improved plasma retention that does not show binding to pre-existing ADA.

[0015] Antibody-dependent cell cytotoxicity (hereinafter shown as "ADCC"), complement-dependent cell cytotoxicity (hereinafter shown as "CDC"), and antibody-dependent cell phagocytosis (ADCP) (which is the phagocytosis of target cells mediated by IgG antibodies) have been reported as effector functions of IgG antibodies. 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 as "Fcγ receptor", "FcgR", "Fcγ receptor", or "FcγR" within the scope of the disclosure A described herein) present on the surface of effector cells such as killer cells, natural killer cells, or activated macrophages. In humans, the FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isotypes have been reported as FcγR family proteins, and their respective allotypes have also been reported (Jefferis et al., Immunol. Lett. 82: 57-65 (2002) (NPL13)). The balance of the respective affinities of an antibody for activating receptors including FcγRIa, FcγRIIa, FcγRIIIa, or FcγRIIIb and inhibitory receptors including FcγRIIb is an important factor in optimizing antibody effector functions.

[0016] Various techniques for increasing or improving the activity of therapeutic antibodies against antigens have been reported to date. For example, the activity of antibodies to bind activating FcγR(s) plays an important role in the cytotoxicity of antibodies, and thus, antibodies targeting membrane-type antigens and having increased cytotoxicity due to enhanced binding of activating FcγR(s) have been developed. See, e.g., 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. U SA 95:652 - 656 (1998) (NPL16); Clynes et al., Nat. Med. 6:443 - 446 (2000) (NPL17)). Similarly, the binding activity to inhibitory FcγR (FcγRIIb in humans) plays an important role in immunosuppressive activity and agonist activity, and thus, there has been research on antibodies targeting membrane-type antigens and having increased inhibitory FcγR-binding activity (Li et al., Proc. Nat. Acad. Sci. USA. 109(27):10966 - 10971 (2012) (NPL18)). In addition, the effect of FcγR binding of antibodies that bind soluble antigens has 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 FcγRIIb binding is used as a drug, a reduced risk may be expected from the production of anti-drug antibodies (Desai et al., J. Immunol. 178(10):6217 - 6226 (2007) (NPL20)).

[0017] Recently, it has been reported that introducing amino acid modifications into the Fc region of IgG antibodies to increase the activity of antibodies targeting soluble antigens to bind activating and / or inhibitory FcγRs 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 FcγRIIb-binding activity from the Fc region of natural IgG antibodies but have reduced activity against other activating FcγRs (WO2014 / 163101 (PTL12)).

[0018] Compared with an antibody having an FcRn-mediated recycling mechanism, the plasma retention of a soluble antigen is very short, and thus the soluble antigen can exhibit increased plasma retention and plasma concentration by binding to an antibody having such a recycling mechanism (e.g., an antibody not having the characteristics of a pH / Ca concentration-dependent antibody). 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 still exacerbate the pathological symptoms caused by other physiological functions resulting from the increased plasma retention and / or plasma concentration of the antigen due to antibody binding. In this case, in addition to the above-described exemplary methods of modifying an antibody to accelerate antigen removal, for example, the formation of multivalent immune complexes using multiple pH / Ca concentration-dependent antibodies and multiple antigens, and methods of increasing the binding to FcRn, FcγR(s), and 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 antibody variable region, the isoelectric point (pI) of the antibody can be increased or decreased regardless of the type of antigen or antibody, and the half-life of the antibody in the blood can be controlled without substantially reducing the antigen-binding activity of the antibody (WO2007 / 114319 (PTL14): a technique of substituting amino acids mainly in FR; WO2009 / 041643 (PTL15): a technique of substituting amino acids mainly in CDR). These documents indicate that it may be possible to extend the plasma half-life of an antibody by decreasing the pI of the antibody, and conversely, to shorten the plasma half-life of an antibody by increasing the pI of the antibody.

[0020] Regarding the modification of the charge of amino acid residues in the constant region of an antibody, it has been reported that the uptake of an antigen by cells can be promoted by modifying the charge of specific one or more amino acid residues, particularly in the CH3 domain, thereby increasing the pI of the antibody, and it has also been described that this modification preferably does not interfere with the binding to FcRn (WO2014 / 145159 (PTL16)). It has also been reported that modifying the charge of amino acid residues in the constant region (mainly the CH1 domain) of an antibody to decrease the pI can extend the half-life of the antibody in plasma, and in combination with amino acid residue mutations that increase the binding to FcRn, it 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 that increases or decreases the binding to FcRn or FcγR(s), it is unclear whether there is an effect in promoting the plasma retention of the antibody or the removal of antigen from the plasma.

[0022] The extracellular matrix (ECM) is a structure that covers cells in vivo and is mainly composed of glycoproteins such as collagen, proteoglycan, 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 proteins administered to living organisms. The blood concentration of the VEGF-Trap molecule (which is a fusion protein between a VEGF receptor and Fc) was detected when administered subcutaneously (Holash et al., Proc. Natl. Acad. Sci., 99(17): 11393-11398 (2002) (NPL21)). The plasma concentration of the VEGF-Trap molecule with a high pI administered subcutaneously was low, and thus its bioavailability was low. The modified VEGF-Trap molecule (whose pI was lowered by amino acid substitution) had a higher plasma concentration, and its bioavailability might be improved. In addition, the change in bioavailability was 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 (PTL18) reported that there is an inverse correlation between antibody binding to ECM and plasma retention, and thus, when compared with an antibody that binds to ECM, an antibody molecule that does not bind to ECM has better plasma retention.

[0025] Thus, a technique for reducing extracellular matrix binding in order to improve the in vivo bioavailability and plasma retention of proteins has been reported. In contrast, the advantages of increasing the binding of an antibody to ECM have not been identified to date.

[0026] Human IL-8 (interleukin 8) is a member of the chemokine family and has a length of 72 or 77 amino acid residues. The term "chemokine" is a general term for a family of proteins with a molecular weight of 8-12 kDa and containing 4 cysteine residues that form intermolecular disulfide bonds. Chemokines are classified into CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines according to the characteristics of cysteine arrangement. 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 antiparallel β-sheet and has a structure in which the C-terminal α-helix passes through and covers the β-sheet. The IL-8 monomer, in the case of the 72-amino acid form of IL-8, includes two disulfide cross-links 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 β-sheets of the two monomers, as there is no covalent binding between the molecules of the homodimer.

[0028] In response to stimulation by inflammatory cytokines, IL-8 expression is induced in various cells such as peripheral blood monocytes, tissue macrophages, NK cells, fibroblasts, and vascular endothelial cells (Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619(2014)(NPL22)).

[0029] In normal tissues, chemokines are usually undetectable or only weakly detectable, but are strongly detectable at sites of inflammation and participate in the induction of inflammation by promoting lymphocyte infiltration into the inflamed tissue site. IL-8 is a pro-inflammatory chemokine known to activate neutrophils, promote the expression of cell adhesion molecules, and enhance neutrophil adhesion to vascular endothelial cells. IL-8 also has neutrophil chemotactic ability and IL-8 is produced at sites of damaged tissue to promote the chemotaxis of neutrophils adhered to vascular endothelial cells into the tissue and to induce inflammation with neutrophil infiltration. IL-8 is also known to be an effective angiogenic factor for endothelial cells and to participate in promoting tumor angiogenesis.

[0030] Inflammatory diseases associated with elevated (e.g., excessive) IL-8 levels include inflammatory diseases of the skin such as inflammatory keratosis (e.g., psoriasis), atopic dermatitis, contact dermatitis; chronic inflammatory disorders (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, aphthous ulcers, chorditis, and inflammation associated with the use of artificial organs and / or artificial blood vessels. Elevated (e.g., excessive) IL-8 levels are also associated with malignancies such as ovarian cancer, lung cancer, prostate cancer, gastric cancer, breast cancer, melanoma, head and neck cancer, and kidney cancer; sepsis caused by infection; cystic fibrosis; and pulmonary fibrosis. (See, e.g., Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014) (NPL22), 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) (NPL23)), however, they have not been launched yet. So far, only one pharmaceutical composition containing an IL-8 antibody is available, which is a murine anti-IL-8 antibody used as an external drug for psoriasis. There is a need for new anti-IL-8 antibodies for treating diseases.

[0032] [Citation List]

[0033] [Patent Document]

[0034] [PTL1]WO2009 / 125825

[0035] [PTL2]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. U SA 95: 652 - 656 (1998)

[0069] [NPL17] Clynes et al., Nat. Med. 6:443 - 446 (2000)

[0070] [NPL18] Li et al., Proc. Nat. Acad. Sci. USA. 109(27):10966 - 10971 (2012)

[0071] [NPL19] Scappaticci et al., J. Natl. Cancer Inst. 99(16):1232 - 1239 (2007)

[0072] [NPL20] Desai et al., J. Immunol. 178(10):6217 - 6226 (2007)

[0073] [NPL21] Holash et al., Proc. Natl. Acad. Sci., 99(17):11393 - 11398 (2002)

[0074] [NPL22] Russo et al., Exp. Rev. Clin. Immunol. 10(5):593 - 619 (2014)

[0075] [NPL23] Desai et al., J. Immunol. 178(10):6217 - 6226 (2007) Summary of the Invention

[0076] In a non - exclusive aspect, a non - limiting objective of embodiments of Disclosure A is to provide molecules that have improved pharmacokinetic properties relative to antibodies, such as improved antibody half - life and / or ion - concentration - dependent antigen - binding properties for clearing antigen from plasma.

[0077] In a non - exclusive aspect, a non - limiting objective of embodiments of Disclosure B is to provide safe and more beneficial Fc region variants that have an increased half - life and reduced binding to pre - existing anti - drug antibodies (ADAs).

[0078] In a non-exclusive aspect, a non-limiting objective of embodiments of disclosure C is to provide anti-IL-8 antibodies having pH-dependent binding affinity for IL-8. Other embodiments relate to anti-IL-8 antibodies that, when administered to an individual, have the effect of rapidly removing IL-8 compared to a reference antibody. In another embodiment, disclosure C relates to anti-IL-8 antibodies that can stably maintain their IL-8-neutralizing activity when administered to an individual. In some embodiments, the anti-IL-8 antibodies exhibit reduced immunogenicity. In other embodiments, disclosure C relates to methods of producing and using the above anti-IL-8 antibodies. Another alternative non-limiting objective of disclosure C is to provide new anti-IL-8 antibodies that can be included in pharmaceutical compositions.

[0079] In a non-exclusive aspect, within the scope of disclosure A provided herein, the inventors unexpectedly 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 according to ion concentration conditions)) to remove an antigen from plasma can be promoted by modifying at least one amino acid residue exposed on the surface of the antibody to increase its isoelectric point (pI). In another non-exclusive aspect, the inventors found that an ion concentration-dependent antibody having an increased pI can further increase the extracellular matrix binding of the antibody. Thus, without being limited to a particular theory, the inventors found that antigen removal from plasma can be increased by increasing the binding of the antibody to the extracellular matrix.

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

[0081] In a non-exclusive aspect, within the scope of the disclosure C provided herein, the inventors have developed a number of pH-dependent anti-IL-8 antibodies (anti-IL-8 antibodies that bind IL-8 in a pH-dependent manner). From the results of various validations, the inventors have identified pH-dependent anti-IL-8 antibodies that, when administered to an individual, have the effect of rapidly removing IL-8 compared to reference antibodies. 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] Furthermore, within the scope of the disclosure C, the inventors have successfully obtained anti-IL-8 antibodies comprising an Fc region with an FcRn-binding affinity relative to the native Fc region, the FcRn-binding affinity of which increases at acidic pH. In an alternative aspect, the inventors have successfully obtained anti-IL-8 antibodies comprising an Fc region with a binding affinity for pre-existing ADA relative to the native Fc region, the binding affinity of which for pre-existing ADA is reduced. In an alternative aspect, the inventors have successfully obtained anti-IL-8 antibodies comprising an Fc region with a plasma half-life that is increased relative to the plasma half-life of the native Fc region. In an alternative aspect, the inventors have successfully obtained pH-dependent anti-IL-8 antibodies comprising an Fc region with a binding affinity for effector receptors that is reduced relative to the binding affinity of the native Fc region for effector receptors. In different aspects, the inventors have identified nucleic acids encoding the above anti-IL-8 antibodies. In another aspect, the inventors have also obtained hosts comprising the above nucleic acids. In another aspect, the inventors have developed a method for producing the above anti-IL-8 antibodies, which comprises culturing the above host. In another aspect, the inventors have developed a method for promoting the removal of IL-8 from an individual relative to a reference antibody, which comprises administering the above anti-IL-8 antibody to the individual.

[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 changes according to ionic concentration conditions, wherein its isoelectric point (pI) is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody;

[0085] [2][1] the antibody as described above, wherein the antigen is a soluble antigen;

[0086] [3][1] or [2] the antibody as described above, wherein the antigen-binding domain is a domain whose antigen-binding activity is higher under high ionic concentration conditions than under low ionic concentration conditions;

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

[0088] The antibody according to [4], wherein the KD ratio in the acidic pH range to that in the neutral pH range, KD(acidic pH range) / KD(neutral pH range), is 2 or higher for the antigen;

[0089] The antibody according to any one of [1] to [5], wherein in the antigen-binding domain, at least one amino acid residue is replaced with histidine, or at least one histidine is inserted;

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

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

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

[0093] 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) replacing a negatively charged amino acid residue with an uncharged amino acid residue;

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

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

[0097] 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] The antibody according to

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

[0099] 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 selected from the group consisting of in the CDR or FR according to

[0100] Kabat numbering

[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]

[13] The antibody as described above, wherein at least one amino acid residue is modified at 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] The antibody according to any one of

[11] to

[14] , wherein at least one amino acid residue is modified at a position 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 in the constant region;

[0111] The antibody according to

[15] , wherein at least one amino acid residue is modified at a position 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 in the constant region;

[0112] The antibody according to

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

[0113] The antibody according to any one of [1] to

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

[0114] The antibody according to

[18] , wherein the FcγR is FcγRIIb;

[0115] The antibody according to 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 binding activity for FcγRIIb, and wherein the FcγRIIb-binding activity is maintained or enhanced and the binding activity for activating FcγRs is reduced compared to a reference antibody that differs only in that its constant region is the constant region of natural IgG;

[0116] The antibody according to any one of [1] to

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

[0117] The antibody according to any one of [1] to

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

[0118] The antibody according to any one of [1] to

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

[0119] A pharmaceutical composition comprising the antibody according to any one of [1] to

[23] ;

[0120] The pharmaceutical composition according to

[24] , which is used for promoting the removal of antigens from plasma;

[0121] The pharmaceutical composition according to

[24] or

[25] , which is used for enhancing the binding of the antibody to the extracellular matrix;

[0122] A nucleic acid encoding the antibody according to any one of [1] to

[23] ;

[0123] A vector comprising the nucleic acid according to

[27] ;

[0124] A host cell comprising the vector according to

[28] ;

[0125] A method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ionic concentration conditions, wherein the method comprises culturing the host cell according to

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

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

[0127] The method according to [30B][30A], wherein at least one amino acid residue is modified at a position selected from the following

[0128] (I) Select positions from the group consisting of the following in CDR or FR: 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 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; or

[0129] (II) Select positions from the group consisting of the following in the constant region: 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] The method according to

[31] [30A] or [30B], wherein the amino acid residue modification includes modifications selected from the group consisting of:

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

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

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

[0134] (d) Replacing or inserting histidine in CDR or FR.

[0135]

[32]

[30] , or the method according to any one of [30A] to [30C], which further optionally includes any one or more of the following:

[0136] Compared with a reference antibody,

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

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

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

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

[0141] (e) selecting an antibody having maintained or enhanced FcγRIIb-binding activity and reduced binding activity to one or more activating FcγRs preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa;

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

[0143] (g) selecting an antibody having an increased isoelectric point (pI);

[0144] (h) determining the isoelectric point (pI) of the collected antibody and subsequently selecting an antibody having an increased isoelectric point (pI); and

[0145] (i) selecting an antibody whose antigen-binding activity changes or increases according to ionic concentration conditions.

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

[0147] [A1] An antibody having a constant region, wherein at least one amino acid residue selected from the group of modification sites identical to the group of modification sites defined in

[15] or

[16] in the constant region is modified;

[0148] [A2] The antibody of [A1], which further has 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 selected from the group of modification sites identical to the group of modification sites defined in

[13] or

[14] in the CDR and / or FR is modified;

[0149] [A3]An antibody having a constant region, wherein at least one amino acid residue in a group of modification sites selected from the same group of modification sites as those defined in

[15] or

[16] in the constant region is modified to increase its pI;

[0150] [A4][A3]The antibody as described above, which further has 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 a group of modification sites selected from the same group of modification sites as those defined in

[13] or

[14] in the CDR and / or FR is modified;

[0151] [A5]An antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ionic concentration conditions, wherein the antibody has a constant region, and wherein at least one amino acid residue in a group of modification sites selected from the same group of modification sites as those defined in

[15] or

[16] in the constant region is modified;

[0152] [A6][A5]The antibody as described above, which further has 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 a group of modification sites selected from the same group of modification sites as those defined in

[13] or

[14] in the CDR and / or FR is modified;

[0153] [A7][1] To

[23] and the use of the antibody according to any one of [A1] to [A6] in the preparation of a medicament for promoting the removal of an antigen from plasma;

[0154] [A8][1] To

[23] and the use of the antibody according to any one of [A1] to [A6] in the preparation of a medicament for increasing extracellular matrix binding;

[0155] [A9][1] To

[23] and the use of the antibody according to any one of [A1] to [A6] for the removal of an antigen from plasma; and

[0156] [A10][1] To

[23] and the use of the antibody according to any one of [A1] to [A6] for increasing extracellular matrix binding.

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

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

[31] ,

[32] .

[0158] According to various embodiments, Disclosure A includes a combination (partial or full) of one or more elements described in any one of [1] to

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

[31] ,

[32] , and [A1] to [A11] above, provided that the combination is not technically inconsistent with the common general knowledge in the art. For example, in some embodiments, Disclosure A includes a method for producing a modified antibody comprising an antigen-binding domain that promotes the removal of an antigen from plasma compared to before antibody modification, wherein the method comprises:

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

[0160] (I) positions selected from the group consisting of in the CDR or FR at positions: 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 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; or

[0161] (II) positions selected from the group consisting of in the constant region at positions: 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;

[0162] (b) Modifying the antigen-binding domain in such a way that the resulting antigen-binding activity varies according to ionic concentration conditions, where (a) and (b) can be carried out simultaneously or sequentially;

[0163] (c) Culturing a host cell to express a 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 before modification,

[0167] (e) Selecting an antibody capable of promoting antigen removal from plasma;

[0168] (f) Selecting an antibody having enhanced binding activity to the extracellular matrix;

[0169] (g) Selecting an antibody having enhanced FcγR-binding activity at neutral pH conditions (e.g., pH 7.4);

[0170] (h) Selecting an antibody having enhanced FcγRIIb-binding activity at neutral pH conditions (e.g., pH 7.4);

[0171] (i) Selecting an antibody having retained or enhanced FcγRIIb-binding activity and reduced binding activity to one or more activating FcγRs, preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa;

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

[0173] (k) Selecting an antibody having an increased isoelectric point (pI);

[0174] (l) Confirming the isoelectric point (pI) of the collected antibody and subsequently selecting an antibody having an increased isoelectric point (pI); and

[0175] (m) Selecting an antibody whose antigen-binding activity varies or increases according to ionic concentration conditions.

[0176] Another embodiment of Disclosure A relates to, for example, but not limited to:

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

[0178] (a) Modify the nucleic acid encoding the antibody before modification to change the charge of at least one amino acid residue at a position 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;

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

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

[0181] [D2] A method for prolonging 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: 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.

[0182] In one 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 Ala at position 434 according to EU numbering; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440;

[0184]

[34]

[33] The Fc region variant as described above, wherein the FcRn-binding domain comprises Ala at position 434 according to EU numbering; Arg or Lys at position 438; and Glu or Asp at position 440;

[0185] The Fc region variant as described in

[35] ,

[33] or

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

[0186]

[36] The Fc region variant as described in

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

[0187] The Fc region variant according to any one of

[33] to

[36] , wherein the FcRn-binding domain comprises a combination of amino acid substitutions selected from the group consisting of: according to EU numbering, N434A / Q438R / S440E; N434A / Q438R / S440D; N434A / Q438K / S440E; N434A / Q438K / S440D; N434A / Y436T / Q438R / S440E; N434A / Y436T / Q438R / S440D; N434A / Y436T / Q438K / S440E; N434A / Y436T / Q438K / S440D; N434A / Y436V / Q438R / S440E; N434A / Y436V / Q438R / S440D; N434A / Y436V / Q438K / S440E; N434A / Y436V / Q438K / S440D; N434A / R435H / F436T / Q438R / S440E; N434A / R435H / F436T / Q438R / S440D; N434A / R435H / F436T / Q438K / S440E; N434A / R435H / F436T / Q438K / S440D; N434A / R435H / F436V / Q438R / S440E; N434A / R435H / F436V / Q438R / S440D; N434A / R435H / F436V / Q438K / 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;and L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E;

[0188]

[38]

[37] The Fc region variant as described above, 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]

[33] to

[38] The Fc region variant as described in any one of the above, wherein compared with the Fc region of natural IgG, its FcRn-binding activity is enhanced under acidic pH conditions (e.g., pH 5.8);

[0191]

[40]

[33] to

[39] The Fc region variant as described in any one of the above, wherein compared with the Fc region of natural IgG, its binding activity to anti-drug antibody (ADA) is not significantly enhanced under neutral pH conditions;

[0192]

[41]

[40] The Fc region variant as described above, wherein the anti-drug antibody (ADA) is rheumatoid factor (RF);

[0193]

[42]

[33] to

[41] The Fc region variant as described in any one of the above, wherein compared with the Fc region of natural IgG, its plasma clearance rate (CL) is reduced, plasma retention time is increased, or plasma half-life (t1 / 2) is increased;

[0194]

[43]

[33] to

[42] The Fc region variant as described in any one of the above, wherein compared with a reference Fc region variant comprising a combination of amino acid substitutions N434Y / Y436V / Q438R / S440E according to EU numbering, its plasma retention is increased;

[0195]

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

[33] to

[43] ;

[0196]

[45]

[44] The antibody as described above, wherein the antibody is an IgG antibody;

[0197]

[46] A pharmaceutical composition comprising the antibody as described in

[44] or

[45] ;

[0198]

[47] The pharmaceutical composition as described in

[46] , which is used to increase the retention of the antibody in plasma;

[0199]

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

[33] to

[43] or the antibody as described in

[44] or

[45] ;

[0200]

[49] A vector comprising the nucleic acid of

[48] ;

[0201]

[50] A host cell comprising the vector as described in

[49] ;

[0202]

[51] A method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the variant, which comprises culturing the host cell as described in

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

[0203]

[52] The method as described in

[51] , which may also optionally include any one or more of the following:

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

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

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

[0207] (d) Selecting an antibody comprising an Fc region variant that can promote the removal of antigen from plasma compared to a reference antibody comprising the Fc region of natural 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 according to EU numbering; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440.

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

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

[33] to

[43] or an antibody as described in

[44] or

[45] in the preparation of a medicament for increasing retention in plasma;

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

[33] to

[43] or an antibody as described in

[44] or

[45] in the preparation of a medicament for not significantly increasing the binding activity against an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of natural IgG;

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

[33] to

[43] or an antibody as described in

[44] or

[45] for increasing retention in plasma;

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

[33] to

[43] or an antibody as described in

[44] or

[45] for not significantly increasing the binding activity against an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of natural IgG; and

[0214] [B5]An Fc region variant or an antibody comprising the variant, which is obtained by any one of the methods described in

[51] ,

[52] , and

[53] .

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

[33] to

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

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

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

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

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

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

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

[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 to human IL-8, which contains at least one amino acid substitution in at least one of the following (a) to (f) and binds to IL-8 in a pH-dependent manner:

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

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

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

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

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

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

[0230]

[55]

[54] The anti-IL-8 antibody described above, which contains amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO: 68, arginine at position 11 of the amino acid sequence of SEQ ID NO: 68, and tyrosine at position 3 of the amino acid sequence of SEQ ID NO: 69;

[0231] The anti-IL-8 antibody according to

[56] ,

[54] or

[55] , which comprises 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 according to any one of

[54] to

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

[0233]

[58] The anti-IL-8 antibody according to any one of

[54] to

[57] , which comprises (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 according to any one of

[54] to

[58] , which comprises (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 according to any one of

[54] to

[59] , which comprises 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 according to any one of

[54] to

[60] , which comprises an Fc region having at least one property selected from the following properties (a) to (f):

[0237] (a) An Fc region having an increased binding affinity for FcRn at acidic pH relative to the native Fc region;

[0238] (b) An Fc region having a reduced binding affinity for pre-existing ADA relative to the native Fc region;

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

[0240] (d) An Fc region having a reduced plasma clearance relative to the native Fc region; and

[0241] (e) a binding affinity of the Fc region for effector receptors that is reduced relative to the binding affinity of the native Fc region for effector receptors; and

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

[0243]

[62]

[61] The anti-IL-8 antibody as described above, 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]

[62] The anti-IL-8 antibody as described above, which comprises an Fc region containing one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E;

[0245]

[64]

[63] The anti-IL-8 antibody as described above, wherein the Fc region comprises the amino acid substitutions of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E;

[0246]

[65]

[63] The anti-IL-8 antibody as described above, wherein the Fc region comprises the amino acid substitutions of L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E;

[0247]

[66] An anti-IL-8 antibody, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 81 and a light chain containing the amino acid sequence of SEQ ID NO: 82;

[0248]

[67] An anti-IL-8 antibody, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 80 and a light chain containing the amino acid sequence of SEQ ID NO: 82;

[0249]

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

[54] to

[67] ;

[0250]

[69] A vector comprising the nucleic acid as described in

[68] ;

[0251]

[70] A host cell comprising the vector as described in

[69] ;

[0252]

[71] A method for producing an anti-IL-8 antibody, which comprises culturing the host described in

[70] ;

[0253]

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

[71] , which comprises separating the antibody from the culture supernatant;

[0254]

[73] A pharmaceutical composition, which comprises the anti-IL-8 antibody described in any one of

[54] to

[67] , and a pharmaceutically acceptable carrier;

[0255]

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

[54] to

[67] , which is for use in a pharmaceutical composition;

[0256]

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

[54] to

[67] , which is for treating a disorder in which there is an excess of IL-8;

[0257]

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

[54] to

[67] in the preparation of a pharmaceutical composition for a disorder in which there is an excess of IL-8;

[0258]

[77] A method for treating a patient suffering from a disorder in which there is an excess of IL-8, which comprises administering to the individual the anti-IL-8 antibody described in any one of

[54] to

[67] ;

[0259]

[78] A method for promoting the removal of IL-8 from an individual, which comprises administering to the individual the anti-IL-8 antibody described in any one of

[54] to

[67] ;

[0260]

[79] A pharmaceutical composition, which comprises the anti-IL-8 antibody described in any one of

[54] to

[67] , wherein the antibody binds to IL-8 and binds to the 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) evaluating the binding of an anti-IL-8 antibody to the extracellular matrix,

[0263] (b) selecting an anti-IL-8 antibody that strongly binds to the extracellular matrix,

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

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

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

[0267] Use of the anti-IL-8 antibody according to any one of

[54] to

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

[0268] [C2] Use of the anti-IL-8 antibody according to any one of

[54] to

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

[0269] [C3] Use of the anti-IL-8 antibody according to any one of

[54] to

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

[0270] [C4] Use of the anti-IL-8 antibody according to any one of

[54] to

[67] for inhibiting angiogenesis;

[0271] [C5] Use of the anti-IL-8 antibody according to any one of

[54] to

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

[0272] [C6] Use of the anti-IL-8 antibody according to any one of

[54] to

[67] for inhibiting the promotion of neutrophil migration;

[0273] [C7] The anti-IL-8 antibody according to any one of

[54] to

[67] , which is used for inhibiting the accumulation of biologically active IL-8;

[0274] [C8] A method for inhibiting the accumulation of biologically active IL-8, wherein the method comprises administering to an individual the anti-IL-8 antibody according to any one of

[54] to

[67] ;

[0275] [C9] A pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, which comprises the anti-IL-8 antibody according to any one of

[54] to

[67] ;

[0276] [C10] The anti-IL-8 antibody according to any one of

[54] to

[67] , which is used for inhibiting angiogenesis;

[0277] [C11] A method for inhibiting angiogenesis in an individual, wherein the method comprises administering to the individual the anti-IL-8 antibody according to any one of

[54] to

[67] ;

[0278] [C12] A pharmaceutical composition for inhibiting angiogenesis, which comprises the anti-IL-8 antibody according to any one of

[54] to

[67] ;

[0279] [C13] The anti-IL-8 antibody according to any one of

[54] to

[67] , which is used for inhibiting the promotion of neutrophil migration;

[0280] [C14]A method for inhibiting the 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 inhibiting the promotion of neutrophil migration, the pharmaceutical composition 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] , which is used for treating a disease with excessive IL-8;

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

[54] to

[67] in the preparation of a pharmaceutical composition for treating a disease with excessive IL-8;

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

[54] to

[67] for treating a disease with excessive IL-8;

[0285] [C19]A method for treating a disease with excessive 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 treating a disease with excessive IL-8, which comprises 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] , which is used for promoting the removal of IL-8;

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

[54] to

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

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

[54] to

[67] for promoting the removal of IL-8;

[0290] [C24]A method for promoting the 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 promoting the removal of IL-8, which comprises 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: according to EU numbering positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440.

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

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

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

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

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

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

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

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

[0301] [C29][C28] The anti-IL-8 antibody as described above, comprising an Fc region comprising one or more amino acid substitutions selected from the group consisting of: according to EU numbering (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E; or (b) L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E.

[0302] [C30][C26]The anti-IL-8 antibody described above, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 81 and a light chain containing the amino acid sequence of SEQ ID NO: 82.

[0303] [C31][C26]The anti-IL-8 antibody described above, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 80 and a light chain containing the amino acid sequence of SEQ ID NO: 82.

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

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

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

[0307] [C35]A method for producing an anti-IL-8 antibody, which comprises culturing the host cell described in [C34].

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

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

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

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

[0312] According to various embodiments, disclosure C includes a combination (partial or whole) of one or more elements described in the above

[54] to

[80] and [C1] to [C39], provided that the combination is not technically inconsistent with the common general knowledge in the art. Brief Description of the Drawings

[0313] Figure 1

[0314] Figure 1 ​​Shows the change in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with an antibody that binds to the human IL-6 receptor in a pH-dependent manner and whose constant region is the constant region of native IgG1 (low_pI-IgG1), or an antibody in which the pI of the variable region in the antibody has been increased (high_pI-IgG1).

[0315] Figure 2

[0316] Figure 2 Shows the change in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered separately with an antibody that binds to the human IL-6 receptor in a pH-dependent manner and is endowed with binding to FcRn under neutral pH conditions (low_pI-F939), and an antibody in which the pI of the variable region in the antibody has been increased (mid_pI-F939, high_pI-F939).

[0317] Figure 3

[0318] Figure 3 Shows the change in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered separately with an antibody that binds to the human IL-6 receptor in a pH-dependent manner and whose FcγR binding at neutral pH is increased (low_pI-F1180), and an antibody in which the pI of the variable region in the antibody has been increased (mid_pI-F1180, high_pI-F1180).

[0319] Figure 4

[0320] Figure 4 Shows the change in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered separately with an antibody that binds to the human IL-6 receptor in a pH-dependent manner and whose constant region is the constant region of native IgG1 (low_pI-IgG1), an antibody containing an Fc region variant in which the Fc region in the antibody has increased FcRn binding under neutral pH conditions (low_pI-F11), and an antibody in which the pI of the variable region in these antibodies has been increased (high_pI-IgG1, high_pI-F11), where the plasma concentration of soluble human IL-6 receptor remains in a steady state.

[0321] Figure 5

[0322] Figure 5 ​​​​​​​​Show the degree of extracellular matrix binding for each of three types of antibodies with different pIs that bind to the human IL-6 receptor in a pH-dependent manner (low_pI-IgG1, medium_pI-IgG1, and high_pI-IgG1) and two types of antibodies with different pIs that do not bind to the human IL-6 receptor in a pH-dependent manner (low_pI(NPH)-IgG1 and high_pI(NPH)-IgG1). "NPH" means pH-independent within the scope of Disclosure A described herein.

[0323] Figure 6

[0324] Figure 6 Show the relative value of the soluble human FcγRIIb binding (measured by BIACORE®) of antibodies containing Fc region variants whose respective pIs are 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, with the value of Ab1H-P600 set to 1.00.

[0325] Figure 7

[0326] Figure 7 Show the relative value of the rate of antibody uptake into cells of a cell line expressing hFcγRIIb for antibodies containing Fc region variants whose respective pIs are increased by modifying one amino acid residue in the constant region of Ab1H-P600, each evaluated with the value of Ab1H-P600 set to 1.00.

[0327] Figure 8

[0328] Figure 8 Show the degree of binding of Fv4-IgG1 (which has the Fc region of natural human IgG1) to rheumatoid factor in the serum of each RA patient.

[0329] Figure 9

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

[0331] Figure 10

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

[0333] Figure 11 ​​​​​​​​​​​

[0334] Figure 11 Show the binding degree of Fv4-N434H (which contains an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[0335] Figure 12

[0336] Figure 12 Show the binding degree of Fv4-F1847m (which contains an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[0337] Figure 13

[0338] Figure 13 Show the binding degree of Fv4-F1848m (which contains an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[0339] Figure 14

[0340] Figure 14 Show the binding degree of Fv4-F1886m (which contains an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[0341] Figure 15

[0342] Figure 15 Show the binding degree of Fv4-F1889m (which contains an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[0343] Figure 16

[0344] Figure 16 Show the binding degree of Fv4-F1927m (which contains an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[0345] Figure 17

[0346] Figure 17 Show the binding degree of Fv4-F1168m (which contains an Fc region variant with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[0347] Figure 18

[0348] Figure 18 ​​​​​​​​​​​​​​​Show the mean binding of Fv4-IgG1 (which has the Fc region of native human IgG1 and each antibody contains a new Fc region variant where the Fc region has an increased binding to each FcRn) to rheumatoid factor in the sera of RA patients.

[0349] Figure 19

[0350] Figure 19 Show the change in plasma concentration of each anti-human IgE antibody in cynomolgus monkeys when administered with OHB-IgG1 (which is an anti-human IgE antibody and has the Fc region of native human IgG1 and each antibody contains a new Fc region variant (where each Fc region contains 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 Show the change in plasma concentration of anti-human IL-6 receptor antibody in human FcRn transgenic mice when administered with Fv4-IgG1 (which is an anti-human IL-6 receptor antibody and has the Fc region of native human IgG1), or Fv4-F1718 (which has increased antibody binding to FcRn at acidic pH conditions).

[0353] Figure 21

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

[0355] Figure 22

[0356] Figure 22 Show the change in human IL-8 concentration in mouse plasma when H998 / L63 or H89 / L118 is administered to mice at 2 mg / kg (in a mixture with human IL-8).

[0357] Figure 23

[0358] Figure 23 Show the change in human IL-8 concentration in mouse plasma when H89 / L118 is administered to mice at 2 mg / kg or 8 mg / kg (in a mixture with human IL-8).

[0359] ​​​​​​​​​​Figure 24

[0360] Figure 24 Show the change in the concentration of human IL-8 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 Show the change in the relative value of antibody concentration-dependent chemiluminescence of antibody Hr9, H89 / L118 or H553 / L118 before storage in plasma.

[0363] Figure 25B

[0364] Figure 25B Show the change in the relative value of antibody concentration-dependent chemiluminescence of antibody Hr9, H89 / L118 or H553 / L118 after storage in plasma for one week.

[0365] Figure 25C

[0366] Figure 25C Show the change in the relative value 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 Show 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 Show the predicted frequency of ADA occurrence for each anti-IL-8 antibody (H496 / L118, H496v1 / L118, H496v2 / L118, H496v3 / L118, H1004 / L118 or H1004 / L395) and the predicted frequency of ADA occurrence for other pre-existing therapeutic antibodies ADA, predicted by EpiMatrix.

[0371] Figure 28A ​​​​​​​​​​​​​​

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

[0373] Figure 28B

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

[0375] Figure 28C

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

[0377] Figure 29

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

[0379] Figure 30

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

[0381] Figure 31

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

[0383] Figure 32

[0384] Figure 32 Shows the predicted frequencies of ADA occurrence for H1009 / L395 and H1004 / L395 and the predicted frequencies of ADA occurrence for other pre-existing therapeutic antibodies, predicted by EpiMatrix.​​​​​​​​​​​​

[0385] Figure 33

[0386] Figure 33 Shows the change in the concentration of each anti-human IL-8 antibody in cynomolgus monkey plasma when administered with H89 / L118-IgG1 (which has the variable region of H89 / L118 and the Fc region of native human IgG1), and each antibody (H89 / L118-F1168m, H89 / L118-F1847m, H89 / L118-F1848m, H89 / L118-F1886m, H89 / L118-F1889m, and H89 / L118-F1927m) containing an Fc region variant with increased binding to FcRn.

[0387] Figure 34

[0388] Figure 34 Shows the binding of antibodies having the variable region of H1009 / L395 and whose Fc region is a variant (F1886m, F1886s, or F1974m) for each FcγR.

[0389] Figure 35

[0390] Figure 35 Shows the change in the concentration of human IL-8 in mouse plasma when an anti-IL-8 antibody is administered to human FcRn transgenic mice as a mixture with human IL-8. In this case, the anti-IL-8 antibody is H1009 / L395-IgG1 (2 mg / kg) containing the variable region of H1009 / L395 and the Fc region of native human IgG1, or H1009 / L395-F1886s (2, 5, or 10 mg / kg) containing the variable region of H1009 / L395 and a modified Fc region.

[0391] Figure 36

[0392] Figure 36 Shows the change in antibody concentration in cynomolgus monkey plasma when administered with Hr9-IgG1 or H89 / L118-IgG1 (both containing the Fc region of native human IgG1), or H1009 / L395-F1886s or H1009 / L395-F1974m (both containing a modified Fc region).

[0393] Figure 37

[0394] Figure 37 Shows the IgE plasma concentration-time curves of some anti-IgE antibodies in C57BL6J mice with respect to antibody variable region modification.​​​​​​​​​​

[0395] Figure 38A

[0396] Figure 38( Figure 38A - 38D ) shows the Octet sensorgrams of the 25 selected pH-dependent and / or calcium-dependent antigen-binding clones.

[0397] Figure 38B

[0398] Figure 38B is Figure 38A a continuation of.

[0399] Figure 38C

[0400] Figure 38C is Figure 38B a continuation of.

[0401] Figure 38D

[0402] Figure 38D is Figure 38C a continuation of.

[0403] Figure 39

[0404] Figure 39 Shows the C5 plasma concentration-time curves in C57BL6J mice for some anti-C5 bispecific antibodies in terms of antibody variable region modifications.

[0405] Figure 40

[0406] Figure 40 Shows the IgE plasma concentration-time curves in C57BL6J mice for some anti-IgE antibodies in terms of antibody variable region modifications. Specific Embodiments

[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 understood correctly also in the "Detailed" section, without being limited by any patent practice, regulations, guidelines, etc. that may attempt to interpret narrowly what is described in the Examples in the country where this patent application is desired to be granted.

[0409] Disclosure A or Disclosure B

[0410] ​​​​​​​​​​​​In some embodiments, Disclosure A relates to antibodies comprising an antigen-binding domain whose antigen-binding activity changes according to ionic concentration conditions, wherein the isoelectric point (pI) is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody (herein, also referred to as "ionic concentration-dependent antibody with increased pI" within the scope of Disclosure A; and the antigen-binding domain of the antibody is also referred to as "ionic concentration-dependent antigen-binding domain with increased pI"). This invention is partly based on the unexpected discovery by the inventors that antigen removal from plasma can be facilitated by ionic concentration-dependent antibodies whose isoelectric point (pI) is increased by modifying at least one amino acid residue that may 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 increase with ionic concentration-dependent antibodies having an increased (elevated) pI. This invention is also partly based on the unexpected discovery by the inventors that this beneficial effect is brought about by combining the following two completely different concepts: an ionic concentration-dependent antigen-binding domain or an ionic concentration-dependent antibody; and an antibody whose pI is increased by modifying at least one amino acid residue that may be exposed on the surface (herein, also referred to as "antibody with increased pI" within the scope of Disclosure A; and an antibody whose pI is decreased (lowered) by modifying at least one amino acid residue that may be exposed on the surface is also referred to as "antibody with decreased pI" within the scope of Disclosure A). This invention is thus classified as a pioneering research that may lead to significant technological innovations in the field to which Disclosure A belongs (e.g., the medical field).

[0411] Generally, for example, antibodies that comprise an antigen-binding domain and whose pI is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody and that are further modified such that the antigen-binding activity of the antigen-binding domain changes according to ionic concentration conditions are also included within the scope of Disclosure A described herein (herein, the antibody is also referred to as "ionic concentration-dependent antibody with increased pI" within the scope of Disclosure A).

[0412] Generally, for example, antibodies containing an ionic concentration-dependent antigen-binding domain (where at least one amino acid residue that may be exposed on the surface of the antibody has a different charge from the corresponding position in the antibody before modification (natural antibody (e.g., natural Ig antibody, preferably natural IgG antibody), or reference or parental antibody (e.g., antibody before modification, or antibody before or during library construction, etc.)) and whose net antibody pI is increased) are also included in Disclosure A described herein (the antibody is also referred to as "ionic concentration-dependent antibody with increased pI" within the scope of Disclosure A described herein).

[0413] Generally, for example, antibodies containing an ion concentration-dependent antigen-binding domain (the pI of which is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody before modification (natural antibody (e.g., natural Ig antibody, preferably natural IgG antibody, or reference or parental antibody (e.g., antibody before modification, or antibody before or during library construction, etc.))) are also included in the disclosure A described herein (such antibodies 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 an ion concentration-dependent antigen-binding domain (wherein at least one amino acid residue that may be exposed on the surface of the antibody is modified to increase the pI of the antibody) are also included in the disclosure A described herein (such antibodies 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 disclosures A and B described herein, "amino acid" includes not only natural amino acids but also non-natural amino acids. Within the scope of the disclosures A and B described herein, an amino acid or amino acid residue may be represented by a single letter (e.g., A), a three-letter code (e.g., Ala), or both (e.g., Ala(A)).

[0416] When used within the scope of Disclosures A and B, "modification of an amino acid", "modification of an amino acid residue", or equivalent terms can be understood to include, but are not limited to, chemically modifying one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) specific amino acids (residues) in the antibody amino acid sequence, or adding, deleting, substituting, or inserting one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) amino acids in the antibody amino acid sequence. The nucleic acid encoding the amino acid sequence can be modified, for example, by site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)) or overlap extension PCR; via affinity maturation of the antibody, or by chain shuffling of the antibody heavy or light chain; or by antigen-based selection using a phage display library (Smith et al., Methods Enzymol. 217:228-257 (1993)) for amino acid addition, deletion, substitution, or insertion; and these can be performed individually or in suitable combinations. The amino acid modifications are preferably performed by adding, deleting, substituting, or inserting amino acids by replacing one or more amino acid residues in the antibody amino acid sequence with different amino acids (respectively), and amino acid sequence modification by humanization or chimerization can be performed by methods known in the art. Alterations or modifications of amino acids (residues), such as amino acid addition, deletion, substitution, or insertion, can also be performed on the antibody variable region or antibody constant region of a recombinant antibody to be used for preparing an antibody against Disclosure A or B.

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

[0418] Amino acid residues are classified into groups such as the following based on the nature of the side chains in the structure: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, and Ile; (2) neutral, hydrophilic: Cys, Ser, Thr, Asn, and Gln; (3) acidic: Asp and Glu; (4) basic: His, Lys, and Arg; (5) residues affecting chain orientation: Gly and Pro; and (6) aromatic: Trp, Tyr, and Phe.

[0419] A substitution of an amino acid residue within each group is called a conservative substitution, while a substitution of an amino acid residue between different groups is called a non-conservative substitution. A substitution of an amino acid residue can be a conservative substitution, a non-conservative substitution, or a combination thereof. A variety of known suitable methods can be used to substitute amino acids with those different from 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 may be used, in which a non-natural amino acid is ligated to a suppressor tRNA complementary to the UAG codon (amber codon), which is a stop codon (Clover Direct (ProteinExpress)).

[0420] Within the scope of Disclosures A and B described herein, it is to be understood that the structure of an "antigen" is not limited to a specific structure, provided that the antigen includes an epitope that binds an antibody. The antigen can be an inorganic or organic substance. The antigen can be any ligand, including various cytokines, e.g., interleukins, chemokines, and cell growth factors. Alternatively, generally, for example, a receptor that exists 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). In addition, 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 antibodies of Disclosures A and B is preferably a soluble antigen present in the biological fluid of a subject (e.g., the biological fluids described in WO2013 / 125667, preferably plasma, interstitial fluid, lymph, ascites, or pleural fluid) (within the scope of Disclosures A and B described herein, the subject to which the antibody is to be administered (applied) can actually be any animal, e.g., a human, a mouse, etc.); however, the antigen can also be a membrane antigen.

[0422] Within the scope of the disclosures 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" (wherein the target molecule may be an antigen or an antibody), or a comparable term may also be more specifically expressed using any other parameter in addition to the half-life parameter (t1 / 2) in plasma, such as the mean residence time in plasma, the clearance rate (CL) in plasma, and the area under the concentration curve (AUC) (Pharmacokinetics: Enshuniyoru Rikai (Understanding through Practice), Nanzando). These parameters can be specifically evaluated, for example, by performing noncompartmental analysis according to the protocol attached to the in vivo kinetics analysis software WinNonlin (Pharsight). Those skilled in the art know that these parameters are generally related to each other.

[0423] Within the scope of the disclosures A and B described herein, an "epitope" refers to an antigenic determinant in an antigen and means the site on the antigen to which the 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 the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues constituting the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be specified based on its specific sugar chain structure. The antigen-binding domains of disclosures A and B can bind to a single epitope or different epitopes on the 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 usually constitute the epitope do not exist continuously as a primary sequence. An antibody recognizes a conformational epitope in the three-dimensional structure of a peptide or protein. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-directed spin labeling, and electron paramagnetic resonance (Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.)).

[0426] Within the scope of Disclosures A and B described herein, "antibody" is not particularly limited and is used in the broadest sense as long as it can bind to a target antigen. Non-limiting examples of antibodies broadly include known common antibodies (e.g., natural immunoglobulins (abbreviated as "Ig")), and molecules and variants derived therefrom, such as Fab, Fab', F(ab')2, diabodies (Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; WO93 / 11161; Peer et al., Nature Nanotechnology 2: 751-760 (2007)), minibodies (Orita et al., Blood 105: 562-566 (2005)), scaffold proteins, single-arm antibodies (including all embodiments of the single-arm antibodies described in WO2005 / 063816), multispecific antibodies (e.g., bispecific antibodies: antibodies specific for two different epitopes, including antibodies that recognize different antigens and antibodies that recognize different epitopes on the same antigen). Within the scope of Disclosures A and B described herein, "bispecific antibody" is not limited to but can be prepared as, for example, an antibody molecule having a common L chain as described in WO2005 / 035756, or by the method described in WO2008 / 119353, where two general types of antibodies having IgG4-like constant regions are mixed, causing an exchange reaction between the two types of said antibodies (referred to as the "Fab-arm exchange" method by those skilled in the art). In an alternative embodiment, they can be structural antibodies having a structure in which the heavy chain variable region and the light chain variable region are linked together into a single chain (e.g., sc(Fv)2). Alternatively, they can be antibody-like molecules (e.g., scFv-Fc) resulting from linking an Fc region (a constant region lacking the CH1 domain) to an scFv (or sc(Fv)2) (where the heavy chain variable region (VH) is linked to the light chain variable region (VL)). Multispecific antibodies composed of scFv-Fc have an (scFv)2-Fc structure, where 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) (US2013 / 0171138), functional fragments thereof, substances equivalent in function thereto, and glycan-modified variants thereof.In this text, natural IgG (such as natural IgG1) refers to a polypeptide containing the same amino acid sequence as naturally occurring IgG (such as natural IgG1) and belonging to the antibody type encoded essentially by immunoglobulin gamma genes. Natural IgG can be its spontaneous mutants, etc.

[0427] Generally, when an antibody has a structure substantially the same as or similar to natural IgG, the 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 linked by disulfide bonds (SS bonds) and form a heterodimer. The heterodimers can be linked together by disulfide bonds and form a Y-shaped heterotetramer. The two heavy chains or light chains can be the same as or different from each other.

[0428] For example, an IgG antibody can be cleaved by papain into two Fab units (regions) and a single Fc unit (region), where the papain cleaves the hinge region (also referred to as "hinge" within the scope of the disclosure A and B described herein), and the heavy chain Fab region is linked to the Fc region. Generally, the Fab region contains an antigen-binding domain. Since phagocytes such as lymphocytes and macrophages have receptors (Fc receptors) capable of binding to the Fc region and can recognize and phagocytose antigens (opsonization) via the Fc receptors. At the same time, the Fc region is involved in mediating immune responses such as ADCC or CDC and has effector functions that induce responses after the antibody binds to the antigen. It is known that antibody effector functions vary according to the type (isotype) of immunoglobulin. The Fc region of the IgG type will show, for example, the region from cysteine at position 226 or proline at position 230 (EU numbering) across to the C-terminus; however, the Fc region is not limited thereto. The Fc region can be appropriately obtained by partially digesting monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies, etc. with a protease such as pepsin, and then eluting the adsorbed fraction from a protein A or protein G column.

[0429] Within the scope of the disclosure A and B described herein, the positions of amino acid residues in the antibody variable region (one or more CDRs and / or one or more FRs) are shown according to Kabat, while the positions of amino acid residues in the constant region or Fc region are shown according to EU numbering based on the amino acid positions of 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, a "library" can refer to a collection of molecules (population) such as a variety of antibodies with sequence variations, where their respective sequences can be the same or different from each other; a variety of fusion polypeptides containing said antibodies; or nucleic acids or oligonucleotides encoding these amino acid sequences, as detailed in WO2013 / 125667 (for example, 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 antibodies that can have different antigen - binding activities and account for a certain proportion among many independent clones with different sequences in the library. In one embodiment, an immune library constructed based on lymphocytes from an animal immunized with a specific antigen, an infected patient, a person with elevated antibody levels in the blood due to immunity, or the antibody genes of a patient with cancer or an autoimmune disease can be suitably used as a random variable - region library. In an alternative embodiment, a naive library containing naive sequences (antibody sequences without bias in the library construction) (which is constructed from antibody genes derived from lymphocytes of healthy individuals) can also be suitably used as a randomized variable - region library (Gejima et al., Human Antibodies 11:121 - 129 (2002)); Cardoso et al., Scand. J. Immunol. 51:337 - 344 (2000)). The amino acid sequences containing naive sequences can refer to those obtained from the naive library. In an alternative embodiment, a synthetic library of CDR sequences in which the V gene from genomic DNA or a reconstructed functional V gene is replaced with oligonucleotides containing a set of sequences encoding codon groups of appropriate lengths can also be suitably used as a random variable - region library. In this case, it may also be possible to replace only the CDR3 sequence of the heavy chain because sequence alterations are observed in the CDR3 gene. The standard way to generate amino acid diversity in the antibody variable region can be to increase the alterations of amino acid residues at positions that are likely to be exposed on the antibody surface.

[0431] In one embodiment, in the case of antibodies of Disclosure A or B, for example, having a structure that is substantially the same as or similar to the structure of a native Ig antibody, they generally have a variable region ("V region") [heavy chain variable region ("VH region") and light chain variable region ("VL region")] and a constant region ("C region") ["heavy chain constant region ("CH region") and light chain constant region ("CL region")]. The CH region is further divided into three: CH1 to CH3. Generally, the Fab region of the heavy chain contains the VH region and CH1, and generally the Fc region of the heavy chain contains CH2 and CH3. Generally, the hinge region is located between CH1 and CH2. In addition, the variable region generally has complementarity determining regions ("CDRs") and framework regions ("FRs"). Generally, each of the VH region and the VL region has 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 and light chains interact and form the antigen-binding domain of the antibody. On the other hand, in the case where only a single CDR is present, while having a lower antigen-binding affinity compared to the case where six CDRs are present, it has the ability to recognize and bind an antigen.

[0432] Ig antibodies are classified into multiple types (isotypes) based on the structural differences in their constant regions. In many mammals, they are classified into five immunoglobulin types based on the structural differences in their constant regions: IgG, IgA, IgM, IgD, and IgE. In addition, in the case of humans, IgG has four types: IgG1, IgG2, IgG3, and IgG4; and IgA has two subclasses: IgA1 and IgA2. Heavy chains are classified into γ chains, μ chains, α chains, δ chains, and ε chains based on the differences in their 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 chains and κ chains, and all immunoglobulins have one of these two.

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

[0434] Within the scope of Disclosures A and B described herein, an "antigen-binding domain" can have any structure as long as it binds the antigen under study. The domain can include, for example, the variable regions of the heavy and light chains of an antibody (e.g., 1 to 6 CDRs); a module of about 35 amino acids called the A domain, which is contained in Avimers (cell membrane proteins present in vivo) (WO2004 / 044011 and WO2005 / 040229); Adnectin containing 10Fn3 domains that bind to proteins in the glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925); Affibody, which has a scaffold IgG-binding domain (a 58-amino acid triple helix bundle that constitutes protein A) (WO1995 / 001937); designed ankyrin repeat proteins (DARPins), which are regions exposed on the molecular surface of ankyrin repeats (ARs), the ankyrin repeats having a structure in which subunits with a 33-amino acid residue turn, two antiparallel helices, and loops are stacked repeatedly (WO2002 / 020565); Anticalins, etc., which are four-ring regions on one side of a centrally twisted barrel structure of eight antiparallel strands highly conserved in a supporting molecule such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462); and a concave region formed by a parallel sheet structure inside a horseshoe structure formed by stacking repeats of leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs), which do not have an immunoglobulin structure and are used in the acquired immune system of jawless vertebrates such as lampreys and hagfishes (WO2008 / 016854). Preferred antigen-binding domains of Disclosure A or B can include those having the variable regions of IgG heavy and light chains of an antibody, and more specifically, ScFv, single-chain antibody, Fv, scFv2 (single-chain Fv2), Fab, and F(ab')2.

[0435] In one embodiment of Disclosure A, the "ion concentration" is not particularly limited and refers to the hydrogen ion concentration (pH) or the metal ion concentration. Herein, a "metal ion" can be any of the ions in Group I elements other than hydrogen, such as alkali metals and copper group elements, Group II elements such as alkaline earth metals and zinc group elements, Group III elements other than boron, Group IV elements other than carbon and silicon, Group VIII elements such as iron group and platinum group elements, elements belonging to Subgroup A of Groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. A metal atom has the property of releasing valence electrons to become a cation. This is called the ionization tendency. A metal with a strong ionization tendency is considered to be chemically active.

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

[0437] In one embodiment of Disclosure A, "one or more ion concentration conditions" may be conditions that focus on the difference in the biological behavior of ion concentration-dependent antibodies between low ion concentrations and high ion concentrations. Additionally, "antigen-binding activity changes according to the ion concentration condition" may mean that the antigen-binding activity of the ion concentration-dependent antigen-binding domain or ion concentration-dependent antibody of Disclosure A or B changes between low ion concentrations and high ion concentrations. Such cases include, for example, those having higher (stronger) or lower (weaker) antigen-binding activity at high ion concentrations than at low ion concentrations, but are not limited thereto.

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

[0439] In one embodiment in the case of disclosure A, an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain having an increased pI, and an ion concentration-dependent antibody having an increased pI can be obtained from a library mainly composed of antibodies that differ in sequence (are variable) 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 according to ion concentration conditions. The antigen-binding domain can preferably be located within the light chain variable region (which can be modified) and / or the heavy chain variable region (which can be modified). In addition, to construct the library, the light chain or heavy chain variable region can be combined with the 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 hydrogen or calcium ion concentration, 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 in a 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 antigen-binding activity according to ion concentration. In addition, in the case where the ion concentration is the calcium ion concentration, the library includes, for example, those in which the heavy chain variable region sequence of SEQ ID NO: 5 (6RL#9-IgG1) or SEQ ID NO: 6 (6KC4-1#85-IgG1) is combined with the light chain variable region constructed as a library of random variable region sequences or the light chain variable region having a germline sequence.

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

[0441] Whether the antigen-binding activity of an antigen-binding domain or an antibody containing the domain changes according to 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 by the methods described in WO2012 / 073992. For example, the antigen-binding activity of an antigen-binding domain or an antibody containing the domain can be measured at low and high calcium ion concentrations and compared. In this case, conditions other than the calcium ion concentration can preferably be the same. In addition, conditions other than the calcium ion concentration in the determination of antigen-binding activity can be appropriately selected by those skilled in the art. The antigen-binding activity can be determined, for example, under the conditions of HEPES buffer at 37°C, or using BIACORE (GE Healthcare), etc.

[0442] In one embodiment in the case of Disclosure A, preferably, the antigen-binding activity of an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain having an increased pI, or an ion concentration-dependent antibody having an increased pI is higher 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 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 curve, or flow cytometer.

[0444] Alternatively, for example, the dissociation rate constant (kd) can also be used as another index representing the binding activity ratio. When using the dissociation rate constant (kd) instead of the dissociation constant (KD) as the index representing the antigen binding activity ratio, the ratio of the dissociation rate constant (kd) under low-calcium-ion-concentration conditions to the dissociation rate constant (kd) under high-calcium-ion-concentration conditions, i.e., kd (low calcium ion concentration 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 additionally more preferably 30 or more. The upper limit of the kd (low calcium ion concentration condition) / kd (high calcium ion concentration condition) value 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 the 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 known methods, for example, by BIACORE (GE healthcare) or flow cytometry.

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

[0447] The method can include, for example:

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

[0449] (b) determining the antigen binding activity of the antigen binding domain or antibody under high calcium ion concentration conditions; and

[0450] (c) selecting an antigen binding domain or antibody whose antigen binding activity is lower under low calcium ion concentration conditions than under high calcium ion concentration conditions.

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

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

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

[0454] (c) Isolate the antigen-binding domain or antibody dissociated in step (b).

[0455] Alternatively, the method may include, for example:

[0456] (a) Contact an antigen with an antigen-binding domain or antibody, or a library thereof, under conditions of low calcium ion concentration;

[0457] (b) Select an antigen-binding domain or antibody that does not bind to the antigen or has low antigen-binding ability in step (a);

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

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

[0460] Alternatively, the method may include, for example:

[0461] (a) Contact an antigen-binding domain or antibody, or a library thereof, with a column immobilizing the antigen under conditions of high calcium ion concentration;

[0462] (b) Elute the antigen-binding domain or antibody bound to the column in step (a) from the column under conditions of low calcium ion concentration; and

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

[0464] Alternatively, the method may include, for example:

[0465] (a) Pass an antigen-binding domain or antibody, or a library thereof, through a column immobilizing the antigen under conditions of low calcium ion concentration to collect the antigen-binding domain or antibody that is eluted without binding to the column;

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

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

[0468] Alternatively, the method may include, for example:

[0469] (a) Contact an antigen with an antigen-binding domain or antibody, or a library thereof, under conditions of high calcium ion concentration;

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

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

[0472] (d) Isolate 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 obtained thereby.

[0474] In the case of 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 with an increased pI obtained by modifying the charge of at least one amino acid residue capable of being exposed on its surface. In an alternative embodiment, in the case of introducing amino acids that change the binding activity of the ion concentration-dependent antigen-binding domain into the sequence, they can be introduced together with the modification of the charge of at least one amino acid residue that may be exposed on the surface of the antigen-binding domain or antibody to increase the pI.

[0475] Alternatively, in the case of Invention A, for example, a pre-existing antigen-binding domain or antibody, a pre-existing library (such as a phage library); antibodies prepared from hybridomas obtained by immunizing animals or from B cells of immunized animals, or their libraries; or antigen-binding domains, antibodies, or libraries obtained by introducing natural or non-natural amino acid mutations capable of chelating calcium therein (described below) (for example, a library having an increased content of amino acids capable of chelating calcium, or a library in which amino acids capable of chelating calcium are introduced at specific sites) can be used.

[0476] In one embodiment in the case of disclosure A, when the ionic concentration is the calcium ion concentration, there is no limitation on the amino acid type for changing the ionic concentration-dependent antigen-binding domain having an increased pI or the binding activity of the ionic concentration-dependent antigen-binding domain, as long as they can form 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, when the antigen-binding domain has any calcium-binding motif such as the calcium-binding motif of C-type lectin, e.g., ASGPR, CD23, MBR, or DC-SIGN, the antigen-binding activity of the domain can be changed according to the calcium ion concentration conditions. The calcium-binding motif may include (in addition to those mentioned 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 case of disclosure A, when the ionic concentration is the calcium ion concentration, amino acids having metal chelating activity can be used as the amino acids for changing the ionic concentration-dependent antigen-binding domain having an increased pI or the binding activity of the ionic concentration-dependent antigen-binding domain. For example, any amino acid can be appropriately used as the amino acid having metal chelating activity as long as they can form a calcium-binding motif. Specifically, the amino acids include those having an electron-donating property. The amino acids preferably include, but are not limited to, Ser (S), Thr (T), Asn (N), Gln (Q), Asp (D), and Glu (E).

[0478] The position of the amino acid with metal chelating activity in the antigen-binding domain is not limited to a specific position. In one embodiment, the amino acid may be located at any position in the variable region of the heavy chain and / or the variable region of the light chain that can form the antigen-binding domain. At least one amino acid residue that causes a calcium ion concentration-dependent change in the antigen-binding activity of the antibody may be included, for example, in one or more of the CDRs (CDR1, CDR2, and CDR3) and / or FRs (FR1, FR2, FR3, and FR4) of the heavy chain and / or the light chain. One or more amino acid residues may be placed, for example, at one or more of positions 95, 96, 100a, and 101 in the heavy chain CDR3 according to Kabat numbering; at one or more of positions 30, 31, and 32 in the light chain CDR1 according to Kabat numbering; at position 50 in the light chain CDR2 according to Kabat numbering; and / or at position 92 in the light chain CDR3 according to Kabat numbering. Those amino acid residues may be placed individually 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 may be designed to contain their binding motifs. For the above purposes, for example, cadherin domains; EF hands contained in calmodulin; C2 domains contained in protein kinase C; Gla domains contained in blood coagulation factor IX; C-type lectins of asialoglycoprotein receptor or mannose-binding receptor; A domains contained in LDL receptor; Annexin; thrombospondin type 3 domains; and EGF-like domains may be appropriately used.

[0480] In one embodiment, when the ionic concentration is the hydrogen ion concentration (pH), the concentration condition of protons, that is, the nuclei of hydrogen atoms, is used synonymously with the condition of the hydrogen index (pH). The amount of hydrogen ion activity in an aqueous solution is represented by aH + and in this case, pH is defined as -log10aH + . When the ionic strength of the aqueous solution is low (e.g., lower than 10 -3 ), aH + is almost equal to the hydrogen ion strength. For example, at 25 °C and 1 atmosphere, the ion product of water is Kw = aH + *aOH = 10 -14 ; thus, for pure water, aH + = aOH = 10 -7In this case, pH = 7 is neutral, an aqueous solution with a pH less than 7 is acidic, and an aqueous solution with a pH greater than 7 is alkaline. Thus, the hydrogen ion concentration condition can be a condition that focuses on the difference in the biological behavior of pH-dependent antibodies at high hydrogen ion concentrations (acidic pH range) and at low hydrogen ion concentrations (neutral pH range) for hydrogen ion concentration conditions or pH conditions. For example, in the case of Disclosure A, "the antigen-binding activity under high hydrogen ion concentration (acidic pH range) conditions is lower than the antigen-binding activity under low hydrogen ion concentration (neutral pH range) conditions" can mean that the antigen-binding activity of an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with an increased pI, or an ion concentration-dependent antibody with an increased pI is weaker at a pH selected from pH 4.0 to pH 6.5, preferably pH 4.5 to pH 6.5, more preferably pH 5.0 to pH 6.5, and still more preferably pH 5.5 to pH 6.5 than at a pH selected from pH 6.7 to pH 10.0, preferably pH 6.7 to pH 9.5, more preferably pH 7.0 to pH 9.0, and still more preferably pH 7.0 to pH 8.0. Preferably, the above expression can mean that the antigen-binding activity at the pH of early endosomes in vivo is weaker than the antigen-binding activity in vivo at plasma pH; and specifically means that the antigen-binding activity of an antibody, for example, at pH 5.8 is weaker than the antigen-binding activity at, for example, pH 7.4.

[0481] Whether the antigen-binding activity of an antigen-binding domain or an antibody containing the domain changes according to the hydrogen ion concentration condition can be easily evaluated by known methods, for example, by the assay methods described in the case of Disclosure A herein or in WO2009 / 125825. For example, the antigen-binding activity of an antigen-binding domain or an antibody containing the domain against the antigen under study can be measured and compared at low and high hydrogen ion concentrations. In this case, it is preferred that the conditions other than the hydrogen ion concentration are the same. In determining the antigen-binding activity, those skilled in the art can appropriately select the conditions other than the hydrogen ion concentration, and for example, the measurement can be carried out at 37 °C under the conditions of HEPES buffer, or using BIACORE (GE Healthcare), etc.

[0482] Within the scope of Disclosure A described herein, unless specifically indicated otherwise 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 may preferably be 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 may preferably be pH 7.4, which is close to the in vivo pH in plasma (blood), but for convenience of measurement, for example, pH 7.0 may be used.

[0483] Within the scope of Disclosure A described herein, unless specifically indicated otherwise 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 may preferably be 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 may preferably be pH 5.8, which is close to the in vivo hydrogen ion concentration in early endosomes, but for convenience, for example, pH 6.0 may be used.

[0484] In one embodiment in the case of Disclosure A, when the ionic concentration is the hydrogen ion concentration, preferably, the antigen-binding activity of the ion concentration-dependent antigen-binding domain, ion concentration-dependent antibody, ion concentration-dependent antigen-binding domain with increased pI, or ion concentration-dependent antibody with increased pI is higher under neutral pH conditions than under acidic pH conditions. In this case, the ratio of the antigen-binding activity under neutral pH conditions to the antigen-binding activity under acidic pH conditions is not limited; however, for the ratio of the dissociation constant (KD) of the antigen under acidic pH conditions to the KD under neutral pH conditions, i.e., KD(acidic pH range) / KD(neutral pH range), (e.g., KD(pH 5.8) / KD(pH 7.4)), it may 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 may 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 binding activity ratio. In the case of using the dissociation rate constant (kd) instead of the dissociation constant (KD) as an index to represent the binding activity ratio, for 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, that is, kd(acidic pH range) / kd(neutral pH range), it can be 2 or more, 5 or more, 10 or more, or 30 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 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), and 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 a flow cytometer.

[0487] In one embodiment, the method for generating 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 methods include, for example, those described in WO2009 / 125825 (for example, paragraphs 0158 - 0190).

[0488] The method may include, for example:

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

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

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

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

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

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

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

[0496] Alternatively, the method may include, for example:

[0497] (a) contacting an antigen with an antigen-binding domain or an antibody, or a library thereof, at an acidic pH;

[0498] (b) selecting an antigen-binding domain or an antibody that does not bind to the antigen or has low antigen-binding ability in step (a);

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

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

[0501] Alternatively, the method may include, for example:

[0502] (a) contacting an antigen-binding domain or an antibody, or a library thereof, with a column immobilizing an antigen at a neutral pH;

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

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

[0505] Alternatively, the method may include, for example:

[0506] (a) passing an antigen-binding domain or an antibody, or a library thereof, through a column immobilizing an antigen at an acidic pH to collect the eluted antigen-binding domain or antibody that does not bind to the column;

[0507] (b) binding the antigen-binding domain or the antibody collected in step (a) to the antigen at a neutral pH; and

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

[0509] Alternatively, the method may include, for example:

[0510] (a) contacting an antigen with an antigen-binding domain or an antibody, or a library thereof, at a neutral pH;

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

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

[0513] (d) Isolate an antigen-binding domain or antibody whose antigen-binding activity is weaker in step (c) than the criterion 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 obtained thereby.

[0515] In the case of 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 due to modifying the charge of at least one amino acid residue capable of being exposed on its surface. In an alternative embodiment, in the case of introducing an amino acid that changes the binding activity of an ion concentration-dependent antigen-binding domain into the sequence, they can be introduced together with a 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 so as to increase the pI.

[0516] Alternatively, in the case of Invention A, for example, a pre-existing antigen-binding domain or antibody, a pre-existing library (such as a phage library); an antibody prepared from a hybridoma obtained by immunizing an animal or from B cells of an immunized animal, or its library; or an antigen-binding domain, antibody, or library obtained by introducing a natural or unnatural amino acid mutation having a side-chain pKa of 4.0 - 8.0 (described below) therein (for example, a library having an increased number of natural or unnatural amino acid mutations 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 replaced with an amino acid having a side-chain pKa of 4.0 - 8.0 and / or an amino acid having a side-chain of 4.0 - 8.0 is inserted therein, as described in WO2009 / 125825.

[0517] In one embodiment in the case of disclosure A, the site where an amino acid mutation with a side chain pKa of 4.0 - 8.0 is introduced 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 (the KD(acidic pH range) / KD(neutral pH range) value increases or the kd(acidic pH range) / kd(neutral pH range) value increases) compared to before the substitution or insertion. In the case where the antibody has 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 to be substituted or inserted can be appropriately determined by those skilled in the art; and the number can be one or more. In addition, other amino acids (in addition to the above substitution or insertion) may be deleted, added, inserted, and / or substituted, or modified. Substitution or insertion of an amino acid with 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 carried out randomly by a scanning method, such as histidine scanning, where histidine is used to replace alanine in alanine scanning known to those 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 antigen-binding domains or antibodies obtained by randomly substituting or randomly inserting these amino acids to mutate these amino acids, or a library thereof.

[0518] Furthermore, the antigen-binding domain or antibody can preferably be those whose antigen-binding activity does not significantly decrease, basically does not decrease, is basically the same, or increases in the neutral pH range before and after these mutations; and in other words, their activity can be maintained at at least 10% or higher, preferably 50% or higher, more preferably 80% or higher, and even more preferably 90% or higher, or even higher. In the case where the binding activity of the antigen-binding domain or antibody decreases due to substitution or insertion of an amino acid with 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, inserting one or more amino acids at a site other than the above substitution or insertion site.

[0519] In an alternative embodiment, an amino acid having a side chain pKa of 4.0 - 8.0 can be placed at any position within the variable region of the heavy and / or light chain that forms the 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 one or more of the CDRs (CDR1, CDR2, and / or CDR3) and / or FRs (FR1, FR2, FR3, and / or FR4) of the heavy and / or light chain. The amino acid residues include, but are not limited to, amino acid residues at one or more of positions 24, 27, 28, 31, 32, and 34 in CDR1 of the light chain variable region according to Kabat numbering; amino acid residues at one or more of positions 50, 51, 52, 53, 54, 55, and 56 in CDR2 of the light chain variable region according to Kabat numbering; and / or amino acid residues at one or more of positions 89, 90, 91, 92, 93, 94, and 95A in CDR3 of the light chain variable region according to Kabat numbering. Those amino acid residues can be included individually or in combination, as long as the antigen-binding activity of the antibody changes according to the hydrogen ion concentration conditions.

[0520] In one embodiment within the scope of Disclosure A, any amino acid residue can be suitably used as an amino acid residue for which the antigen-binding activity of the antigen-binding domain or antibody changes according to the hydrogen ion concentration conditions. Specifically, the amino acid residues can include those having a side chain pKa of 4.0 - 8.0. The amino acids having an electron-donating property can include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (US2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17): 3761 - 3768 (2003)). 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 Disclosure A described herein, unless otherwise specified and unless there is an inconsistency in the context, it is to be understood that the isoelectric point (pI) can be the 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 with an increased pI or an antibody of Disclosure A is increased compared to an 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 antibody modification, or prior to or during library construction)) can be determined by conducting (in addition to or in place of the above methods) antibody pharmacokinetic testing using plasma (e.g., from mice, rats, rabbits, dogs, monkeys, or humans) in combination with methods such as BIACORE, cell proliferation assays, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), or fluorescence immunoassay.

[0524] Within the scope of 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 constituting the antibody. "Amino acid residues located on the surface of the polypeptide" may refer to amino acid residues whose side chains may come into contact with solvent molecules (which can generally be mainly 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 residue is defined as an "amino acid 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 thus may be affected by the shared charge of one or more other amino acid residues from their side chains, even partially in contact with the solvent molecules. A person skilled in the art can prepare a homology model of a polypeptide or an antibody by, for example, using commercially available software for homology modeling. Alternatively, methods such as X-ray crystallography may be used. Amino acid residues that may be exposed on the surface can be determined, for example, by integrating from a three-dimensional model of the antibody using computer software such as the InsightII program (Accelrys). Surface-exposed sites can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. 55: 379-400 (1971)); Connolly (J. Appl. Cryst. 16: 548-558 (1983)). Surface-exposed sites 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 the user to input size parameters, the "size" of the probe used in the calculation can be set to a radius of about 1.4 angstroms (Å) or less. In addition, methods for determining surface-exposed regions and areas using software for personal computers are 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 constituting the antibody can be selected.

[0525] The method for increasing the pI of a protein is, for example, to reduce the number of amino acids having a negatively charged side chain (e.g., aspartic acid and glutamic acid) under neutral pH conditions and / or increase the number of amino acids having a positively charged side chain (e.g., arginine, lysine, and histidine). It is a well-known theory to those skilled in the art that an amino acid residue having a negatively charged side chain has a negative charge represented as -1 under pH conditions sufficiently higher than the pKa of its side chain. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the side chain has a negative charge represented as -1 under neutral pH conditions (e.g., in a solution at pH 7.0). In contrast, an amino acid residue having a positively charged side chain has a positive charge represented as +1 under pH conditions sufficiently lower than the pKa of its side chain. For example, the theoretical pKa of the side chain of arginine is 12.5, and the side chain has a positive charge represented as +1 under neutral pH conditions (e.g., in a solution at pH 7.0). Amino acid residues known to have an uncharged side chain under neutral pH conditions (e.g., in a solution at pH 7.0) include 15 types of natural amino acids, namely, alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Generally, it is to be understood that the amino acids used to change 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 aspartic acid (residue) or glutamic acid (residue) in the protein amino acid sequence (whose side chain has a negative charge of -1), the charge of the protein under study can be changed by +1 by replacing the amino acid (residue) with an amino acid having an uncharged side chain. In addition, for example, for an amino acid (residue) having an uncharged side chain, the charge of the protein can be changed by +1 by replacing it with arginine or lysine (whose side chain has a positive charge of +1). In addition, for aspartic acid or glutamic acid (whose side chain has a negative charge of -1), the charge of the protein can be changed by +2 each time by replacing it with arginine or lysine (whose side chain has a positive charge of +1). Alternatively, to increase the pI of a protein, an amino acid having an uncharged side chain and / or an amino acid having a positively charged side chain can be added or inserted into the amino acid sequence of the protein, or an amino acid having an uncharged side chain and / or an amino acid having a negatively charged side chain 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 its side chain, the N-terminal and C-terminal amino acid residues of a protein have charges derived from the main chain (the NH of the amino group at the N-terminal and the COO of the carboxyl group at the C-terminal). 3+ and the COO of the carboxyl group at the C-terminal - ). Therefore, the pI of a protein can also be increased by making some additions, deletions, replacements, or insertions to the functional groups derived from the main chain.

[0527] Those skilled in the art will understand that the effect of altering the net charge or pI of a protein (obtained by modifying one or more amino acids (residues) in the amino acid sequence, where the modification is directed at the presence or magnitude of the charge of the amino acid (residue)) depends not only (or substantially) on the amino acid sequence of the antibody itself or the type of target antigen, but also on the type and number of amino acid residues added, deleted, substituted or inserted.

[0528] Antibodies modified to have an increased pI by modification of at least one amino acid residue that may be exposed on the surface of the antibody ("antibodies having an increased pI" or "pI-increased antibodies") may be taken up into cells more rapidly or may be able to facilitate the removal of antigen from the plasma, as described or suggested, for example, in 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 major metabolic pathway is not via renal excretion. It is known that IgG antibodies (which have an Fc region as part of the molecule) recycle via the salvage pathway through FcRn and thus have a long in vivo half-life. It is thought that IgG antibodies are metabolized mainly via the metabolic pathway in endothelial cells (He et al., J. Immunol. 160(2):1029-1035 (1998)). Specifically, it is believed that when non-specifically internalized into endothelial cells, IgG antibodies recycle by binding to FcRn, while IgG antibodies that cannot bind are metabolized. When its Fc region is modified such that its FcRn-binding activity is reduced, the plasma half-life of the IgG antibody can be shortened. On the other hand, it has been shown that the plasma half-life of antibodies having an increased pI depends in a highly correlated manner on the pI, as described, for example, in WO2007 / 114319 and WO2009 / 041643. Specifically, the plasma half-life of the pI-increased antibodies described in the above-mentioned documents is reduced without modifying the amino acid sequence constituting Fc (which may potentially lead to the acquisition of immunogenicity), and this result suggests that the technique of increasing pI can be widely applied even to any type of antibody molecule whose major metabolic pathway is renal excretion, 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 a pI-increased antibody increases due to the increased pI, and thus, compared to an antibody whose pI is not increased, the antibody more strongly adsorbs to the endothelial cell surface with a negative net charge through physicochemical Coulomb interactions; through non-specific binding, the antibody binds to and is internalized by the cell, which results in a shortened half-life of the antibody in plasma or enhanced removal of the antigen from plasma. In addition, increasing the pI of an antibody enhances the internalization of the antibody (or antigen / antibody complex) into cells and / or intracellular permeability, which is considered to result in a decreased antibody concentration in plasma, reduced antibody bioavailability, and / or a shortened half-life of the antibody in plasma; and these phenomena are expected to occur in vivo frequently, regardless of cell type, tissue type, organ type, etc. In addition, in the case where an antibody forms a complex with an antigen and is internalized by a cell, not only the pI of the antibody but also the pI of the antigen may affect the decrease or increase in internalization into the cell.

[0531] In one embodiment, methods for generating or screening antibodies with increased pI can include, for example, those described in WO2007 / 114319 (e.g., paragraphs 0060 - 0087), WO2009 / 041643 (e.g., paragraph 0115 -), WO2014 / 145159, and WO2012 / 016227. The methods can include, for example:

[0532] (a) Modifying a nucleic acid encoding an antibody that includes at least one amino acid residue that may be exposed on the surface of the antibody such that the charge of one or more amino acid residues is modified to increase the pI of the antibody;

[0533] (b) Culturing a host cell to express the nucleic acid; and

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

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

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

[0537] (b') Culturing a host cell to express the nucleic acid;

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

[0539] (d') Optionally confirm or measure and select an antibody with an increased pI compared to the antibody before modification. Here, the antibody as the starting material, or the antibody before modification, or the reference antibody can be, for example, an ion concentration-dependent antibody. Alternatively, when modifying one or more amino acid residues, 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.

[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 for producing a multispecific antibody comprising a first polypeptide and a second polypeptide, and optionally a third polypeptide and a fourth polypeptide, the method comprising:

[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 of them comprises at least one amino acid residue that may be exposed on the surface of the polypeptide, such that the charge of one or more amino acid residues is modified to increase the pI of the antibody;

[0543] (B) Culturing a host cell to express the nucleic acid; and

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

[0545] Alternatively, the method can include, 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 of them comprises at least one amino acid residue that may be exposed on the surface of the polypeptide, such that the charge of one or more amino acid residues is changed;

[0547] (B') Culturing a host cell to express the nucleic acid;

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

[0549] (D') Optionally confirm and select an antibody with an increased pI compared to the antibody before modification.

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

[0551] Alternatively, the method may simply comprise 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 may preferably be a homopolymer of the first polypeptide, a homopolymer of the second polypeptide, or a heteropolymer of the first and second polypeptides (and optionally, a homopolymer of the third polypeptide, a homopolymer of the fourth polypeptide, or a heteropolymer of the third and fourth polypeptides).

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

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

[0554] (I') modifying at least one amino acid residue that may 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 the constant region to one or more amino acid residues having a different charge from one or more amino acid residues present at the corresponding position before modification; and

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

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

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

[0558] In one embodiment of the antibody of Disclosure A, compared with the antibody before modification or alteration (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody), or a reference or parental antibody (e.g., the antibody before antibody modification, or the antibody before library construction or during library construction)), the pI value can preferably 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 at least 0.6, 0.7, 0.8, 0.9, or more, and the antibody half-life in plasma can be 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 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 3.0 or more. Depending on the purpose, considering the pharmacological effect and toxicity, and, for example, the balance between the number of antigen-binding domains of the antibody and the pI of the antigen, those skilled in the art can appropriately and routinely determine the optimal pI value of the antibody of Disclosure A. Without being limited by a particular theory, it is believed that in one embodiment, the antibody of Disclosure A is beneficial because, in addition to shuttling between plasma and endosomes and the characteristic of repetitive binding of multiple antigens with a single antibody molecule due to the presence of ion concentration-dependent antigen-binding domains, the net positive charge of the antibody increases due to the increase in pI, and this enables rapid cellular uptake of the antibody. These properties shorten the antibody half-life in plasma, increase the extracellular matrix-binding activity of the antibody, or enhance the removal of antigen from plasma. Those skilled in the art can determine the optimal pI value to utilize these properties.

[0559] In one embodiment in the case of disclosure A, when compared with an antibody (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody), or a reference or parental antibody (e.g., an antibody modification, or an antibody before or during library construction), which can be an ion concentration-dependent antibody) that modifies or changes at least one amino acid residue to increase the pI, the ion concentration-dependent antibody with increased pI of disclosure A can preferably enhance the 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.

[0560] In one embodiment in the case of disclosure A, when compared with an antibody before the introduction of an ion concentration-dependent antigen-binding domain (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody), or a reference or parental antibody (e.g., an antibody modification, or an antibody before or during library construction), which can be an antibody with increased pI), the ion concentration-dependent antibody with increased pI of disclosure A can preferably enhance the 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.

[0561] In one embodiment, the method for determining whether the extracellular matrix binding activity of the antibody of Disclosure A is increased compared to an antibody before modification or alteration (a native antibody (e.g., a native Ig antibody, which may be a native IgG antibody), or a reference or parental antibody (e.g., an antibody before antibody modification or during library construction), which may be an ion concentration-dependent antibody or antibody with an increased pI) is not limited. For example, the determination can be carried out using an ELISA system, which detects the binding between the antibody and the extracellular matrix, wherein the antibody is added to a plate immobilized with the extracellular matrix, and a labeled antibody against the antibody is added thereto. Alternatively, as described in Examples 1 to 4 herein and WO2012 / 093704, it is also possible to use electrochemiluminescence (ECL), which enables highly sensitive detection of the extracellular matrix binding ability. The method can, for example, be carried out using an ECL system, wherein a mixture of the antibody and a ruthenium antibody is added to a plate immobilized with the extracellular matrix, 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 set appropriately; the added concentration can be high to increase the sensitivity of detecting the extracellular matrix binding. The extracellular matrix can be derived from animals or plants as long as they contain glycoproteins such as collagen, proteoglycan, fibronectin, laminin, nidogen, fibrin, and basement membrane proteoglycan (perlecan); and the extracellular matrix of animal origin is preferably used. For example, it is possible to use an extracellular matrix derived from animals such as humans, mice, rats, monkeys, rabbits, or dogs. For example, a native extracellular matrix of human origin can be used as an indicator of the pharmacokinetics of the antibody in human plasma. The conditions for evaluating the extracellular matrix binding of the antibody can preferably be in the neutral pH range of about pH 7.4, which is a physiological condition; however, the conditions do not have to be in the neutral range, and the binding can also be evaluated in an acidic pH range (e.g., about pH 6.0). Alternatively, when evaluating the extracellular matrix binding of the antibody, the determination can be carried out in the co-presence of the 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 disclosure A can (substantially) retain antigen-binding activity as compared to an antibody (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody) or a reference antibody (e.g., an antibody before antibody modification, or during library construction)) before modifying or altering at least one amino acid residue to increase the pI. In this case, "to (substantially) retain antigen-binding activity" may 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 as compared to the binding activity of the antibody before modification or alteration. Alternatively, the antibody of disclosure A only needs to retain binding activity to an extent that allows them to retain their function when binding to an antigen; thus, the affinity determined under physiological conditions at 37°C 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 disclosure A, the statement "modifying at least one amino acid residue that may be exposed on the surface of the antibody" or an equivalent statement may mean performing one or more of addition, deletion, substitution, and insertion on at least one amino acid residue that may be exposed on the surface of the antibody. The modification may preferably include substituting at least one amino acid residue.

[0564] Substitution of an amino acid residue may include, for example, in the amino acid sequence of the target antibody, substituting an amino acid residue with an uncharged side chain with an amino acid residue with a negatively charged side chain, substituting an amino acid residue with a positively charged side chain with an amino acid residue with an uncharged side chain, and substituting an amino acid residue with a positively charged side chain with an amino acid residue with a negatively charged side chain, which may be performed alone or in a suitable combination. Insertion or addition of an amino acid residue may include, for example, in the amino acid sequence of the target antibody, inserting or adding an amino acid with an uncharged side chain and / or inserting or adding an amino acid with a positively charged side chain, which may be performed alone or in a suitable combination. Deletion of an amino acid residue may include, for example, in the amino acid sequence of the target antibody, deleting an amino acid residue with an uncharged side chain and / or deleting an amino acid residue with a negatively charged side chain, which may be performed alone or in a suitable combination.

[0565] One or more of these additions, deletions, substitutions, and insertions in the amino acid sequence of the target antibody can be appropriately combined by those skilled in the art. Modifications that cause a decrease in the local charge of an amino acid residue are also acceptable, since only the net pI of the antibody of Disclosure A must increase. For example, if desired, an antibody whose pI has been increased (too much) can be modified to decrease the pI (slightly). Also acceptable is a decrease in the local charge of an amino acid residue resulting from modification of at least one amino acid residue, either simultaneously or not, for other purposes (e.g., increasing antibody stability or decreasing immunogenicity). The antibodies include those from libraries constructed for specific purposes.

[0566] In one embodiment, among the amino acids (residues) for modifying at least one amino acid residue that may be exposed on the surface of the antibody, the natural amino acids are as follows: Amino acids with negatively charged side chains can be Glu (E) or Asp (D); Amino acids with uncharged side chains can be Ala (A), Asn (N), Cys (C), Gln (Q), Gly (G), His (H), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), or Val (V); and Amino acids with positively charged side chains can be His (H), Lys (K), or Arg (R).

[0567] As detailed in Examples 1 to 4, in a solution at neutral pH (e.g., pH 7.0), lysine and arginine are almost 100% positively charged when present as residues in the antibody, while histidine is only about 9% positively charged when present as a residue in the antibody, and the remaining major portion is considered to have no charge. Therefore, Lys (K) or Arg (R) is preferably selected as the amino acid with a positively charged side chain.

[0568] In one embodiment, the antibody of Disclosure A preferably has a variable region and / or a constant region. Further, 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, human IgG2, human IgG3, or human IgG4-type constant region, a human κ-chain constant region, and a human λ-chain constant region). Modified variants of these constant regions may be used.

[0569] In one embodiment, the modification of at least one amino acid residue that may 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. A preferred method can be introducing a combination of multiple amino acid substitutions at sites where the amino acids may be exposed on the surface of the antibody. Additionally, without limitation, the multiple amino acid substitutions are preferably introduced at positions that are close 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 may 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 using a pre-existing positively charged amino acid (e.g., Lys (K) or Arg (R)), for example, one or more amino acids that are close to the amino acid in three dimensions (which may include amino acids embedded within the antibody molecule depending on the circumstances) can also be substituted with positively charged amino acids, thereby correspondingly creating a dense state of local positive charges at three-dimensionally adjacent positions. Herein, the definition of "three-dimensionally close positions" is not particularly limited; but it can mean a state where one or more amino acid substitutions are introduced, for example, within 20 Å, preferably within 15 Å, and more preferably within 10 Å. Whether the target amino acid substitution site is exposed on the surface of the antibody molecule or whether the amino acid substitution site is close to other amino acid substitution sites or the aforementioned pre-existing amino acids can be evaluated by known methods such as X-ray crystallography.

[0570] In addition to those described above, methods for imparting multiple positive charges at sites that are close to each other in three dimensions can include using those amino acids that originally have positive charges in the native IgG constant region. Such amino acids include, for example: arginine at positions 255, 292, 301, 344, 355, and 416, according to EU numbering; and lysine 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 EU numbering. Multiple positive charges can be imparted at three-dimensionally close positions by substituting positively charged amino acids at sites that are close 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 that are not covered by antigen binding (i.e., are still exposed on the surface) can be modified, and / or amino acid modifications can be made that do not introduce amino acid modifications at sites covered by antigen binding or that (substantially) do not inhibit antigen binding. In the case where amino acid residues that may be exposed on the surface of the antibody molecule and are present in an ion concentration-dependent binding domain are modified, the amino acids of the antigen binding domain can be modified in such a way that the modification (substantially) does not reduce the binding activity of amino acid residues (such as those in a calcium-binding motif, or in a histidine insertion site and / or a site of histidine substitution) that may change the antigen binding activity of the antibody according to ion concentration conditions, or the amino acid residues can be modified at sites different from those of amino acid residues that change the antigen binding activity of the antibody according to ion concentration conditions. On the other hand, when amino acid residues that may be exposed on the surface of the antibody molecule and are present in an ion concentration-dependent binding domain have been modified, the type or position of amino acid residues that may change the antigen binding activity of the antibody according to ion concentration conditions can be selected such that the pI of the antibody is not reduced to below an acceptable level. In the case where the pI of the antibody is reduced to below an acceptable level, the pI of the whole antibody can be increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody molecule.

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

[0573] In the case where the antibody of disclosure A has a constant region (which can be modified) containing 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, if desired, the sites for modifying at least one amino acid residue that may 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, sites can preferably be selected that (substantially) do not affect the binding activity or binding affinity for FcγR and / or FcRn, or, if they do affect, sites that are biologically or pharmacologically acceptable.

[0574] In one embodiment, the sites of at least one amino acid residue modified to generate the antibody of Disclosure A, whose pI is increased by modifying at least one amino acid residue that may be exposed on the surface of the variable region and that can be modified, are not limited; however, the sites may be 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 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 may be selected from any of the above amino acids in terms of side chain charge after modification, such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N), 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, among the positions to be modified, the following positions may be combined with other positions that may themselves have a sufficient effect on increasing the pI of the antibody to assist in increasing the pI of the antibody of Disclosure A. The positions for assisting in pI increase may be, for example, for the light chain variable region, selected from the group consisting of positions 27, 52, 56, 65, and 69 (according to Kabat numbering).

[0576] In addition, the sites of at least one amino acid residue modified in the CDR and / or FR are not limited; however, the sites 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 modification site of at least one amino acid residue is selected from, for example, a group comprising the above group, the type of amino acid modified in the heavy chain variable region is, for example:

[0578] (a) for position 8 is 8K, 8R, 8Q, 8G, 8S, or 8N; (b) for position 13 is 13K, 13R, 13Q, 13G, 13S, or 13N; (c) for position 15 is 15K, 15R, 15Q, 15G, 15S, or 15N; (d) for position 16 is 16K, 16R, 16Q, 16G, 16S, or 16N; (e) for position 18 is 18K, 18R, 18Q, 18G, 18S, or 18N; (f) for position 39 is 39K, 39R, 39Q, 39G, 39S, or 39N; (g) for position 41 is 41K, 41R, 41Q, 41G, 41S, or 41N; (h) for position 43 is 43K, 43R, 43Q, 43G, 43S, or 43N; (i) for position 44 is 44K, 44R, 44Q, 44G, 44S, or 44N; (j) for position 63 is 63K, 63R, 63Q, 63G, 63S, or 63N; (k) for position 64 is 64K, 64R, 64Q, 64G, 64S, or 64N; (l) for position 77 is 77K, 77R, 77Q, 77G, 77S, or 77N; (m) for position 82 is 82K, 82R, 82Q, 82G, 82S, or 82N; (n) for position 82a is 82aK, 82aR, 82aQ, 82aG, 82aS, or 82aN; (o) for position 82b is 82bK, 82bR, 82bQ, 82bG, 82bS, or 82bN; (p) for position 83 is 83K, 83R, 83Q, 83G, 83S, or 83N; (q) for position 84 is 84K, 84R, 84Q, 84G, 84S, or 84N; (r) for position 85 is 85K, 85R, 85Q, 85G, 85S, or 85N; or (s) for position 105 is 105K, 105R, 105Q, 105G, 105S, or 105N. 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 (according to Kabat numbering): position 77, 82a, and 82b; position 77 and 85; position 41 and 44; position 82a and 82b; position 82 and 82b; position 82b and 83; or position 63 and 64, wherein the amino acid at each modified position can be selected from any of the above amino acids in terms of side-chain charge, such as Lys (K), Arg (R), Gln (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; (l) 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 (according to Kabat numbering), wherein each position can be selected from any of the above amino acids in terms of side chain charge of the modified amino acid, such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N), but not limited thereto.;

[0584] The 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 WO2007 / 114319 or WO2009 / 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 does not depend solely (or substantially) on the amino acid sequence of the antibody itself or the type of target antigen, but rather on the type and number of amino acid residues being substituted. It has also been demonstrated that even after modifying some amino acids, the antigen-binding activity (substantially) remains for several types of antigens, or at least one of ordinary skill in the art can expect with a high likelihood that it will remain.

[0586] For example, WO2009 / 041643 specifically shows that in the heavy chain FR of a humanized glypican 3 antibody as shown in SEQ ID NO: 8, the preferred modification sites of amino acid residues that may be exposed on the surface 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. WO2009 / 041643 also shows that the amino acid residues at positions 52, 54, 62, 63, 65, and 66 in the heavy chain CDR of the antibody are preferred. It also indicates that in the light chain FR of the humanized glypican 3 antibody as shown 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 CDR of the antibody are preferred. In addition, WO2009 / 041643 specifically shows that the amino acid residues at positions 31, 64, and 65 according to Kabat numbering in the heavy chain CDR of the anti-human IL-6 receptor antibody as shown in SEQ ID NO: 10 are preferred sites that allow modification of amino acid residues that may be exposed on the surface while maintaining antigen-binding activity. It also shows that the amino acid residues at positions 24, 27, 53, and 55 according to Kabat numbering in the light chain CDR of the anti-human IL-6 receptor antibody as shown in SEQ ID NO: 11 are preferred. It also specifically shows that the amino acid residue at position 31 according to Kabat numbering in the heavy chain CDR of the anti-human IL-6 receptor antibody as shown in SEQ ID NO: 12 is a preferred site that allows modification of amino acid residues that may be exposed on the surface while maintaining antigen-binding activity. It also shows that the amino acid residues at positions 24, 53, 54, and 55 according to Kabat numbering in the light chain CDR of the anti-human IL-6 receptor antibody as shown in SEQ ID NO: 13 are preferred.WO2009 / 041643 also shows that the amino acid residues at Kabat numbering positions 61, 62, 64, and 65 in the heavy chain CDRs of the anti-human glypican 3 antibody shown in SEQ ID NO: 14 are preferred sites that allow modification of amino acid residues that may be exposed on the surface while maintaining antigen-binding activity. It also shows that the amino acid residues at Kabat numbering positions 24 and 27 in the light chain CDRs of the anti-human glypican 3 antibody shown in SEQ ID NO: 15 are preferred. It also shows that the amino acid residues at Kabat numbering 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 are preferred sites that allow modification of amino acid residues that may be exposed on the surface while maintaining antigen-binding activity. WO2009 / 041643 also shows that the amino acid residues at Kabat numbering positions 24 and 54 in the light chain CDRs of the anti-human IL-31 receptor antibody shown in SEQ ID NO: 17 are preferred. Similarly, WO2007 / 114319 reports antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and hB26-PF, which were generated by modifying the charge of one or more amino acid residues that may be exposed on the surface, showed a change in pI (as demonstrated by isoelectric focusing), and had comparable binding activity to factor IXa or factor X (which are their antigens) compared to the antibody before modification or alteration. It also reports that when these antibodies were administered to mice, the pI of each antibody showed a high correlation with its clearance rate (CL) in plasma, retention in plasma, and half-life (T1 / 2) in plasma. WO2007 / 114319 also shows that the amino acid residues at positions 10, 12, 23, 39, 43, 97, and 105 in the variable region are preferred as sites for modification of amino acid residues that may be exposed on the surface.

[0587] In an alternative or another embodiment, for example, using known methods such as X-ray crystallography or homology models constructed by homology modeling from the antibody constant region (which is preferably a human constant region, more preferably a human Ig-type constant region, and even more preferably a human IgG-type constant region, but not limited thereto), amino acid residues that may be exposed on the surface of the antibody constant region can be identified to determine the modification sites of at least one amino acid residue for generating the antibody with increased pI of Disclosure A. The modification sites of at least one amino acid residue that may be exposed on the surface of the constant region are not limited; however, the sites may preferably be selected from the group consisting of: according to EU numbering, 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, and may 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 may also preferably be selected from the group consisting of: positions 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, and the amino acid at each of these positions after modification can be selected from the above amino acids in terms of side chain charge, such as Lys (K), Arg (R), Gln (Q), or Asn (N), but not limited thereto. When, for example, selecting the modification sites of at least one amino acid residue from the group containing the above groups, for example, the amino acid types 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 amino acid type of at least one modified 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 Disclosure A, the net pI of the antibody can be increased by modifying at least one amino acid residue that may be exposed on the surface of the variable region (which can be modified) as described above and at least one amino acid residue that may be exposed on the surface of the constant region (which can be modified) as described above.

[0607] Within the scope of Disclosures A and B described herein, when the antibody of Disclosure A or B is an IgG-type antibody or a molecule derived therefrom, the heavy chain constant region of the antibody can contain a constant region of IgG1 type, IgG2 type, IgG3 type, or IgG4 type. In Disclosures A or B, the heavy chain constant region can be a human heavy chain constant region, but is not limited thereto. Multiple allotypes are known for human IgG. Specifically, some differences in the amino acid sequences of the human IgG constant regions among individuals have been reported (Methods Mol. Biol. 882: 635 - 80 (2012); Sequences of proteins of immunological interest, NIH Publication No. 91 - 3242). Examples include the human IgG1 constant region (SEQ ID NO: 18), the human IgG2 constant region (SEQ ID NO: 19), the human IgG3 constant region (SEQ ID NO: 20), and the human IgG4 constant region (SEQ ID NO: 21).

[0608] Among these, for example, for human IgG1, allotypes called G1m1,17 and G1m3 are known. 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, no reports have suggested significant differences in the basic antibody functions and properties among the reported allotypes. Thus, those skilled in the art can easily predict that various evaluations are performed using a specific allotype, and the results are not limited to the allotype used to obtain the examples and the same effects are predicted using any allotype. Within the scope of Disclosures A and B described herein, when referred to as "human IgG1", "human IgG2", "human IgG3", or "human IgG4", the allotypes are not limited to specific allotypes and may include all reported allotypes.

[0609] In an alternative or another embodiment of Disclosure A or B, the light chain constant region of the antibody can include any constant region of the κ-chain (IgK) type or λ-chain (IgL1, IgL2, IgL3, IgL6, or IgL7) type. The light chain constant region can preferably be a human light chain constant region, but is not limited thereto. As reported in Sequences of proteins of immunological interest, NIH publication No. 91-3242, there are reports on various allotypic sequences due to gene polymorphisms of the human κ-chain constant region and the human λ-chain constant region. The allotypes include, for example, the human κ-chain constant region (SEQ ID NO: 22) and the human λ-chain constant region (SEQ ID NO: 23). However, no reports have suggested significant differences in the basic antibody functions and properties among the reported allotypes. Thus, those skilled in the art can easily understand that the same effects are expected for any allotype (hereinafter also collectively referred to as the native (human) IgG(type) constant region) when referring to specific allotypes within the scope of Disclosures A and B described herein.

[0610] In addition, since the Fc region of a natural IgG antibody forms part of the constant region of the natural IgG antibody, when the antibody of Disclosure A or B is, for example, an IgG-type antibody or a molecule derived therefrom, the antibody can have an Fc region (hereinafter also collectively referred to as a natural (human) IgG(type) Fc region) contained in the constant region of natural IgG (IgG1, IgG2, IgG3, or IgG4 type). The Fc region of natural IgG can refer to an Fc region consisting of the same amino acid sequence as the Fc region derived from natural IgG. Specific examples of the Fc region of natural human IgG can include the Fc regions 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 can refer to, for example, from the cysteine at EU numbering position 226 to the C-terminus, or from the proline at EU numbering position 230 to the C-terminus).

[0611] In one embodiment, the antibodies of Disclosure A and B can 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 Disclosure A described herein, WO2013 / 081143 reports that, for example, due to the avidity (the sum of the binding strengths between multiple epitopes and multiple paratopes) of at least two or more multivalent constant regions (which may be modified) or Fc regions (which may be modified) contained in an antibody molecule, an ion concentration-dependent antibody capable of forming a multivalent immune complex (multivalent antigen-antibody complex) with a multivalent antigen and a multispecific ion concentration-dependent antibody or multiparatopic ion concentration-dependent antibody capable of forming a multivalent immune complex (multivalent antigen-antibody complex) by recognizing two or more epitopes on a monomeric antigen can bind more strongly to FcγR, FcRn, complement receptors, and thus the antibody is internalized into cells more rapidly. Therefore, when modified to have an increased pI by modifying at least one amino acid residue that may be exposed on the surface of the antibody, the above-described ion concentration-dependent antibody (which is capable of forming a multivalent immune complex with a multivalent antigen or a monomeric antigen) can also be used as an antibody of Disclosure A (an ion concentration-dependent antibody with an increased pI). Those skilled in the art will understand that an ion concentration-dependent antibody with an increased pI that is capable of forming a multivalent immune complex with a multivalent antigen or a monomeric antigen can be internalized into cells more rapidly compared to an ion concentration-dependent antibody with an increased pI that cannot form a multivalent immune complex. Those skilled in the art can also understand that in one embodiment, the activity of the antibody of Disclosure A to bind FcRn and / or FcγR can be increased under neutral pH conditions and in this case, an ion concentration-dependent antibody with an increased pI that is capable of forming a multivalent immune complex with a multivalent antigen or a monomeric antigen can be internalized into cells even more rapidly.

[0613] In one embodiment, the antibody of disclosure A can be a single-arm antibody (including all embodiments of the single-arm antibodies described in WO2005 / 063816). Generally, a single-arm antibody is an antibody that lacks one of the two Fab regions that a normal 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 more N-terminal relative to the hinge (e.g., VH or CH1), the antibody will be expressed in a form containing extra sequences, and when one of the Fab regions is cleaved at a site more C-terminal relative to the hinge (e.g., CH2), the Fc region will have an incomplete form. Thus, without limitation, from the perspective of antibody molecule stability, it is preferred that the single-arm antibody is produced by cleavage in the hinge region (hinge) of one of the two Fab regions of the IgG antibody. More preferably, the heavy chain is linked to the uncleaved heavy chain by an intramolecular disulfide bond after cleavage. WO2005 / 063816 reports that such single-arm antibodies have increased stability compared to Fab molecules. Antibodies with increased or decreased pI can also be produced by preparing such single-arm antibodies. In addition, when an ion concentration-dependent antigen-binding domain is introduced into an antibody with increased pI that is a single-arm antibody, compared to an antibody with increased pI that does not have an ion concentration-dependent antigen-binding domain, the half-life of the antibody in plasma can be further shortened, the cellular uptake of the antibody can be further enhanced, the removal of the antigen from plasma can be further enhanced, or the affinity of the antibody for the extracellular matrix can be further increased.

[0614] Without being limited by a particular theory, it is contemplated that an embodiment in the case where an accelerated cellular uptake effect of a single-arm antibody is expected is, but not limited to such a situation, where the pI of the soluble antigen is lower than that of the antibody. The net pI of the complex composed of the antibody and the antigen can be calculated by known methods, considering the complex as a single molecule. In this case, the lower the pI of the soluble antigen, the lower the net pI of the complex; and the higher the pI of the soluble antigen, the higher the net pI of the complex. When a normal 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 normal antibody is converted to a single-arm antibody, only one antigen can bind to a single molecule of the antibody; the decrease in the pI of the complex caused by binding the second antigen can thus be suppressed. In other words, it is believed that when the pI of the soluble antigen is lower than the pI of the antibody, conversion to a single-arm antibody causes the pI of the complex to increase compared to the normal antibody and accelerates uptake into cells.

[0615] In addition, without limitation, when the Fab of a normal IgG-type antibody molecule (having two Fabs) has a lower pI than the Fc, conversion to a single-arm antibody increases the net pI of the complex composed of the single-arm antibody and the antigen. Further, when such conversion to a single-arm antibody is performed, it is preferred from the viewpoint of the 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 possible to expect an effective increase in pI by selecting a site that will increase the pI of the single-arm antibody to the desired degree.

[0616] Therefore, those skilled in the art will understand that the pI of an antibody can be increased and the cellular uptake of the attached antigen can be accelerated not only (or substantially) depending on the antibody amino acid sequence itself and the type of the soluble antigen, but also by converting the antibody to a single-arm antibody and 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 in their theoretical pI values.

[0617] In one embodiment, the antibody of disclosure A or B can be a multispecific antibody, and the multispecific antibody 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" refers to an antibody that binds at least two or more different types of antigens or at least two or more types of epitopes in the same antigen. The first polypeptide and the second polypeptide preferably may 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 may 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, when the antibody of disclosure A is a multispecific antibody and the multispecific antibody contains a heavy chain constant region, in order to lower its pI, for example, the following sequences can be used: the IgG2 or IgG4 sequence at position 137; the IgG1, IgG2, or IgG4 sequence at position 196; the IgG2 or IgG4 sequence at position 203; the IgG2 sequence at position 214; the IgG1, IgG3, or IgG4 sequence at position 217; the IgG1, IgG3, or IgG4 sequence at position 233; the IgG4 sequence at position 268; the IgG2, IgG3, or IgG4 sequence at position 274; the IgG1, IgG2, or IgG4 sequence at position 276; the IgG4 sequence at position 355; the IgG3 sequence at position 392; the IgG4 sequence at position 419; or the IgG1, IgG2, or IgG4 sequence at position 435. Meanwhile, in order to increase its pI, for example, the following sequences can be used: the IgG1 or IgG3 sequence at position 137; the IgG3 sequence at position 196; the IgG1 or IgG3 sequence at position 203; the IgG1, IgG3, or IgG4 sequence at position 214; the IgG2 sequence at position 217; the IgG2 sequence at position 233; the IgG1, IgG2, or IgG3 sequence at position 268; the IgG1 sequence at position 274; the IgG3 sequence at position 276; the IgG1, IgG2, or IgG3 sequence at position 355; the IgG1, IgG2, or IgG4 sequence at position 392; the IgG1, IgG2, or IgG3 sequence at position 419; or the IgG3 sequence at position 435.

[0619] In one embodiment, when 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 IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions that initially have different pIs. Alternatively, a pI difference may be introduced between the two heavy chain constant regions. The modification site of at least one amino acid residue for introducing such a pI difference in the constant region can be one or more of the above-mentioned positions or one or more positions selected from, for example, the group consisting of positions 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435 (according to EU numbering) in the heavy chain constant region described in WO2009 / 041643. Alternatively, the amino acid residue at position 297, which is a glycosylation site, can be modified to remove the sugar chain, because removing the sugar chain from the heavy chain constant region results in a pI difference.

[0620] In one embodiment, the antibodies of disclosure A or B can be polyclonal or monoclonal antibodies, and monoclonal antibodies of mammalian origin are preferred. Monoclonal antibodies include those produced by hybridomas or those produced by host cells transformed by genetic engineering techniques using an expression vector carrying the antibody gene. The antibodies of disclosure A or B can be, for example, antibodies such as chimeric antibodies, humanized antibodies, or antibodies produced by affinity maturation, or molecules derived therefrom.

[0621] In one embodiment, the antibodies of disclosure A or B can be derived from, without limitation to 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 antibodies are preferably derived from human, monkey or mouse.

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

[0623] Within the scope of disclosure A and B described herein, Fc receptor (also referred to as "FcR") refers to 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, the Fc receptors for IgG, IgA, IgE and IgM are known to be FcγR, FcαR, FcεR 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 Disclosure A described herein, "FcγR" can refer to a receptor protein that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4 antibodies or molecules derived therefrom, or a variant of the Fc region, and can include any one or more or all of the members of the protein family encoded substantially by the FcγR gene. In humans, the family includes, but is not limited to, FcγRI (CD64) (including isotypes FcγRIa, FcγRIb, and FcγRIc); FcγRII (CD32) (including isotypes FcγRIIa ((including allotypes H131 (type H) and R131 (type R))), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc); and FcγRIII (CD16) (including isotypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2)), as well as all uncharacterized human FcγRs and FcγR isotypes and allotypes. In addition, FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb (hFcγRIIb). There are also reports of a splice variant called FcγRIIb3 (Brooks et al., J. Exp. Med, 170: 1369-1385 (1989)). In addition to those described above, hFcγRIIb includes all splice variants, such as those recorded in NCBI under NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. hFcγRIIb also includes all reported genetic polymorphisms, for example, FcγRIIb (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 uncharacterized mouse FcγRs, and FcγR isotypes and allotypes. The preferred FcγRs include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), or FcγRIIIB (CD16). Since FcγRs exist in vivo in membrane form, they can be used in experimental systems after being artificially converted into the 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 FcγRI (CD64), which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16), which includes FcγRIIIa (including allotypes V158 and F158), the α chain that binds to the Fc region of IgG is associated with the common γ chain, which has an ITAM for transmitting intracellular activation signals. FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPI-anchored protein. Meanwhile, the cytoplasmic domain of FcγRII (CD32), which includes FcγRIIa (including allotypes H131 and R131) and the FcγRIIc isotype, contains an ITAM. These receptors are expressed on many immune cells such as macrophages, mast cells, and antigen-presenting cells. The activation signals transduced after these receptors bind to the Fc region of IgG promote the phagocytic ability of macrophages, the production of inflammatory cytokines, the degranulation of mast cells, and the increased function of antigen-presenting cells. FcγRs with the ability to transduce the above activation signals are also referred to as activating FcγRs within the scope of the disclosure A and B described herein.

[0629] Meanwhile, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains an ITIM, which transmits inhibitory signals. In B cells, the cross-linking between FcγRIIb and the B cell receptor (BCR) inhibits the activation signal from the BCR, which results in the inhibition of antibody production through the BCR. In macrophages, the cross-linking of FcγRIII and FcγRIIb inhibits the phagocytic ability and the ability to produce inflammatory cytokines. Within the scope of the disclosure A and B described herein, FcγRs with the ability to transduce the inhibitory signals described above are also referred to as inhibitory Fcγ receptors.

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

[0631] For the pH conditions used to measure the binding activity between the FcγR-binding domain contained in an antibody or Fc region (variant) and FcγR, acidic or neutral pH conditions can be appropriately used. For the temperature used in the measurement conditions, for example, the binding activity (binding affinity) between the FcγR-binding domain and FcγR can be evaluated at any temperature between 10°C and 50°C. A preferred temperature for determining the binding activity (binding affinity) of the human FcγR-binding domain to FcγR is, for example, 15°C to 40°C. More preferably, in order to determine the binding activity (binding affinity) between the FcγR-binding domain and FcγR, any temperature between 20°C and 35°C can be used, such as any one 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, when the antibody of 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 FcγR-binding domain within the scope of Disclosure A and an FcRn-binding domain within the scope of Disclosure A and B described herein.

[0633] In one embodiment, when the antibody of disclosure A has FcγR-binding activity, it may have an FcγR-binding domain, preferably a human FcγR-binding domain. The FcγR-binding domain is not particularly limited as long as the antibody has binding activity or affinity for FcγR at acidic pH and / or neutral pH, and it may be a domain having the activity of directly or indirectly binding to FcγR.

[0634] In one embodiment, when the antibody of disclosure A has FcγR-binding activity, preferably, compared with a reference antibody containing a native IgG constant region, the FcγR-binding activity of the antibody is increased under neutral pH conditions. From the perspective of comparing the FcγR-binding activities of the two, preferably, but not limited to, the antibody of disclosure A and the reference antibody containing a native IgG constant region have the same amino acid sequence in regions other than the constant region of the antibody of disclosure A modified at one or more amino acid residues (e.g., the variable region).

[0635] In one embodiment, when 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 limited by theory, it is considered that the antibody has a combination of the following properties: the property of shuttling between plasma and endosomes and repeatedly binding multiple antigens through an ion concentration-dependent antigen-binding domain as a single antibody molecule; the property of being rapidly internalized into cells by having an increased pI and increased positive charge in the whole antibody; and the property of being rapidly internalized into cells by having increased FcγR-binding activity under neutral pH conditions. Thus, the half-life of the antibody 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; therefore, the antibody of disclosure A is beneficial. Those skilled in the art can routinely determine the optimal pI value for the antibody to utilize these properties.

[0636] In one embodiment, an FcγR-binding domain having higher FcγR-binding activity than the Fc region or constant region of a natural human IgG in which the sugar chain linked at position 297 according to EU numbering is a fucosylated sugar chain can be generated by modifying amino acid residues in the Fc region or constant region of a natural human IgG (see WO2013 / 047752). In addition, a domain of any structure that binds to FcγR can be used as an FcγR-binding domain. In this case, an FcγR-binding domain can be generated without the need to introduce amino acid modifications, and alternatively, its affinity for FcγR can be increased by introducing additional modifications. The FcγR-binding domain can include Fab fragment antibodies that bind to FcγRIIIa described by 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), camelid single-domain antibodies, and single-chain Fv antibodies, as well as cyclic peptides that bind to FcγRI described by Bonetto et al., FASEB J. 23(2): 575-585 (2008). The FcγR-binding activity of the FcγR-binding domain can be appropriately evaluated using the method described above to determine whether it is higher than the Fc region or constant region of a natural human IgG in which the sugar chain linked at position 297 according to EU numbering is a fucosylated sugar chain.

[0637] In one embodiment of Disclosure A, the starting FcγR-binding domain preferably comprises, for example, the (human) IgG Fc region or the (human) IgG constant region. Any Fc region or constant region can be used as the starting Fc region or starting constant region as long as the variant of the starting Fc region or starting constant region is capable of binding human FcγR in the neutral pH range. The Fc region or constant region further obtained from the starting Fc region or starting constant region that has been modified by further modifying one or more of its amino acid residues can also be suitably used as the Fc region or constant region of Disclosure A. The starting Fc region or starting constant region can refer to the polypeptide itself, a composition containing the starting Fc region or starting constant region, or the amino acid sequence encoding the starting Fc region or starting constant region. The starting Fc region or starting constant region can include known Fc regions or known constant regions produced by recombinant techniques. The source of the starting Fc region or starting constant region is not limited, and it can be obtained from any organism or human of non-human animals. In addition, the starting FcγR-binding domain can be obtained from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. The starting Fc region or starting constant region can preferably be obtained from human IgG1; however, it is not limited to a specific IgG type. This means that the Fc regions of human IgG1, IgG2, IgG3, or IgG4 can be used as suitable starting FcγR-binding domains, and it also means that within the scope of Disclosure A described herein, the Fc regions or constant regions of IgG types or subclasses derived from any organism can preferably be used as the starting Fc regions or starting constant regions. Examples of native IgG variants or modified forms are described in well-known literature 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, starting Fc region, or starting constant region can contain, for example, one or more mutations: for example, substitution with amino acid residues different from those in the starting Fc region or starting constant region; insertion of one or more amino acid residues into the amino acid residues in the starting Fc region or starting constant region; or deletion of one or more amino acid residues from those in the starting Fc region or starting constant region. The amino acid sequence of the modified Fc region or constant region preferably contains an amino acid sequence that may be at least a portion of an Fc region or constant region that is not naturally occurring. The variant must have less than 100% sequence identity or similarity to the starting Fc region or 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 starting constant region. In a non-limiting example, there is at least one amino acid difference between the modified Fc region or constant region of Disclosure A and the starting Fc region or starting constant region.

[0639] In one embodiment, an Fc region or constant region having FcγR-binding activity in the acidic pH range and / or in the neutral pH range (which can be included in the antibodies of Disclosure A) can be obtained by any method. Specifically, an Fc region or constant region variant having FcγR-binding activity in the neutral pH range can be obtained by modifying the amino acids of a human IgG antibody that can be used as the starting Fc region or starting constant region. Suitable IgG antibody Fc regions or IgG antibody constant regions for modification can include, for example, the Fc region or constant region of human IgG (IgG1, IgG2, IgG3, or IgG4, or variants thereof), and mutants that occur spontaneously therefrom. For the Fc region or constant region of human IgG1, human IgG2, human IgG3, or human IgG4 antibodies, many allotypic sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, and any of them can be used for 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 disclosure A, the modification of other amino acids is not limited as long as the variant has FcγR-binding activity in the neutral pH range. One or more amino acid positions of the modification are reported, for example, in WO2007 / 024249, WO2007 / 021841, WO2006 / 031370, WO2000 / 042072, WO2004 / 029207, WO2004 / 099249, WO2006 / 105338, WO2007 / 041635, WO2008 / 092117, WO2005 / 070963, WO2006 / 020114, WO2006 / 116260, WO2006 / 023403, WO2013 / 047752, WO2006 / 019447, WO2012 / 115241, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101, WO2013 / 118858, and WO2014 / 030750.

[0641] To increase the FcγR-binding activity in the neutral pH range, the sites of amino acid modification in the constant or Fc region can include, for example, one or more positions selected from the group consisting of the 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. Modification of the amino acid residues can increase the binding of the Fc region or constant region of the IgG antibody to FcγR under neutral pH conditions. WO2013 / 047752 describes, as preferred modifications in the IgG-type constant region or Fc region, for example, modification of one or more amino acid residues selected from the group consisting of: modification of the amino acid at position 221 to Lys or Tyr; modification of the amino acid at position 222 to any one of Phe, Trp, Glu, and Tyr; modification of the amino acid at position 223 to any one of Phe, Trp, Glu, and Lys; modification of the amino acid at position 224 to any one of Phe, Trp, Glu, and Tyr; modification of the amino acid at position 225 to any one of Glu, Lys, and Trp; modification of the amino acid at position 227 to any one of Glu, Gly, Lys, and Tyr; modification of the amino acid at position 228 to any one of Glu, Gly, Lys, and Tyr; modification of the amino acid at position 230 to any one of Ala, Glu, Gly, and Tyr; modification of the amino acid at position 231 to any one of Glu, Gly, Lys, Pro, and Tyr;The amino acid at position 232 is modified to any one of Glu, Gly, Lys, and Tyr; the amino acid at position 233 is modified to any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 is modified to any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 is modified to any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 238 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 239 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 240 is modified to any one of Ala, Ile, Met, and Thr; the amino acid at position 241 is modified to any one of Asp, Glu, Leu, Arg, Trp, and Tyr; the amino acid at position 243 is modified to any one of Glu, Leu, Gln, Arg, Trp, and Tyr; the amino acid at position 244 is modified to His; the amino acid at position 245 is modified to Ala; the amino acid at position 246 is modified to any one of Asp, Glu, His, and Tyr; the amino acid at position 247 is modified to any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; the amino acid at position 249 is modified to any one of Glu, His, Gln, and Tyr; the amino acid at position 250 is modified to Glu or Gln; the amino acid at position 251 is modified to Phe;The amino acid at position 254 is modified to any one of Phe, Met, and Tyr; the amino acid at position 255 is modified to any one of Glu, Leu, and Tyr; the amino acid at position 256 is modified to any one of Ala, Met, and Pro; the amino acid at position 258 is modified to any one of Asp, Glu, His, Ser, and Tyr; the amino acid at position 260 is modified to any one of Asp, Glu, His, and Tyr; the amino acid at position 262 is modified to any one of Ala, Glu, Phe, Ile, and Thr; the amino acid at position 263 is modified to any one of Ala, Ile, Met, and Thr; the amino acid at position 264 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 265 is modified to any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 266 is modified to any one of Ala, Ile, Met, and Thr; the amino acid at position 267 is modified to any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 268 is modified to any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp; the amino acid at position 269 is modified to any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 270 is modified to any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 272 is modified to any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 273 is modified to Phe or Ile;The amino acid at position 274 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 275 is modified to Leu or Trp; the amino acid at position 276 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 278 is modified to any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; the amino acid at position 279 is modified to Ala; the amino acid at position 280 is modified to any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid at position 281 is modified to any one of Asp, Lys, Pro, and Tyr; the amino acid at position 282 is modified to any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 283 is modified to any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid at position 284 is modified to any one of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid at position 285 is modified to any one of Asp, Glu, Lys, Gln, Trp, and Tyr; the amino acid at position 286 is modified to any one of Glu, Gly, Pro, and Tyr; the amino acid at position 288 is modified to any one of Asn, Asp, Glu, and Tyr; the amino acid at position 290 is modified to any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid at position 291 is modified to any one of Asp, Glu, Gly, His, Ile, Gln, and Thr; the amino acid at position 292 is modified to any one of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid at position 293 is modified to any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 294 is modified to any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr;The amino acid at position 296 is modified to any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val; the amino acid at position 297 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 298 is modified to any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 299 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 300 is modified to any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; the amino acid at position 301 is modified to any one of Asp, Glu, His, and Tyr; the amino acid at position 302 is modified to Ile; the amino acid at position 303 is modified to any one of Asp, Gly, and Tyr; the amino acid at position 304 is modified to any one of Asp, His, Leu, Asn, and Thr; the amino acid at position 305 is modified to any one of Glu, Ile, Thr, and Tyr; the amino acid at position 311 is modified to any one of Ala, Asp, Asn, Thr, Val, and Tyr; the amino acid at position 313 is modified to Phe; the amino acid at position 315 is modified to Leu; the amino acid at position 317 is modified to Glu or Gln; the amino acid at position 318 is modified to any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 is modified to any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 322 is modified to any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 323 is modified to Ile; the amino acid at position 324 is modified to any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr;The amino acid at position 325 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 326 is modified to any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 327 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 328 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 329 is modified to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 330 is modified to any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 is modified to any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 332 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 333 is modified to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 is modified to any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; the amino acid at position 335 is modified to any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 is modified to any one of Glu, Lys, and Tyr; the amino acid at position 337 is modified to any one of Glu, His, and Asn; the amino acid at position 339 is modified to any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr;The amino acid at position 376 is modified to Ala or Val; the amino acid at position 377 is modified to Gly or Lys; the amino acid at position 378 is modified to Asp; the amino acid at position 379 is modified to Asn; the amino acid at position 380 is modified to any one of Ala, Asn, and Ser; the amino acid at position 382 is modified to Ala or Ile; the amino acid at position 385 is modified to Glu; the amino acid at position 392 is modified to Thr; the amino acid at position 396 is modified to Leu; the amino acid at position 421 is modified to Lys; the amino acid at position 427 is modified to Asn; the amino acid at position 428 is modified to Phe or Leu; the amino acid at position 429 is modified to Met; the amino acid at position 434 is modified to Trp; the amino acid at position 436 is modified to Ile; and the amino acid at position 440 is modified to any one of Gly, His, Ile, Leu, and Tyr (according to EU numbering). The number of amino acids to be modified is not particularly limited, and it is possible to modify the amino acid at only one position or the amino acids at two or more positions. Combinations of amino acid modifications at two or more positions are shown in Table 5 of WO2013 / 047752. Modifications of these amino acid residues can also be appropriately introduced into the antibodies of Disclosure A.;

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

[0643] In another embodiment, the increased level of binding activity to inhibitory FcγRs (FcγRIIb-1 and / or FcγRIIb-2) (within the neutral pH range) can be greater than the increased level of binding activity to activating FcγRs (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 disclosure A can have binding activity to FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

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

[0646] As described above, activating FcγRs preferably include, 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, inhibitory FcγRs preferably include, for example, FcγRIIb (such as FcγRIIb-1 or FcγRIIb-2).

[0647] In one embodiment, an FcγR-binding domain having higher binding activity for an inhibitory FcγR than for an activating FcγR can be used as the selective FcγR-binding domain included in the antibody of Disclosure A. The selective FcγR-binding domain can include, for example, an FcγR-binding domain having higher binding activity for FcγRIIb (such as FcγRIIb-1 and / or FcγRIIb-2) than for 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] Furthermore, whether the FcγR-binding domain has selective binding activity can be evaluated by comparing the binding activities with each FcγR determined by the above method, for example, by comparing the value (ratio) obtained by dividing the KD value for an activating FcγR by the KD value for an inhibitory FcγR, more specifically by comparing the FcγR selectivity index shown in Equation 1 below:

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

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

[0651] The FcγR selectivity index can be, for example: 1.2 or higher, 1.3 or higher, 1.4 or higher, 1.5 or higher, 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2 or higher, 3 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, 15 or higher, 20 or higher, 25 or higher, 30 or higher, 35 or higher, 40 or higher, 45 or higher, 50 or higher, 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, 95 or higher, 100 or higher, 110 or higher, 120 or higher, 130 or higher, 140 or higher, 150 or higher, 160 or higher, 170 or higher, 180 or higher, 190 or higher, 200 or higher, 210 or higher, 220 or higher, 230 or higher, 240 or higher, 250 or higher, 260 or higher, 270 or higher, 280 or higher, 290 or higher, 300 or higher, 310 or higher, 320 or higher, 330 or higher, 340 or higher, 350 or higher, 360 or higher, 370 or higher, 380 or higher, 390 or higher, 400 or higher, 410 or higher, 420 or higher, 430 or higher, 440 or higher, 450 or higher, 460 or higher, 470 or higher, 480 or higher, 490 or higher, 500 or higher, 520 or higher, 540 or higher, 560 or higher, 580 or higher, 600 or higher, 620 or higher, 640 or higher, 660 or higher, 680 or higher, 700 or higher, 720 or higher, 740 or higher, 760 or higher, 780 or higher, 800 or higher, 820 or higher, 840 or higher, 860 or higher, 880 or higher, 900 or higher, 920 or higher, 940 or higher, 960 or higher, 980 or higher, 1000 or higher, 1500 or higher, 2000 or higher, 2500 or higher, 3000 or higher, 3500 or higher, 4000 or higher, 4500 or higher, 5000 or higher, 5500 or higher, 6000 or higher, 6500 or higher, 7000 or higher, 7500 or higher, 8000 or higher, 8500 or higher, 9000 or higher, 9500 or higher, 10000 or higher, or 100000 or higher; however, it is not limited thereto.

[0652] In one embodiment, an Fc region variant or constant region variant (antibody containing the same) of human IgG (IgG1, IgG2, IgG3, or IgG4) in which the amino acid at position 238 or 328 according to EU numbering is Asp or Glu, respectively, can preferably be used as an antibody of disclosure A containing an Fc region variant or constant region variant, because as specifically described in WO2013 / 125667, WO2012 / 115241, and WO2013 / 047752, it has higher binding activity to FcγRIIb-1 and / or FcγRIIb-2 than to 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), FcγRIIa (including allotype R131), and / or FcγRIIc. In this embodiment, the antibodies of disclosure A have binding activity to all activating FcγRs (herein, which are selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, FcγRIIa) and FcγRIIb, and compared to a reference antibody containing a native IgG constant region or native IgG Fc region, their FcγRIIb-binding activity is maintained or increased, and / or their binding activity to all activating FcγRs is decreased.

[0653] In one embodiment, for an antibody of disclosure A containing an Fc region variant or constant region variant, compared to a reference antibody having a native IgG constant region or Fc region, their binding activity to FcγRIIb can be maintained or increased, and their binding activity to FcγRIIa (type H) and FcγRIIa (type R) can be decreased. The antibody can have an increased binding selectivity for FcγRIIb relative to FcγRIIa.

[0654] Within the scope of Disclosure A described herein, the degree of "reduced binding activity to all activating FcγRs" 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 scope of Disclosure A described herein, the degree to which "FcγRIIb-binding activity is maintained or increased", the degree to which "binding activity to FcγRIIb is maintained or increased", or the degree of "maintained or increased binding activity to FcγRIIb" can be, but is not limited to, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, 100% or higher, 101% or higher, 102% or higher, 103% or higher, 104% or higher, 105% or higher, 106% or higher, 107% or higher, 108% or higher, 109% or higher, 110% or higher, 112% or higher, 114% or higher, 116% or higher, 118% or higher, 120% or higher, 122% or higher, 124% or higher, 126% or higher, 128% or higher, 130% or higher, 132% or higher, 134% or higher, 136% or higher, 138% or higher, 140% or higher, 142% or higher, 144% or higher, 146% or higher, 148% or higher, 150% or higher, 155% or higher, 160% or higher, 165% or higher, 170% or higher, 175% or higher, 180% or higher, 185% or higher, 190% or higher, 195% or higher, 2-fold or higher, 3-fold or higher, 4-fold or higher, 5-fold or higher, 6-fold or higher, 7-fold or higher, 8-fold or higher, 9-fold or higher, 10-fold or higher, 20-fold or higher, 30-fold or higher, 40-fold or higher, 50-fold or higher, 60-fold or higher, 70-fold or higher, 80-fold or higher, 90-fold or higher, 100-fold or higher, 200-fold or higher, 300-fold or higher, 400-fold or higher, 500-fold or higher, 600-fold or higher, 700-fold or higher, 800-fold or higher, 900-fold or higher, 1000-fold or higher, 10000-fold or higher, or 100000-fold or higher.

[0656] Within the scope of Disclosure A described herein, the degree of "reduced binding activity to FcγRIIa (type H) and FcγRIIa (type R)" or "reduced binding activity to FcγRIIa (type H) and FcγRIIa (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 FcγRIIb relative to FcγRIIa (type R) may be preferred, and modifications that increase the binding selectivity for FcγRIIb relative to FcγRIIa (type H) may be more preferred, and as reported in WO2013 / 047752, preferred amino acid substitutions for such modifications may include, for example, according to EU numbering: (a) modification by substituting Gly with Trp at position 237; (b) modification by substituting Gly with Phe at position 237; (c) modification by substituting Pro with Phe at position 238; (d) modification by substituting Asn with Met at position 325; (e) modification by substituting Ser with Ile at position 267; (f) modification by substituting Leu with Asp at position 328; (g) modification by substituting Ser with Val at position 267; (h) modification by substituting Leu with Trp at position 328; (i) modification by substituting Ser with Gln at position 267; (j) modification by substituting Ser with Met at position 267; (k) modification by substituting Gly with Asp at position 236; (l) modification by substituting Ala with Asn at position 327; (m) modification by substituting Asn with Ser at position 325; (n) modification by substituting Leu with Tyr at position 235; (o) modification by substituting Val with Met at position 266; (p) modification by substituting Leu with Tyr at position 328; (q) modification by substituting Leu with Trp at position 235; (r) modification by substituting Leu with Phe at position 235; (s) modification by substituting Ser with Gly at position 239; (t) modification by substituting Ala with Glu at position 327; (u) modification by substituting Ala with Gly at position 327; (v) modification by substituting Pro with Leu at position 238; (w) modification by substituting Ser with Leu at position 239; (x) modification by substituting Leu with Thr at position 328; (y) modification by substituting Leu with Ser at position 328; (z) modification by substituting Leu with Met at position 328; (aa) modification by substituting Pro with Trp at position 331; (ab) modification by substituting Pro with Tyr at position 331; (ac) modification by substituting Pro with Phe at position 331; (ad) modification by substituting Ala with Asp at position 327; (ae) modification by substituting Leu with Phe at position 328;(af) Modification by replacing Pro with Leu at position 271; (ag) Modification by replacing Ser with Glu at position 267; (ah) Modification by replacing Leu with Ala at position 328; (ai) Modification by replacing Leu with Ile at position 328; (aj) Modification by replacing Leu with Gln at position 328; (ak) Modification by replacing Leu with Val at position 328; (al) Modification by replacing Lys with Trp at position 326; (am) Modification by replacing Lys with Arg at position 334; (an) Modification by replacing His with Gly at position 268; (ao) Modification by replacing His with Asn at position 268; (ap) Modification by replacing Ser with Val at position 324; (aq) Modification by replacing Val with Leu at position 266; (ar) Modification by replacing Pro with Gly at position 271; (as) Modification by replacing Ile with Phe at position 332; (at) Modification by replacing Ser with Ile at position 324; (au) Modification by replacing Glu with Pro at position 333; (av) Modification by replacing Tyr with Asp at position 300; (aw) Modification by replacing Ser with Asp at position 337; (ax) Modification by replacing Tyr with Gln at position 300; (ay) Modification by replacing Thr with Asp at position 335; (az) Modification by replacing Ser with Asn at position 239; (ba) Modification by replacing Lys with Leu at position 326; (bb) Modification by replacing Lys with Ile at position 326; (bc) Modification by replacing Ser with Glu at position 239; (bd) Modification by replacing Lys with Phe at position 326; (be) Modification by replacing Lys with Val at position 326; (bf) Modification by replacing Lys with Tyr at position 326; (bg) Modification by replacing Ser with Asp at position 267; (bh) Modification by replacing Lys with Pro at position 326; (bi) Modification by replacing Lys with His at position 326; (bj) Modification by replacing Lys with Ala at position 334; (bk) Modification by replacing Lys with Trp at position 334; (bl) Modification by replacing His with Gln at position 268; (bm) Modification by replacing Lys with Gln at position 326; (bn) Modification by replacing Lys with Glu at position 326; (bo) Modification by replacing Lys with Met at position 326;(bp)Modification by replacing Val with Ile at position 266; (bq)Modification by replacing Lys with Glu at position 334; (br)Modification by replacing Tyr with Glu at position 300; (bs)Modification by replacing Lys with Met at position 334; (bt)Modification by replacing Lys with Val at position 334; (bu)Modification by replacing Lys with Thr at position 334; (bv)Modification by replacing Lys with Ser at position 334; (bw)Modification by replacing Lys with His at position 334; (bx)Modification by replacing Lys with Phe at position 334; (by)Modification by replacing Lys with Gln at position 334; (bz)Modification by replacing Lys with Pro at position 334; (ca)Modification by replacing Lys with Tyr at position 334; (cb)Modification by replacing Lys with Ile at position 334; (cc)Modification by replacing Gln with Leu at position 295; (cd)Modification by replacing Lys with Leu at position 334; (ce)Modification by replacing Lys with Asn at position 334; (cf)Modification by replacing His with Ala at position 268; (cg)Modification by replacing Ser with Asp at position 239; (ch)Modification by replacing Ser with Ala at position 267; (ci)Modification by replacing Leu with Trp at position 234; (cj)Modification by replacing Leu with Tyr at position 234; (ck)Modification by replacing Gly with Ala at position 237; (cl)Modification by replacing Gly with Asp at position 237; (cm)Modification by replacing Gly with Glu at position 237; (cn)Modification by replacing Gly with Leu at position 237; (co)Modification by replacing Gly with Met at position 237; (cp)Modification by replacing Gly with Tyr at position 237; (cq)Modification by replacing Ala with Lys at position 330; (cr)Modification by replacing Ala with Arg at position 330; (cs)Modification by replacing Glu with Asp at position 233; (ct)Modification by replacing His with Asp at position 268; (cu)Modification by replacing His with Glu at position 268; (cv)Modification by replacing Lys with Asp at position 326; (cw)Modification by replacing Lys with Ser at position 326; (cx)Modification by replacing Lys with Thr at position 326; (cy)Modification by replacing Val with Ile at position 323;(cz)Modification by replacing Val with Leu at position 323; (da)Modification by replacing Val with Met at position 323; (db)Modification by replacing Tyr with Asp at position 296; (dc)Modification by replacing Lys with Ala at position 326; (dd)Modification by replacing Lys with Asn at position 326; and (de)Modification by replacing Ala with Met at position 330.;

[0658] The above-mentioned modifications can be made 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 to 15, 17 to 24, and 26 to 28 of WO2013 / 047752, for example, variants of the human constant region or the human Fc region, wherein in human IgG (IgG1, IgG2, IgG3, or IgG4), 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 addition, one or more of the positions according to EU numbering at 233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396 can be replaced. In this case, the variants can include, but are not limited to, variants of the human constant region or the human Fc region containing one or more of the following:

[0659] Asp at position 233, Tyr at position 234, Asp at position 237, Ile at position 264, Glu at position 265, Phe, Met, or Leu at position 266, Ala, Glu, Gly, or Gln at position 267, Asp or Glu at position 268, Asp at position 269, Asp, Phe, Ile, Met, Asn, or Gln 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, Thr, Lys, or Arg at position 333, and Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, or Tyr at position 396 (according to EU numbering).

[0660] In an alternative embodiment, an antibody of disclosure A that contains a variant of the Fc region or constant region can have a maintained or increased binding activity to FcγRIIb and a decreased binding activity to FcγRIIa (type H) and FcγRIIa (type R) compared to a reference antibody that contains the constant region or Fc region of natural IgG. Preferred sites for amino acid substitution of such variants can be as reported in WO2014 / 030728, for example, based on the amino acid at EU numbering position 238 and at least one amino acid at positions selected from the group consisting of: positions 233, 234, 235, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409, and 419 (according to EU numbering).

[0661] More preferably, the variant can have Asp at position 238 according to EU numbering and at least one amino acid from the group consisting of: Asp at position 233, Tyr at position 234, Phe at position 235, Asp at position 237, Ile at position 264, Glu at position 265, Phe, Leu, or Met at position 266, Ala, Glu, Gly, or Gln at position 267, Asp, Gln, or Glu at position 268, Asp at position 269, Gly at position 271, Asp, Phe, Ile, Met, Asn, Pro, or Gln at position 272, Gln at position 274, Asp or Phe at position 296, Ala or Asp at position 326, Gly at position 327, Lys, Arg, or Ser at position 330, Ser at position 331, Lys, Arg, Ser, or Thr at position 332, Lys, Arg, Ser, or Thr at position 333, Arg, Ser, or Thr at position 334, Ala or Gln at position 355, Glu at position 356, Met at position 358, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 396, Arg at position 409, and Glu at position 419 (according to EU numbering).

[0662] In an alternative embodiment, compared to a reference antibody containing the constant region or Fc region of native IgG, the antibody of disclosure A containing an Fc region variant or a constant region variant can have a maintained binding activity to FcγRIIb and a reduced binding activity to all activating FcγRs, FcγRIIa (type R). Preferred sites for amino acid substitutions for such variants can be as reported in WO2014 / 163101, for example, in addition to the amino acid at position 238 (according to EU numbering), at least one amino acid selected from positions 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 according to EU numbering. More preferably, the variant can have Asp at position 238 according to EU numbering and at least one amino acid from the group consisting of: Phe at position 235; Gln or Asp at position 237; Met or Leu at position 241; Pro at position 268; Met or Val at position 295; Glu, His, Asn, or Asp at position 296; Ala or Met at position 298; Ile at position 323; Asn or His at position 324; and His or Tyr at position 330 (according to EU numbering).

[0663] Within the scope of disclosure A described herein, the level of "maintained binding activity to FcγRIIb" can be, but is not limited to, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, 100% or higher, 101% or higher, 102% or higher, 103% or higher, 104% or higher, 105% or higher, 106% or higher, 107% or higher, 108% or higher, 109% or higher, 110% or higher, 120% or higher, 130% or higher, 140% or higher, 150% or higher, 175% or higher, or 2-fold or higher.

[0664] Within the scope of Disclosure A described herein, the level of the foregoing "reduced binding activity to all activating FcγRs, especially FcγRIIa (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 murine constant regions and Fc regions. In one embodiment, an antibody of Disclosure A or B can comprise such variants.

[0666] Within the scope of Disclosure A and B described herein, unlike FcγRs that belong to the immunoglobulin superfamily, "FcRn", especially human FcRn, is structurally similar to the polypeptide of class I major histocompatibility complex (MHC), and exhibits 22% to 29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today 18(12), 592 - 598 (1997)). FcRn is expressed as a heterodimer, which consists 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 attaches the protein to the cell surface. The α1 and α2 domains interact with the FcRn - binding domain of the antibody Fc region (Raghavan et al., Immunity 1:303 - 315 (1994)).

[0667] FcRn is expressed in the maternal placenta and yolk sac of mammals and is involved in the transfer of maternal IgG to the 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 a variety of other tissues and endothelial cell systems in various species. FcRn is also expressed in adult vascular endothelium, the muscle vasculature, and hepatic sinusoidal capillaries. FcRn is thought to play a role in maintaining plasma IgG concentration by binding IgG and recycling it to the serum. Generally, 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 precursors (containing signal sequences) shown in NM_004107.4 and NP_004098.1, respectively (RefSeq accession numbers are shown in parentheses).

[0669] This precursor forms a complex with human β2-microglobulin in vivo. Thus, soluble human FcRn capable of forming a complex with human β2-microglobulin can be prepared using known recombinant expression techniques and is suitable for use in various experimental systems. The soluble human FcRn can be used to evaluate the FcRn-binding activity of antibodies or Fc region variants. In Disclosure A or B, the 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 Disclosures A and B described herein, when an antibody or Fc region variant has FcRn-binding activity, it may have an "FcRn-binding domain", preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited as long as the antibody has binding activity or affinity for FcRn at acidic pH and / or at neutral pH; or it may be a domain having the activity of directly or indirectly binding FcRn. The domains include, but are not limited to, the Fc region of IgG-type immunoglobulins, albumin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides, and anti-FcRn scaffold molecules (which have the activity of directly binding FcRn), and molecules that bind IgG or albumin (which have the activity of indirectly binding FcRn). In Disclosure A or B, it is also possible to use domains having FcRn-binding activity in the acidic pH range and / or in the neutral pH range. If the domains initially have FcRn-binding activity in the acidic pH range and / or in the neutral pH range, they can be used without further modification. If the domain has only weak or no FcRn-binding activity in the acidic pH range and / or in the neutral pH range, the amino acid residues in the FcRn-binding domain of the antibody or Fc region variant can be modified to have FcRn-binding activity in the acidic pH range and / or in the neutral pH range. Alternatively, the amino acids of a domain that initially has FcRn-binding activity in the acidic pH range and / or in the neutral pH range can be modified to further increase its FcRn-binding activity. The FcRn-binding activity in the acidic pH range and / or in the neutral pH range can be compared before and after amino acid modification to find amino acid modifications of interest for the FcRn-binding domain.

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

[0672] Within the scope of the disclosures A and B described herein, whether the FcRn-binding activity of an antibody or an Fc region (variant) increases, (substantially) remains, or decreases compared to the antibody or Fc region (variant) before modification can be evaluated by known methods, such as those described in the examples herein, and for example, BIACORE, Scatchard curves, and flow cytometry (see WO2013 / 046722). The extracellular domain of human FcRn can be used as a soluble antigen in these assays. In the measurement of the FcRn-binding activity of an antibody or an Fc region (variant), a person skilled in the art can appropriately select conditions (other than pH). The assay can be carried out, for example, under the conditions of MES buffer and 37 °C, as described in WO2009 / 125825. The FcRn-binding activity of an antibody or an Fc region (variant) can be evaluated, for example, by loading FcRn as an analyte onto a chip immobilized with the antibody.

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

[0674] For the pH conditions for measuring the binding activity between FcRn and the FcRn-binding domain contained in an antibody or Fc region (variant), acidic pH conditions or neutral pH conditions can be appropriately used. For the temperature conditions for measuring the binding activity (binding affinity) between FcRn and the FcRn-binding domain, any temperature between 10°C and 50°C can be used. To determine the binding activity (binding affinity) between FcRn and the human FcRn-binding domain, a temperature between 15°C and 40°C can be preferably used. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C can be used. A non-limiting example of this temperature can be 25°C.

[0675] In one embodiment, in the case where the antibodies of Disclosure A or B have FcRn-binding activity, they 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 the activity of directly or indirectly binding to FcRn. In a specific embodiment, it can be preferable that, compared with a reference antibody containing the constant region of natural IgG, the antibodies of Disclosure A or B have, for example, increased FcRn-binding activity under neutral pH conditions (see WO2013 / 046722). From the perspective of comparing the FcRn-binding activities of the two, it can be preferable that, without limitation, the antibodies of Disclosure A or B and the reference antibody containing the constant region of natural IgG have the same amino acid sequence in regions other than, preferably, the constant region modified at one or more amino acid residues of the antibodies of Disclosure A or B (e.g., the variable region).

[0676] In one embodiment, within the scope of Disclosure A described herein, in the case where the antibodies of Disclosure A have increased FcRn-binding activity under neutral pH conditions, without being limited by a particular theory, the antibodies of Disclosure A can have any combination of 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 by having an ion concentration-dependent antigen-binding domain; the property of being rapidly internalized into cells by having an increased pI and increased positive charge throughout the antibody; and the property of being rapidly internalized 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 antigens from plasma can be further promoted. Those skilled in the art can determine the optimal pI value of the antibodies of Disclosure A to utilize these properties.

[0677] Within the scope of Disclosures A and B described herein, according to Yeung et al. (J. Immunol. 182: 7663-7671 (2009)), the activity of native human IgG1 binding to human FcRn is KD 1.7 μM within the acidic pH range (pH 6.0), while the activity is almost undetectable within the neutral pH range. Therefore, in order to increase the FcRn-binding activity within the neutral pH range, as an antibody of Disclosure A or B, it is preferable to use: an antibody or constant region variant or Fc region variant whose human FcRn-binding activity is KD 20 μM or stronger within the acidic pH range and whose human FcRn-binding activity is equivalent to or stronger than that of native human IgG within the neutral pH range; preferably an antibody or constant region variant or Fc region variant whose human FcRn-binding activity is KD 2.0 μM or stronger within the acidic pH range and whose human FcRn-binding activity is KD 40 μM or stronger within the neutral pH range; and more preferably an antibody or constant region variant or Fc region variant whose human FcRn-binding activity is KD 0.5 μM or stronger within the acidic pH range and whose human FcRn-binding activity is KD 15 μM or stronger within the neutral pH range. The KD value is determined by the method described by 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 Disclosures A and B described herein, a domain of any structure that binds to FcRn can be used as an FcRn-binding domain. In this case, the FcRn-binding domain can be produced without introducing amino acid modifications, or the affinity for FcRn can be increased by introducing additional modifications.

[0679] Within the scope of the disclosure A and B described herein, the starting FcRn-binding domain can include, for example, the Fc region or constant region of (human) IgG. Any Fc region or constant region can be used as the starting Fc region or starting constant region as long as the variant of the starting Fc region or starting constant region is capable of binding to FcRn within the acidic pH range and / or within the neutral pH range. Alternatively, the Fc region or constant region obtained by further modifying the starting Fc region or starting constant region (whose amino acid residues have been modified from the Fc region or constant region) can also be suitably used as the Fc region or constant region. The starting Fc region or starting constant region can include Fc regions known to be produced recombinantly. The starting Fc region or starting constant region can refer to the polypeptide itself, a composition containing the starting Fc region or starting constant region, or the amino acid sequence encoding the starting Fc region or starting constant region, depending on the context. The source of the starting Fc region or starting constant region is not limited, and it can be obtained from any organism of non-human animals or humans. In addition, the starting FcRn-binding domain can be obtained from cynomolgus monkeys, rhesus, macaques, chimpanzees, and humans. The starting Fc region or starting constant region can be obtained from human IgG1, but is not limited to any specific IgG type. This means that the Fc regions of human IgG1, IgG2, IgG3, or IgG4 can be used as suitable starting FcRn-binding domains, and the Fc regions or constant regions of IgG types or subclasses 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, for example, in Strohl, Curr.Opin. Biotechnol.20(6):685-691(2009); Presta, Curr.Opin.Immunol.20(4): 460-470(2008); Davis et al., Protein Eng.Des.Sel.23(4):195-202(2010), WO2009 / 086320, WO2008 / 092117; WO2007 / 041635; and WO2006 / 105338).

[0680] Within the scope of Disclosures A and B described herein, the amino acid residues of the starting FcRn-binding domain, starting Fc region, or starting constant region can contain, for example, one or more mutations: for example, substitution mutations using amino acid residues different from those in the starting Fc region or starting constant region; insertion of one or more amino acid residues into the amino acid residues in the starting Fc region or starting constant region; or deletion of one or more amino acid residues from the amino acid residues in the starting Fc region or starting constant region. 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 an Fc region or constant region that does not occur in nature. The variant must have less than 100% sequence identity or similarity to the starting Fc region or 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 additionally 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 starting constant region. In a non-limiting example, at least one amino acid is different between the modified Fc region or constant region of Disclosure A or B and the starting Fc region or starting constant region.

[0681] Within the scope of Disclosures A and B described herein, an Fc region or constant region having FcRn-binding activity within the acidic pH range and / or within the neutral pH range can be obtained by any method. Specifically, variants of an Fc region or constant region having FcRn-binding activity within the acidic pH range and / or within the neutral pH range can be obtained by modifying the amino acids of a human IgG-type antibody that can be used as the starting Fc region or starting constant region. IgG-type antibody Fc regions or constant regions suitable for modification include, for example, the Fc regions or constant regions of human IgG (IgG1, IgG2, IgG3, and IgG4, and their variants), and mutants that spontaneously arise therefrom are also included in the IgG Fc region or constant region. For the Fc regions or constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, many allotypic sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, and any of them can be used for Disclosure A or B. In particular, for the human IgG1 sequence, the amino acid sequence according to EU numbering positions 356 to 358 can be DEL or EEM.

[0682] In one embodiment of 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 for modifying amino acids to increase FcRn-binding activity at 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 (according to EU numbering) in the Fc region or constant region of a human IgG antibody, as described in WO2013 / 046722. WO2013 / 046722 also describes, as part of the preferred modifications in the Fc region or constant region, for example, the modification of one or more amino acids selected from the group consisting of: the amino acid at position 256 is modified to Pro, the amino acid at position 280 is modified to Lys, the amino acid at position 339 is modified to Thr, the amino acid at position 385 is modified to His, the amino acid at position 428 is modified to Leu, and the amino acid at position 434 is modified 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 carried out individually at a single position or at two or more positions. Modification of these amino acid residues can enhance the FcRn binding of the Fc region or constant region of an IgG-type antibody at neutral pH conditions. Modification of these amino acid residues can also be appropriately introduced into the antibodies of Disclosure A or B.

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

[0684] In one embodiment, when compared to a reference antibody containing the Fc region or constant region of native IgG or a reference antibody containing the starting Fc region or starting constant region, the FcRn-binding activity of the FcRn-binding domain of the antibody of Disclosure A or B is increased. That is, the FcRn-binding activity of the Fc region variant or constant region variant of Disclosure A or B, or an antibody containing said variant, is greater than the binding activity of the reference antibody. This can mean that when compared to the FcRn-binding activity of the reference antibody, the FcRn-binding activity of the antibody of Disclosure A or B can be, for example: 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 100% or higher, 105% or higher, preferably 110% or higher, 115% or higher, 120% or higher, 125% or higher, more preferably 130% or higher, 135% or higher, 140% or higher, 145% or higher, 150% or higher, 155% or higher, 160% or higher, 165% or higher, 170% or higher, 175% or higher, 180% or higher, 185% or higher, 190% or higher, 195% or higher, 2-fold or higher, 2.5-fold or higher, 3-fold or higher, 3.5-fold or higher, 4-fold or higher, 4.5-fold or higher, or 5-fold or higher.

[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 (the sequence present in a native human antibody), so that the immunogenicity of the antibody is not increased when the antibody is administered in vivo (preferably, into the human body). Alternatively, after modification, mutations can be introduced in such a way into positions outside the amino acid modification sites that one or more of the FRs (FR1, FR2, FR3, and FR4) are replaced with human sequences. Methods for replacing one or more FRs with human sequences are known in the art and include, but are not limited to, those reported by Ono et al., Mol. Immunol. 36(6): 387-395 (1999). Humanization methods 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 sequences (also referred to as "FR sequences") of the heavy and / or light chain variable regions of an antibody of Disclosure A or B can comprise human germline framework region sequences. When the framework region sequences are entirely germline sequences, the antibody is expected to elicit little or no immunogenic response when administered to a human (e.g., for treating or preventing a disease).

[0687] The 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 suitably used for Disclosure A or B. The germline sequences can be classified based on their similarity (Tomlinson et al. (J. Mol. Biol. 227:776-798 (1992)); Williams et al. (Eur. J. Immunol. 23:1456-1461 (1993)); and Cox et al. (Nat. Genet. 7:162-168 (1994))). Preferred germline sequences can be suitably selected from: Vκ, which is classified into seven subgroups; Vλ, which is classified into ten subgroups; and VH, which is classified 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, for example, in Matsuda et al. (J. Exp. Med. 188:1973-1975 (1998)), and those skilled in the art can suitably design based on the information of these sequences. Other fully human FR sequences or sequences of regions equivalent thereto can also preferably be used.

[0689] Fully human Vκ sequences can preferably include, for example: A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14, or O18, which are classified into subgroup Vk1; A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, which are classified into subgroup Vk2; A11, A27, L2, L6, L10, L16, L20, and L25, which are classified into subgroup Vk3; B3, which is classified into subgroup Vk4; B2 (also known as "Vk5-2"), which is classified into subgroup Vk5; or A10, A14, and A26, which are classified into 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)).

[0690] Fully human Vλ sequences can preferably include, for example: V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, and V1-22, which are classified into subgroup VL1; V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19, which are classified into subgroup VL2; V3-2, V3-3, and V3-4, which are classified into subgroup VL3; V4-1, V4-2, V4-3, V4-4, and V4-6, which are classified into subgroup VL4; or V5-1, V5-2, V5-...

Claims

1. An Fc region variant comprising an FcRn-binding domain, wherein the Fc region variant is derived from a human IgG1 antibody, and the combination of amino acid substitutions in the FcRn-binding domain is selected from the group consisting of: According to EU numbering, (a) N434A / Y436T / Q438R / S440E; (b) M428L / N434A / Q438R / S440E; (c) M428L / N434A / Y436T / Q438R / S440E; and (d) M428L / N434A / Y436V / Q438R / S440E, wherein the plasma retention of the Fc region variant is increased relative to a reference Fc region variant comprising the combination of amino acid substitutions N434Y / Y436V / Q438R / S440E according to EU numbering.

2. The Fc region variant according to claim 1, wherein the Fc region variant has any one or more of the following characteristics: (a) Enhanced FcRn-binding activity under acidic pH conditions compared to the Fc region of native IgG; (b) No significant enhancement in binding activity to an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of native IgG, wherein the ADA is optionally a rheumatoid factor (RF); or (c) Reduced plasma clearance (CL), increased plasma retention time, or increased plasma half-life (t1 / 2) compared to the Fc region of native IgG.

3. An antibody comprising the Fc region variant according to claim 1 or 2, wherein the antibody is an IgG1 antibody.

4. A pharmaceutical composition comprising the antibody according to claim 3, optionally wherein the pharmaceutical composition is for increasing the retention of the antibody in plasma.

5. A nucleic acid encoding the Fc region variant according to claim 1 or 2 or the antibody according to claim 3.

6. A vector comprising the nucleic acid according to claim 5.

7. A host cell comprising the vector according to claim 6.

8. A method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the variant, the method comprising culturing the host cell according to claim 7 and subsequently collecting the Fc region variant or the antibody comprising the variant from the cell culture.

9. The method according to claim 8, which further optionally comprises any one or more of the following: (a) Selecting an Fc region variant having enhanced FcRn-binding activity under acidic pH conditions compared to the Fc region of native IgG; (b) Selecting an Fc region variant having no significant enhancement in binding activity to an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of native IgG; (c) Selecting an Fc region variant having increased plasma retention compared to the Fc region of native IgG; and (d) Selecting an antibody comprising an Fc region variant that is capable of promoting antigen removal from plasma compared to a reference antibody comprising the Fc region of native IgG.

10. A method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising said variant, wherein said Fc region variant is derived from a human IgG1 antibody, the method comprising substituting amino acids in such a way that the combination of amino acid substitutions in the resulting Fc region variant or antibody comprising said variant is selected from the group consisting of: According to EU numbering, (a) N434A / Y436T / Q438R / S440E; (b) M428L / N434A / Q438R / S440E; (c) M428L / N434A / Y436T / Q438R / S440E; and (d) M428L / N434A / Y436V / Q438R / S440E, wherein the plasma retention of said Fc region variant is increased relative to a reference Fc region variant comprising the amino acid substitution combination N434Y / Y436V / Q438R / S440E according to EU numbering.

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