CN108271372B

CN108271372B - IL-8-binding antibodies and uses thereof

Info

Publication number: CN108271372B
Application number: CN201680054135.3A
Authority: CN
Inventors: 井川智之; 前田敦彦; 中村元气; 村冈优
Original assignee: Chugai Pharmaceutical Co Ltd
Current assignee: Chugai Pharmaceutical Co Ltd
Priority date: 2015-09-18
Filing date: 2016-08-05
Publication date: 2021-07-09
Anticipated expiration: 2036-08-05
Also published as: CN108271372A; IL258088A; TWI814162B; MA42808A; TW202214688A; JP2021168658A; KR20180125036A; HK1252615A1; AU2016323088B2; IL258088B2; CA2993423C; ZA201800536B; MX2022008781A; JP2023164732A; JP6266164B2; AU2016323088A1; IL285375A; TW201712032A; MX2018003005A; IL258088B1

Abstract

One non-exclusive aspect provides novel IL-8 antibodies that are excellent as medicaments.

Description

IL-8-binding antibodies and uses thereof

Technical Field

Cross reference to related applications

This application relates to and claims the priority of japanese priority patent application No. 2015-185254 filed in japan on 18/9/2015. The contents of this priority application are incorporated by reference in their entirety. Technical Field

In one non-exclusive aspect, the present disclosure provides anti-IL-8 antibodies, pharmaceutical compositions containing the antibodies, nucleic acids encoding the antibodies, and host cells containing the nucleic acids. Methods of producing IL-8 antibodies and pharmaceutical compositions and uses in treating, for example, IL-8 related disorders, are also provided.

Background

Antibodies are attractive as drugs because they are highly stable in plasma and have fewer side effects. Many IgG-type therapeutic antibodies are on the market, and even many therapeutic antibodies are currently in development (Reichert et al, nat. Biotechnol.23: 1073-1078(2005) (NPL 1); Pavlou et al, Eur. J. pharm. Biopharm.59 (3): 389-396(2005) (NPL 2)). Meanwhile, various technologies are being developed for second-generation therapeutic antibodies; including techniques for improving effector function, antigen binding capacity, pharmacokinetics or stability, and reducing the risk of immunogenicity (Kim et al, mol. cells.20 (1): 17-29 (2005)) (NPL 3)). The dosage of therapeutic antibodies is usually very high, and therefore the development of therapeutic antibodies encounters problems such as difficulty in producing subcutaneous formulations and high production costs. Methods of improving the pharmacokinetic, pharmacodynamic, and antigen binding properties of therapeutic antibodies provide a means of reducing the dosage and production costs associated with therapeutic antibodies.

Substitution of amino acid residues in the constant region (hystration) provides a method for improving antibody pharmacokinetics (Hinton et al, J.Immunol.176 (1): 346-640 (2006) (NPL 4); Ghetie et al, nat. Biotechnol.15 (7): 637-640(1997)) (NPL 5). Affinity maturation techniques provide methods for enhancing the antigen neutralizing capacity of antibodies (Rajpal et al, Proc. Natl. Acad. Sci. USA 102 (24): 8466-. Improving the antigen binding properties of antibodies can improve the in vitro biological activity or reduce the dose of the antibody, and can further improve in vivo (in the body) efficacy (Wu et al, j.mol. biol.368: 652-.

The amount of antigen that can be neutralized by an antibody molecule depends on the affinity of the antibody for the antigen; and therefore, it is 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-. Furthermore, if it can be covalently bound to an antigen so that the affinity is infinite, it is theoretically possible to neutralize one antigen molecule with one antibody molecule (2 antigens when the antibody is bivalent). Nevertheless, one limitation of therapeutic antibody development to date is that one antibody molecule typically binds and neutralizes only one antigen molecule (2 antigens when the antibody is bivalent). It has recently been reported that an antibody that binds an antigen in a pH-dependent manner (hereinafter also referred to as "pH-dependent antibody" or "pH-dependent-binding antibody") is used to allow one antibody molecule to bind to and neutralize a plurality of antigen molecules (see, for example, WO2009/125825(PTL 1); Igawa et al, nat. biotechnol.28: 1203-1207(2010) (NPL 9)). The pH-dependent antibody binds strongly to antigen under neutral pH conditions in plasma and dissociates from antigen under acidic pH conditions within the endosome (endosome) of the cell. After dissociation from the antigen, the antibody is recycled to the plasma via FcRn and subsequently free to bind and neutralize another antigenic molecule; and thus one pH-dependent antibody can repeatedly bind to and neutralize multiple antigen molecules.

It has recently been reported that the antibody recycling property can be achieved by focusing on the difference in calcium (Ca) ion concentration between plasma and endosomes, and using an antibody having an antigen-antibody interaction indicating calcium dependence (hereinafter also referred to as "calcium ion concentration-dependent antibody") (WO2012/073992(PTL 2)). (hereinafter, pH-dependent antibody and "calcium ion concentration-dependent antibody" are collectively referred to as "pH/Ca concentration-dependent antibody")

By binding to FcRn, IgG antibodies have a long retention 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 taken up non-specifically into the cells and returned to the cell surface by binding to FcRn in the endosome under acidic pH conditions in the endosome. IgG subsequently dissociates from FcRn under neutral pH conditions in plasma.

pH-dependent antibodies modified to increase FcRn binding under neutral pH conditions are reported to have the ability to repeatedly bind and remove antigenic molecules from plasma; and thus the use of such antibodies allows removal of antigen from plasma (WO2011/122011(PTL 3)). According to this report, a pH-dependent antibody modified to increase FcRn binding thereof under neutral pH conditions (e.g., pH 7.4) can further accelerate antigen removal compared to a pH-dependent antibody comprising an Fc region of a native IgG antibody (WO2011/122011(PTL 3)).

At the same time, when mutations are introduced into the Fc region of IgG antibodies to eliminate their binding to FcRn under acidic pH conditions, they may no longer be recycled from the endosome into the plasma, which significantly impairs the retention of the antibody in the plasma. Thereupon, a method of increasing FcRn binding under acidic pH conditions was reported as a method for improving plasma retention of IgG antibodies. Introduction of amino acid modifications into the Fc region of IgG antibodies to increase their FcRn binding under acidic pH conditions can enhance the efficacy of recycling from endosomes to plasma, which thereby results in improved plasma retention. For example, modifications of M252Y/S254T/T256E (YTE; Dall' Acqua et al, J.biol.chem.281: 23514-.

However, in addition to considering that immunogenicity or aggregation incidence may be deteriorated in antibodies comprising such Fc region variants, the FcRn binding of which is increased under neutral pH conditions or acidic pH conditions, increased binding to anti-drug antibodies (hereinafter also referred to as "Pre-existing ADA") present in patients prior to administration of therapeutic antibodies (e.g., rheumatoid factors) has been further reported (WO2013/046722(PTL4), WO2013/046704(PTL 5)).

WO2013/046704(PTL5) reports that a variant of the Fc region containing specific mutations (represented by two residue modifications of Q438R/S440E according to EU numbering) increases binding to FcRn under acidic pH conditions and also shows a significant decrease in binding to rheumatoid factor compared to unmodified native Fc. However, WO2013/046704(PTL5) does not specifically demonstrate that this variant Fc region has better plasma retention relative to antibodies to the native Fc region.

Therefore, safe and more favorable Fc region variants (which do not show binding to pre-existing ADA) with further improved plasma retention are needed.

Antibody-dependent cellular cytotoxicity (hereinafter referred to as "ADCC"), complement-dependent cytotoxicity (hereinafter referred to as "CDC"), antibody-dependent cellular phagocytosis (ADCP), which is IgG antibody-mediated phagocytosis of target cells, are reported as effector functions of IgG antibodies. In order for an IgG antibody to mediate ADCC activity or ADCP activity, the Fc region of the IgG antibody must bind to an antibody receptor (referred to as "Fc γ receptor", "FcgR", "Fc γ receptor" or "Fc γ R" in the context of publication a described herein) present on the surface of effector cells such as killer cells, natural killer cells or activated macrophages. In humans, Fc γ RIa, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa and Fc γ RIIIb isoforms have been reported as Fc γ R family proteins, and their respective allotypes have also been reported (Jefferis et al, immunol. lett.82: 57-65(2002) (NPL 13)). The balance of the affinity of an antibody for each of an activating receptor comprising Fc γ RIa, Fc γ RIIa, Fc γ RIIIa or Fc γ RIIIb and an inhibitory receptor comprising Fc γ RIIb is an important element in optimizing antibody effector function.

Various techniques for increasing or improving the activity of a therapeutic antibody against an antigen have been reported so far. For example, the activity of antibodies binding to activating Fc γ r(s) plays an important role in the cytotoxicity of antibodies, and thus, antibodies targeting membrane-type antigens and having increased cytotoxicity due to enhanced activating Fc γ r(s) binding have been developed. See, for example, WO2000/042072(PTL 6); WO2006/019447(PTL 7); lazar et al, proc.nat.acad.sci.usa.103: 4005 and 4010(2006) (NPL 14); shinkawa et al, J.biol.chem.278, 3466-; clynes et al, Proc.Natl.Acad.Sci.U SA 95: 652-656(1998) (NPL 16); clynes et al, nat. med.6: 443-446(2000) (NPL 17)). Similarly, the binding activity to inhibitory Fc γ R (Fc γ RIIb in humans) plays an important role in immunosuppressive activity, agonist activity, and thus there has been research into antibodies with increased inhibitory Fc γ R-binding activity that target membrane-type antigens (Li et al, proc. nat. acad. sci. usa.109 (27): 10966-. Furthermore, the effect of Fc γ R binding of antibodies that bind soluble antigens was examined mainly from the point of view of side effects (Scappathicii et al, J.Natl.cancer Inst.99 (16): 1232-1239(2007) (NPL 19)). For example, when antibodies with increased Fc γ RIIb binding are used as drugs, one might expect a reduced risk from the production of anti-drug antibodies (Desai et al, J.Immunol.178 (10): 6217-6226(2007) (NPL 20)).

Recently, it has been reported that introduction of amino acid modifications into the Fc region of IgG antibodies to increase the activity of antibody binding activation and/or inhibitory fcyr targeting soluble antigens may further accelerate antigen removal from serum (WO2012/115241(PTL8), WO2013/047752(PTL9), WO2013/125667(PTL10), WO2014/030728(PTL 11)). Furthermore, Fc region variants have been identified which show little change in their Fc γ RIIb-binding activity from the native IgG antibody Fc region, but have reduced activity on other activating Fc γ Rs (WO2014/163101(PTL 12)).

Plasma retention of soluble antigen is very short compared to antibodies with an FcRn-mediated recycling mechanism, and thus soluble antigen can show increased plasma retention and plasma concentration by binding to antibodies with such a recycling mechanism (e.g., antibodies that do not have the characteristics of pH/Ca concentration-dependent antibodies). Thus, for example, when soluble antigens in plasma have multiple types of physiological functions, even if one type of physiological function is blocked by antibody binding, the plasma concentration of the antigen may worsen the pathological symptoms caused by increased plasma retention of the antigen due to antibody binding and/or other physiological functions resulting from plasma concentration. In this case, in addition to the above-described exemplary modification of antibodies to accelerate antigen removal, for example, methods using formation of multivalent immune complexes from various pH/Ca concentration-dependent antibodies and various antigens, and increasing binding to FcRn, Fc γ r(s), complement receptors have been reported (WO2013/081143(PTL 13)).

Even when the Fc region is not modified, it is reported that the isoelectric point (pI) of an antibody can be increased or decreased irrespective of the type of antigen or antibody by modifying one or more amino acid residues so as to change the charge of the one or more amino acid residues that can be exposed on the surface of the variable region of the antibody, and the half-life of the antibody in blood can be controlled without substantially decreasing the antigen-binding activity of the antibody (WO2007/114319(PTL 14): a technique of replacing amino acids mainly in the FR; WO2009/041643(PTL 15): a technique of replacing amino acids mainly in the CDRs). These documents show that it is possible to prolong 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.

With regard to modification of the charge of amino acid residues in the constant region of an antibody, it has been reported that antigen uptake in cells can be facilitated by modifying the charge of a particular amino acid residue or residues, particularly in the CH3 domain, to increase the pI of the antibody, and it also describes that this modification preferably does not interfere with binding to FcRn (WO2014/145159(PTL 16)). It has also been reported that modifying the charge of amino acid residues in the constant region of an antibody (the main CH1 domain) to reduce pI can prolong the half-life of the antibody in plasma, and that in combination with mutations in the amino acid residues that increase FcRn binding, can enhance its binding to FcRn and prolong the plasma half-life of the antibody (WO2012/016227(PTL 17)).

Meanwhile, when such modification techniques designed to increase or decrease the pI of an antibody are combined with techniques other than those that increase or decrease binding to FcRn or fcyr(s), it is unclear whether there is an effect in promoting plasma retention of the antibody or removal of antigen from plasma.

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

ECM has been reported to be involved in the in vivo kinetics of proteins administered to living organisms. The blood concentration of VEGF-Trap molecules, which are fusion proteins between VEGF receptors and Fc, was measured at the time of subcutaneous administration (Holash et al, Proc. Natl. Acad. Sci., 99 (17): 11393-11398(2002) (NPL 21)). The plasma concentration of subcutaneously administered VEGF-Trap molecules with high pI is low and therefore their bioavailability is low. The modified VEGF-Trap molecule, whose pI is reduced by amino acid substitution, has higher plasma concentrations and its bioavailability is likely to be improved. Furthermore, the change in bioavailability correlates with the strength of binding to ECM, and it becomes apparent therefore that the bioavailability of the VEGF-Trap molecule upon subcutaneous administration is dependent on its strength of binding to ECM at the subcutaneous site.

WO2012/093704(PTL18) reports that there is an inverse correlation between antibody binding to ECM and plasma retention, and thus, antibody molecules that do not bind ECM have better plasma retention when compared to antibodies that bind ECM.

Thus, techniques have been reported to reduce extracellular matrix binding in order to improve protein bioavailability and plasma retention in vivo. In contrast, the advantage of increasing antibody binding to the ECM has not been identified to date.

Human IL-8 (interleukin 8) is a member of the chemokine family, which is 72 or 77 amino acid residues in length. The term "chemokine" is a generic term for a family of proteins with molecular weights of 8-12kDa and containing 4 cysteine residues that form intermolecular disulfide bonds. Chemokines are classified into CC chemokines, CXC chemokines, C chemokines, CA3C chemokines according to the features of the cysteine arrangement. IL-8 is classified as a CXC chemokine and is also known as CXCL 8.

IL-8 in solution in monomer or homodimer form. IL-8 monomers contain antiparallel beta sheets and have a structure in which the C-terminal alpha helix passes through and covers the beta sheet. The IL-8 monomer, in the case of the 72 amino acid form of IL-8, includes two disulfide linkages between cysteine 7 and cysteine 34 and between cysteine 9 and cysteine 50. IL-8 homodimers are stabilized by non-covalent interactions between the beta sheets of the two monomers, since there is no covalent binding between the molecules of the homodimers.

IL-8 expression is induced in various cells such as peripheral blood mononuclear cells, tissue macrophages, NK cells, fibroblasts and vascular endothelial cells in response to stimulation by inflammatory cytokines (Russo et al, exp. Rev. Clin. Immunol.10 (5): 593-619(2014) (NPL 22)).

In normal tissues, chemokines are usually undetectable, or only weakly detectable, but strongly detectable at sites of inflammation and participate in inducing inflammation by promoting lymphocyte infiltration into sites of inflamed tissue. IL-8 is a proinflammatory chemokine that is 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 activity and produces IL-8 at damaged tissues to promote the chemotaxis of neutrophils adhered to vascular endothelial cells into the tissues and induce inflammation with neutrophil infiltration. IL-8 is also known to be a potent angiogenic factor of endothelial cells and is involved in promoting tumor angiogenesis.

Inflammatory diseases associated with elevated (e.g., excessive) IL-8 levels include inflammatory diseases of the skin such as inflammatory keratoses (e.g., psoriasis), atopic dermatitis (atopic dermatitis), contact dermatitis (contact dermatitis); chronic inflammatory disorders (which are autoimmune diseases), such as rheumatoid arthritis (rhematoid arthritis), Systemic Lupus Erythematosus (SLE), and Behcet's disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis (ulcerative colitis); inflammatory liver diseases such as hepatitis B (hepatitis B), hepatitis C (hepatitis C), alcoholic hepatitis (alcoholic hepatitis), drug-induced allergic hepatitis; inflammatory renal diseases such as glomerulonephritis (glorulephritis); inflammatory respiratory diseases such as bronchitis and asthma; inflammatory chronic vascular diseases such as atherosclerosis; multiple sclerosis, aphtha, vocal cords inflammation (choritis), 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, lung, prostate, gastric, breast, melanoma, head and neck, and renal cancers; sepsis due to 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).

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 proposed yet. To date, only one pharmaceutical composition comprising an IL-8 antibody is available, which is a murine anti-IL-8 antibody for use as an external medicament for psoriasis. New anti-IL-8 antibodies for the treatment of disease are desired.

[ list of references ]

[ patent document ]

[PTL1]WO2009/125825

[PTL2]WO2012/073992

[PTL3]WO2011/122011

[PTL4]WO2013/046722

[PTL5]WO2013/046704

[PTL6]WO2000/042072

[PTL7]WO2006/019447

[PTL8]WO2012/115241

[PTL9]WO2013/047752

[PTL10]WO2013/125667

[PTL11]WO2014/030728

[PTL12]WO2014/163101

[PTL13]WO2013/081143

[PTL14]WO2007/114319

[PTL15]WO2009/041643

[PTL16]WO2014/145159

[PTL17]WO2012/016227

[PTL18]WO2012/093704

[ non-patent document ]

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

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

[ NPL3] Kim et al, mol. cells.20 (1): 17-29(2005)

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

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

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

[ NPL7] Wu et al, J.mol.biol.368: 652(2007)

[ NPL8] Wu et al, J.mol.biol.368: 652-665(2007)

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

[ NPL10] Dall' Acqua et al, J.biol.chem.281: 23514-235249(2006)

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

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

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

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

[ NPL15] Shinkawa et al, J.biol.chem.278, 3466-

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

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

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

[ NPL19] Scappacici et al, J.Natl.cancer Inst.99 (16): 1232-1239(2007)

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

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

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

[ NPL23] Desai et al, J.Immunol.178 (10): 6217-6226(2007)

Summary of The Invention

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

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

In one non-exclusive aspect, a non-limiting object of embodiments of disclosure C is to provide anti-IL-8 antibodies with pH-dependent binding affinity for IL-8. Other embodiments relate to anti-IL-8 antibodies that have the effect of rapidly removing IL-8 when administered to an individual as compared to a reference antibody. In another embodiment, the disclosure C relates to an anti-IL-8 antibody that is capable of stably maintaining its IL-8-neutralizing activity when administered to an individual. In some embodiments, the anti-IL-8 antibody exhibits reduced immunogenicity. In other embodiments, the disclosure C relates to methods of making and using the above anti-IL-8 antibodies. Another alternative non-limiting object of disclosure C is to provide novel anti-IL-8 antibodies that can be included in pharmaceutical compositions.

In one non-exclusive aspect, within the scope of disclosure a as provided herein, the inventors have surprisingly found that the ability of an ion concentration-dependent antibody, which is an antibody comprising an ion concentration-dependent antigen binding domain ("antigen binding domain whose antigen binding activity changes depending on the ion concentration conditions"), to remove antigen from plasma can be facilitated by modifying at least one amino acid residue exposed on the surface of the antibody, thereby increasing its isoelectric point (pI). In another non-exclusive aspect, the inventors have found that an ion concentration-dependent antibody with an increased pI can further increase extracellular matrix binding of the antibody. Thus, without being bound to a particular theory, the inventors found that removal of antigen from plasma can be increased by increasing the binding of antibodies to the extracellular matrix.

In one non-exclusive aspect, the present inventors conducted extensive studies on safe and more advantageous Fc region variants that do not show binding to anti-drug antibodies (pre-existing ADA) and that can further improve plasma retention, within the scope of disclosure B as provided herein. As a result, the present inventors surprisingly found that Fc region variants comprising a combination of replacement of amino acid 434 by EU numbering and two specific residue mutations (represented by Q438R/S440E by ala (a) as amino acid residue mutations are preferred for maintaining a significant reduction in binding to rheumatoid factor, together with achieving plasma retention of the antibody.

In one non-exclusive aspect, within the scope of disclosure C as 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 verifications, the present inventors identified a pH-dependent anti-IL-8 antibody having the effect of rapidly removing IL-8 when administered to an individual compared to a reference antibody. In some embodiments, the disclosure C relates to can stably maintain its IL-8-neutralizing activity of pH-dependent anti IL-8 antibody. In other non-limiting embodiments, the pH-dependent anti-IL-8 antibody has reduced immunogenicity and excellent expression levels.

Furthermore, within the scope of disclosure C, the inventors succeeded in obtaining anti-IL-8 antibodies comprising an Fc region with increased FcRn-binding affinity at acidic pH relative to the FcRn-binding affinity of the native Fc region. In an alternative aspect, the inventors have succeeded in obtaining anti-IL-8 antibodies comprising an Fc region having reduced binding affinity for pre-existing ADA relative to the binding affinity of the native Fc region for pre-existing ADA. In an alternative aspect, the present inventors have succeeded in obtaining anti-IL-8 antibodies comprising an Fc region having an increased plasma half-life relative to that of the native Fc region. In an alternative aspect, the inventors have succeeded in obtaining a pH-dependent anti-IL-8 antibody comprising an Fc region having a reduced binding affinity for an effector receptor relative to the binding affinity of the native Fc region for the effector receptor. In various aspects, the inventors have identified nucleic acids encoding the above anti-IL-8 antibodies. In another aspect, the present inventors also obtained a host comprising the above nucleic acid. In another aspect, the present inventors have developed a method for producing the above anti-IL-8 antibody, which comprises culturing the above host. In another aspect, the present inventors have developed a method of promoting removal of IL-8 from an individual relative to a reference antibody, comprising administering to the individual an anti-IL-8 antibody as described above.

In one embodiment, disclosure a, relates to, but is not limited to,

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

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

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

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

[5] [4] the antibody, wherein the ratio of KD in an acidic pH range to a neutral pH range, KD (acidic pH range)/KD (neutral pH range), for an antigen, is 2 or higher;

[6] the antibody of any one of [1] to [5], wherein in the antigen binding domain, at least one amino acid residue is substituted with histidine, or at least one histidine is inserted;

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

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

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

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

(a) replacing a negatively charged amino acid residue with an uncharged amino acid residue;

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

(c) replacement of an uncharged amino acid residue with a positively charged amino acid residue;

[11] the antibody of 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;

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

[13] the antibody of [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 in a position selected from the group consisting of a CDR or a FR:

Numbering according to Kabat

(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 CDRs of the heavy chain variable region;

(c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in the FR of the light chain variable region; and

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

[14] [13] the antibody according to, wherein at least one amino acid residue is modified in a position selected from the group consisting of CDR or FR:

(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;

(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

[15] the antibody of any one of [11] to [14], wherein at least one amino acid residue is modified in a position in the constant region selected from the group consisting of: positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443;

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

[17] the antibody of [16], wherein at least one amino acid residue is modified in the constant region 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;

[18] The antibody of any one of [1] to [17], wherein the constant region has fcgamma receptor (fcyr) -binding activity, and wherein fcyr-binding activity is enhanced under neutral pH conditions as compared to a reference antibody comprising a constant region of native IgG;

[19] [18] the antibody of [ Fc γ R, wherein the Fc γ R is Fc γ RIIb;

[20] the antibody of any one of [1] to [17], wherein the constant region has binding activity against one or more activating Fc γ rs selected from the group consisting of Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb and Fc γ RIIa, and binding activity against Fc γ RIIb, and the Fc γ RIIb-binding activity is maintained or enhanced and the binding activity against activating Fc γ rs is reduced as compared to a reference antibody differing only in that its constant region is a constant region of native IgG;

[21] the antibody of any one of [1] to [20], wherein the constant region has FcRn-binding activity, and wherein the FcRn-binding activity is enhanced under neutral pH conditions (e.g., pH 7.4) as compared to a reference antibody differing only in that its constant region is that of a native IgG;

[22] the antibody of any one of [1] to [21], which is a multispecific antibody that binds at least two antigens;

[23] The antibody of any one of [1] to [22], wherein the antibody is an IgG antibody;

[24] a pharmaceutical composition comprising the antibody of any one of [1] to [23 ];

[25] [24] the pharmaceutical composition for promoting removal of an antigen from plasma;

[26] the pharmaceutical composition of [24] or [25], for enhancing binding of an antibody to an extracellular matrix;

[27] a nucleic acid encoding the antibody of any one of [1] to [23 ];

[28] a vector comprising the nucleic acid of [27 ];

[29] a host cell comprising the vector of [28 ];

[30] a method for producing an antibody comprising an antigen binding domain whose antigen binding activity changes depending on ion concentration conditions, wherein the method comprises culturing the host cell of [29] and collecting the antibody from the cell culture;

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

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

(I) A position in the CDR or FR selected from the group consisting of: according to Kabat numbering, (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 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 CDRs of the light chain variable region; or

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

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

(b) replacing a negatively charged amino acid residue with a positively charged amino acid residue;

(c) replacement of an uncharged amino acid residue with a positively charged amino acid residue; and

(d) substitution or insertion with histidine in the CDR or FR.

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

in contrast to the reference antibody,

(a) selecting antibodies capable of facilitating removal of antigen from plasma;

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

(c) selecting an antibody having enhanced Fc γ R-binding activity under neutral pH conditions (e.g., pH 7.4);

(d) selecting an antibody having enhanced Fc γ RIIb-binding activity under neutral pH conditions (e.g., pH 7.4);

(e) selecting an antibody having maintained or enhanced Fc γ RIIb-binding activity and reduced binding activity to one or more activating Fc γ rs, preferably selected from the group consisting of Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb and Fc γ RIIa;

(f) Selecting an antibody having enhanced FcRn-binding activity under neutral pH conditions (e.g., pH 7.4);

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

(h) confirming the isoelectric point (pI) of the collected antibodies, and then selecting antibodies having an increased isoelectric point (pI); and

(i) antibodies are selected whose antigen binding activity changes or increases depending on the ion concentration conditions.

In an alternative embodiment, disclosure a relates to, but is not limited to:

[A1] an antibody having a constant region in which at least one amino acid residue in the constant region selected from the group of modification sites identical to the modification sites in the group defined in [15] or [16] is modified;

[A2] the antibody of [ A1], further comprising a heavy chain variable region and/or a light chain variable region, wherein the variable region has one or more CDRs and/or one or more FRs, and wherein at least one amino acid residue in the CDR and/or FR selected from the group of modification sites identical to the modification sites in the group defined in [13] or [14] is modified;

[A3] an antibody having a constant region in which at least one amino acid residue in the constant region selected from the group of modification sites identical to those in the group defined in [15] or [16] is modified to increase its pI;

[A4] The antibody of [ A3], further comprising a heavy chain variable region and/or a light chain variable region, wherein the variable region has one or more CDRs and/or one or more FRs, and wherein at least one amino acid residue in the CDR and/or FR selected from the group of modification sites identical to the modification sites in the group defined in [13] or [14] is modified;

[A5] an antibody comprising an antigen binding domain whose antigen binding activity changes depending on ion concentration conditions, wherein the antibody has a constant region, and wherein at least one amino acid residue in the constant region selected from the group of modification sites identical to the modification sites in the group defined in [15] or [16] is modified;

[A6] the antibody of [ A5], further comprising a heavy chain variable region and/or a light chain variable region, wherein the variable region has one or more CDRs and/or one or more FRs, and wherein at least one amino acid residue in the CDR and/or FR selected from the group of modification sites identical to the modification sites in the group defined in [13] or [14] is modified;

[A7] use of the antibody of any one of [1] to [23] and [ A1] to [ A6] in the manufacture of a medicament for promoting removal of antigen from plasma;

[A8] use of the antibody of any one of [1] to [23] and [ A1] to [ A6] in the manufacture of a medicament for increasing extracellular matrix binding;

[A9] Use of the antibody of any one of [1] to [23] and [ A1] to [ A6] for removing an antigen from plasma; and

[A10] use of the antibody of any one of [1] to [23] and [ A1] to [ A6] for increasing extracellular matrix binding.

[A11] [30], [30A ], [30B ], [31], [32] ].

According to various embodiments, disclosure a includes a combination (in part or in whole) of one or more elements described in any one of [1] to [30], [30A ], [30B ], [31], [32] and [ a1] to [ a11] above, so long as the combination is not technically inconsistent with ordinary skill in the art. For example, in some embodiments, disclosure a includes a method for producing a modified antibody comprising an antigen binding domain that facilitates removal of an antigen from plasma as compared to prior to modification of the antibody, wherein the method comprises:

(a) the modification may expose at least one amino acid residue on the surface of the antibody at a position:

(b) modifying the antigen binding domain in such a way that the resulting antigen binding activity varies depending on the ionic concentration conditions, wherein said (a) and (b) can be performed simultaneously or sequentially;

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

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

In another embodiment, the method optionally further comprises one or more of:

compared with the antibody before the modification, the modified antibody,

(e) selecting antibodies capable of facilitating removal of antigen from plasma;

(f) selecting an antibody having enhanced binding activity to the extracellular matrix;

(g) selecting an antibody having enhanced Fc γ R-binding activity under neutral pH conditions (e.g., pH 7.4);

(h) selecting an antibody having enhanced Fc γ RIIb-binding activity under neutral pH conditions (e.g., pH 7.4);

(i) Selecting an antibody having maintained or enhanced Fc γ RIIb-binding activity and reduced binding activity to one or more activating Fc γ rs, preferably an Fc γ R selected from the group consisting of Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb and Fc γ RIIa;

(j) selecting an antibody having enhanced FcRn-binding activity under neutral pH conditions (e.g., pH 7.4);

(k) selecting an antibody having an increased isoelectric point (pI);

(l) Confirming the isoelectric point (pI) of the collected antibodies, and then selecting antibodies having an increased isoelectric point (pI); and

(m) selecting an antibody whose antigen-binding activity is changed or increased depending on the ion concentration condition.

Another embodiment of disclosure a relates to, for example, but not limited to:

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

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

(b) Culturing the host cell to express the nucleic acid; and

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

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

In one embodiment, disclosure B relates to, for example, but not limited to:

[33] a variant Fc region 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;

[34] [33] the Fc region variant of, 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;

[35] the Fc region variant of [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;

[36] [35] the Fc region variant of, wherein the FcRn-binding domain comprises a Leu at position 428 according to EU numbering; and/or Val or Thr at position 436;

[37] the Fc region variant of any one of [33] to [36], wherein the FcRn-binding domain comprises a combination of amino acid substitutions selected from the group consisting of: 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;

[38] [37] the Fc region variant of, 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/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;

[39] the Fc region variant of any one of [33] to [38], wherein the FcRn-binding activity thereof is enhanced under acidic pH conditions (e.g., pH 5.8) as compared to the Fc region of a native IgG;

[40] the Fc region variant of any one of [33] to [39], wherein the binding activity to an anti-drug antibody (ADA) is not significantly enhanced under neutral pH conditions as compared to the Fc region of a native IgG;

[41] [40] the Fc region variant of, wherein the anti-drug antibody (ADA) is Rheumatoid Factor (RF);

[42] the Fc region variant of any one of [33] to [41], wherein the plasma Clearance (CL), plasma retention time, or plasma half-life (t1/2) is decreased, compared to the Fc region of a native IgG;

[43] the Fc region variant of any one of [33] to [42], wherein plasma retention thereof is increased as compared to a reference Fc region variant comprising a combination of amino acid substitutions N434Y/Y436V/Q438R/S440E according to EU numbering;

[44] An antibody comprising the Fc region variant of any one of [33] to [43 ];

[45] [44] the antibody of, wherein the antibody is an IgG antibody;

[46] a pharmaceutical composition comprising the antibody of [44] or [45 ];

[47] [46] the pharmaceutical composition for increasing the retention of antibodies in plasma;

[48] a nucleic acid encoding the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45 ];

[49] a vector comprising the nucleic acid of [48 ];

[50] a host cell comprising the vector of [49 ];

[51] a method for producing a variant Fc region comprising an FcRn-binding domain or an antibody comprising said variant, comprising culturing the host cell of [50] and subsequently collecting the variant Fc region or the antibody comprising said variant from the cell culture;

[52] [51] the method of, which further optionally comprises any one or more selected from the group consisting of:

(a) selecting a variant Fc region having enhanced FcRn-binding activity under acidic pH conditions as compared to the Fc region of native IgG;

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

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

(d) Selecting an antibody comprising a variant Fc region capable of promoting removal of antigen from plasma as compared to a reference antibody comprising an Fc region of a native IgG; and

[53] a method for producing a variant Fc region comprising an FcRn-binding domain or an antibody comprising said variant, wherein said method comprises replacing amino acids in such a way that: the resulting variant Fc region or antibody comprising said 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.

In one embodiment, disclosure B relates to, for example, but not limited to:

[B1] use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] for the manufacture of a medicament for increasing retention in plasma;

[B2] use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] for the manufacture of a medicament for not significantly increasing the binding activity to an anti-drug antibody (ADA) under neutral pH conditions as compared to the Fc region of a native IgG;

[B3] use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] for increasing retention in plasma;

[B4] Use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] for not significantly increasing the binding activity to an anti-drug antibody (ADA) under neutral pH conditions as compared to the Fc region of a native IgG; and

[B5] a variant of an Fc region or an antibody comprising said variant, obtained by the method of any one of [51], [52], and [53 ].

According to various embodiments, disclosure B includes a combination (in part or in whole) of one or more elements described in any of [33] to [53] and [ B1] to [ B5] above, so long as the combination is not inconsistent with ordinary skill in the art. For example, in some embodiments, disclosure B includes Fc region variants comprising an FcRn-binding domain, wherein the FcRn-binding domain may include:

(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;

(b) ala at position 434, according to EU numbering; arg or Lys at position 438; and Glu or Asp at position 440;

(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;

(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;

(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

(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.

In one embodiment, disclosure C relates to, for example, but not limited to:

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

(a) HVR-H1 comprising SEQ ID NO: 67;

(b) HVR-H2 comprising SEQ ID NO: 68;

(c) HVR-H3 comprising SEQ ID NO: 69;

(d) HVR-L1, comprising SEQ ID NO: 70;

(e) HVR-L2, comprising SEQ ID NO: 71; and

(f) HVR-L3, comprising SEQ ID NO: 72;

[55] [54] the anti-IL-8 antibody of SEQ ID NO: 68, tyrosine at position 9 of the amino acid sequence of SEQ ID NO: 68, and the arginine at position 11 of the amino acid sequence of SEQ ID NO: 69 at position 3 of the amino acid sequence of seq id no;

[56] [54] or [55] the anti-IL-8 antibody, which comprises the amino acid sequence of SEQ ID NO: 68 and alanine at position 6 of the amino acid sequence of SEQ ID NO: 68 at position 8 of the amino acid sequence;

[57] the anti-IL-8 antibody of any one of [54] to [56], which comprises the amino acid sequence of SEQ ID NO: 71, asparagine at position 1 of the amino acid sequence of SEQ ID NO: 71, and the leucine at position 5 of the amino acid sequence of SEQ ID NO: 72, an amino acid substitution of glutamine at position 1 of the amino acid sequence of;

[58] the anti-IL-8 antibody as described in any one of [54] to [57], which comprises (a) a heavy chain variable region comprising SEQ ID NO: 67, (b) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73, and (c) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, HVR-H3;

[59] the anti-IL-8 antibody as described in any one of [54] to [58], which comprises (a) a heavy chain variable region comprising SEQ ID NO: 70, (b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 75, and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 76, HVR-L3 of the amino acid sequence of seq id no;

[60] The anti-IL-8 antibody as described in any one of [54] to [59], which comprises the amino acid sequence of SEQ ID NO: 78 and SEQ ID NO: 79, a light chain variable region;

[61] the anti-IL-8 antibody as described in any one of [54] to [60], which comprises an Fc region of at least one property selected from the following properties (a) to (f):

(a) binding affinity of the Fc region to FcRn that is increased relative to the binding affinity of the native Fc region to FcRn at acidic pH;

(b) a binding affinity of the Fc region for the pre-existing ADA that is reduced relative to the binding affinity of the native Fc region for the pre-existing ADA;

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

(d) reduced plasma clearance of the Fc region relative to plasma clearance of the native Fc region; and

(e) a reduced binding affinity of the Fc region for the effector receptor relative to the binding affinity of the native Fc region for the effector receptor; and

(f) increased binding to the extracellular matrix.

[62] The anti-IL-8 antibody of [61], wherein the Fc region comprises one or more amino acid substitutions at one or more positions selected from the group consisting of: positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438, and 440 according to EU numbering;

[63] [62] the anti-IL-8 antibody, which comprises an Fc region comprising one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, a327G, a330S, P331S, M428L, N434A, Y436T, Q438R and S440E;

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

[65] the anti-IL-8 antibody of [63], wherein the Fc region comprises amino acid substitutions of L235R, G236R, a327G, a330S, P331S, M428L, N434A, Y436T, Q438R, and S440E;

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

[67] an anti-IL-8 antibody comprising a heavy chain comprising SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 82;

[68] an isolated nucleic acid encoding the anti-IL-8 antibody described in any one of [54] to [67 ];

[69] a vector comprising the nucleic acid of [68 ];

[70] a host cell comprising the vector of [69 ];

[71] a method for producing an anti-IL-8 antibody, which comprises culturing the host of [70 ];

[72] a method for producing the anti-IL-8 antibody of [71], comprising isolating the antibody from a culture supernatant;

[73] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of [54] to [67], and a pharmaceutically acceptable carrier;

[74] the anti-IL-8 antibody as described in any one of [54] to [67], for use in a pharmaceutical composition;

[75] an anti-IL-8 antibody as described in any one of [54] to [67], for use in treating a disorder in which an excess of IL-8 is present;

[76] use of an anti-IL-8 antibody as described in any one of [54] to [67] for the preparation of a pharmaceutical composition for a disorder in which an excess of IL-8 is present;

[77] a method for treating a patient suffering from a disorder in which there is excess IL-8, comprising administering to the individual an anti-IL-8 antibody as described in any one of [54] to [67 ];

[78] a method for facilitating removal of IL-8 from a subject, comprising administering to the subject an anti-IL-8 antibody as described in any one of [54] to [67 ];

[79] a pharmaceutical composition comprising an anti-IL-8 antibody as described in any one of [54] to [67], wherein the antibody binds IL-8 and binds extracellular matrix; and

[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:

(a) evaluating the binding of anti-IL-8 antibodies to the extracellular matrix,

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

(c) Culturing a host comprising a vector comprising a nucleic acid encoding said antibody, and

(d) isolating the antibody from the culture solution.

In an alternative embodiment, disclosure C relates to:

[C1] use of an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31] for the preparation of a pharmaceutical composition for inhibiting the accumulation of IL-8 having biological activity;

[C2] use of an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31] for inhibiting the accumulation of IL-8 having biological activity;

[C3] use of an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31] for the preparation of a pharmaceutical composition for inhibiting angiogenesis;

[C4] use of an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31] for inhibiting angiogenesis;

[C5] use of the anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31] for the preparation of a pharmaceutical composition for inhibiting the promotion of neutrophil migration;

[C6] use of the anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31] for inhibiting promotion of neutrophil migration;

[C7] the anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31], for use in inhibiting the accumulation of IL-8 having biological activity;

[C8] A method for inhibiting the accumulation of IL-8 having biological activity, wherein the method comprises administering to a subject an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ];

[C9] a pharmaceutical composition for inhibiting the accumulation of IL-8 having biological activity, comprising an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ];

[C10] the anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31], for use in inhibiting angiogenesis;

[ C11 case J A method of producing angiogenesis in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ];

[C12] a pharmaceutical composition for inhibiting angiogenesis, comprising the anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ];

[C13] the anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31], for use in inhibiting promotion of neutrophil migration;

[C14] a method for inhibiting promotion of neutrophil migration in an individual, wherein the method comprises administering to the individual an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ];

[C15] a pharmaceutical composition for inhibiting promotion of neutrophil migration, comprising an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ];

[C16] An anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31], for use in the treatment of a disorder in which an excess of IL-8 is present;

[C17] use of an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31] in the preparation of a pharmaceutical composition for the treatment of a disorder in which an excess of IL-8 is present;

[C18] use of an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31] for the treatment of a disorder in which an excess of IL-8 is present;

[C19] a method of treating a disorder in which there is excess IL-8 in a subject, wherein the method comprises administering to the subject an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31 ];

[C20] a pharmaceutical composition for treating a disorder in which an excess of IL-8 is present, comprising an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ];

[C21] the anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31], for use in promoting removal of IL-8;

[C22] use of the anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31] for the preparation of a pharmaceutical composition for promoting removal of IL-8;

[C23] use of an anti-IL-8 antibody as described in any one of [54] to [67] and [ C26] to [ C31] for promoting removal of IL-8;

[C24] A method for promoting removal of IL-8 in a subject, wherein the method comprises administering to the subject an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ]; and

[C25] a pharmaceutical composition for promoting removal of IL-8, comprising the anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31 ].

[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 locations 235, 236, 239, 327, 330, 331, 428, 434, 436, 438, and 440.

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

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

(b) a reduced binding affinity of the Fc region for the pre-existing ADA relative to the binding affinity of the native Fc region for the pre-existing ADA;

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

(d) reduced plasma clearance of the Fc region relative to plasma clearance of the native Fc region;

(e) a reduced binding affinity of the Fc region for an effector receptor relative to the binding affinity of the native Fc region for the effector receptor; and

(f) Increased binding to the extracellular matrix.

[C28] The anti-IL-8 antibody of [ C26] or [ C27], comprising an Fc region comprising one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, a327G, a330S, P331S, M428L, N434A, Y436T, Q438R and S440E according to EU numbering.

[C29] [ C28] the anti-IL-8 antibody, which comprises 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.

[C30] [ C26] the anti-IL-8 antibody, which comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence of SEQ ID NO: 82.

[C31] [ C26] the anti-IL-8 antibody, which comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 82.

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

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

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

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

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

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

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

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

[C40] A method for inhibiting IL-8, wherein the method comprises contacting an anti-IL-8 antibody described in any one of [54] to [67] and [ C26] to [ C31] with IL-8.

[C41] [ C40] wherein the method inhibits the biological activity of IL-8.

According to various embodiments, disclosure C includes a combination (in part or in whole) of one or more elements described in any of [54] to [80] and [ C1] to [ C41] above, so long as the combination is not technically inconsistent with ordinary skill in the art.

Brief Description of Drawings

[ FIG. 1]

Figure 1 shows the change in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with an antibody that binds human IL-6 receptor in a pH-dependent manner and whose constant region is that of native IgG1 (low pI-IgG1), or an antibody that has increased the pI of the variable region in the antibody (high pI-IgG 1).

[ FIG. 2]

Figure 2 shows the change in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with antibody binding to human IL-6 receptor (low pI-F939) which binds human IL-6 receptor in a pH-dependent manner and is conferred to bind FcRn under neutral pH conditions, and antibody which has increased the pI of the variable region in the antibody (medium pI-F939, high pI-F939), respectively.

[ FIG. 3]

Figure 3 shows the change in plasma concentration of human IL-6 receptor in human FcRn transgenic mice administered with antibody that binds human IL-6 receptor in a pH-dependent manner and whose Fc γ R binding under neutral pH conditions is increased (low _ pI-F1180), and antibody that has increased the pI of the variable region in the antibody (medium _ pI-F1180, high _ pI-F1180), respectively.

[ FIG. 4]

Fig. 4 shows changes in plasma concentration of human IL-6 receptor in human IL-6 transgenic mice to which soluble human IL-6 receptor concentration in plasma was maintained in a steady state, an antibody that binds human IL-6 receptor in a pH-dependent manner and whose constant region is that of native IgG1 (low _ pI-IgG1), an antibody comprising a variant of the Fc region in which the Fc region in the antibody has increased FcRn binding under neutral pH conditions (low _ pI-F11), and an antibody that has increased the pI of the variable region in these antibodies (high _ pI-IgG1, high _ pI-F11) were administered, respectively.

[ FIG. 5]

Figure 5 shows the extent of extracellular matrix binding of each of three types of antibodies with different pI that bind 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 pI that do not bind human IL-6 receptor in a pH-dependent manner (low pI (nph) -IgG1 and high pI (nph) -IgG 1). "NPH" means pH independent within the scope of disclosure A as described herein.

[ FIG. 6]

Fig. 6 shows the relative values of the degree of soluble human Fc γ RIIb binding (measured by BIACORE (registered trademark)) of antibodies comprising Fc region variants whose respective pis were increased by modification of one amino acid residue in the constant region of Ab1H-P600 antibody that binds IgE in a pH-dependent manner, setting the value of Ab1H-P600 to 1.00.

[ FIG. 7]

Figure 7 shows the relative values of the rate of antibody uptake into cells of the hfcyriib-expressing cell line of Fc region variants comprising Fc region variants whose respective pI was increased by modification of one amino acid residue in the constant region of Ab1H-P600, each assessed at the value of Ab1H-P600 set at 1.00.

[ FIG. 8]

FIG. 8 shows the extent of binding of Fv4-IgG1 (which has the Fc region of native human IgG1) to rheumatoid factor in the serum of each RA patient.

[ FIG. 9]

Figure 9 shows the extent of binding of Fv4-YTE (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 10]

Figure 10 shows the extent of binding of Fv4-LS (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 11]

Figure 11 shows the extent of binding of Fv4-N434H (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 12]

Figure 12 shows the extent of binding of Fv4-F1847m (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 13]

Figure 13 shows the extent of binding of Fv4-F1848m (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 14]

Figure 14 shows the extent of binding of Fv4-F1886m (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 15]

Figure 15 shows the extent of binding of Fv4-F1889m (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 16]

Figure 16 shows the extent of binding of Fv4-F1927m (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 17]

Figure 17 shows the extent of binding of Fv4-F1168m (which comprises a variant Fc region with increased FcRn binding) to rheumatoid factor in the serum of each RA patient.

[ FIG. 18]

Figure 18 shows the average binding of Fv4-IgG1 (which has the Fc region of native human IgG1, and each antibody comprises a novel Fc region variant in which the Fc region has an Fc region variant with increased binding to each FcRn) to rheumatoid factor in the serum of RA patients.

[ FIG. 19]

Figure 19 shows 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 an Fc region of native human IgG1, and each antibody comprises a novel Fc region variant (wherein each Fc region comprises an Fc region variant with increased binding to FcRn)) (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927 m).

[ FIG. 20]

Figure 20 shows the change in plasma concentration of anti-human IL-6 receptor antibody in human FcRn transgenic mice when administered with either 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.

[ FIG. 21]

FIG. 21 shows sensorgrams obtained for IL-8 binding of H998/L63 and Hr9 measured with Biacore at pH 7.4 and pH 5.8.

[ FIG. 22]

FIG. 22 shows the change in the concentration of human IL-8 in the plasma of mice when either H998/L63 or H89/L118 was administered to the mice at 2mg/kg (in admixture with human IL-8).

[ FIG. 23]

FIG. 23 shows the change in the concentration of human IL-8 in the plasma of mice when H89/L118 was administered to the mice at 2mg/kg or 8mg/kg (in admixture with human IL-8).

[ FIG. 24]

FIG. 24 shows the change in the concentration of human IL-8 in the plasma of mice when either H89/L118 or H553/L118 is administered to the mice at 2mg/kg or 8mg/kg (in admixture with human IL-8).

[ FIG. 25A ]

FIG. 25A shows the change in relative values of antibody concentration-dependent chemiluminescence for antibodies Hr9, H89/L118 or H553/L118 prior to storage in plasma.

[ FIG. 25B ]

FIG. 25B shows the change in relative values of antibody concentration-dependent chemiluminescence for antibodies Hr9, H89/L118 or H553/L118 after one week of storage in plasma.

[ FIG. 25C ]

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

[ FIG. 26]

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

[ FIG. 27]

FIG. 27 shows the predicted frequency of occurrence of ADA for each anti-IL-8 antibody (H496/L118, H496v1/L118, H496v2/L118, H496v3/L118, H1004/L118, or H1004/L395) and the predicted frequency of occurrence of ADA for other pre-existing therapeutic antibodies, as predicted by EpiMatrix.

[ FIG. 28A ]

FIG. 28A shows the change in the relative values of antibody concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H1009/L395-F1886s prior to storage in plasma.

[ FIG. 28B ]

FIG. 28B shows the change in relative values of antibody concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H1009/L395-F1886s after one week of storage in plasma.

[ FIG. 28C ]

FIG. 28C shows the change in the relative values of antibody concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H1009/L395-F1886s after two weeks of storage in plasma.

[ FIG. 29]

FIG. 29 shows the change in human IL-8 concentration in the plasma of mice when each of H1009/L395, H553/L118 and H998/L63 (in admixture with human IL-8) was administered to the mice.

[ FIG. 30]

FIG. 30 shows the degree of extracellular matrix binding when Hr9, H89/L118 or H1009/L395 were added to the extracellular matrix alone, and when they were added in admixture with human IL-8.

[ FIG. 31]

Figure 31 shows the change in mouse plasma antibody concentration when an antibody having the variable region of H1009/L395 and an Fc region that does not bind FcRn (F1942m) was administered to human FcRn transgenic mice alone or in admixture with human IL-8.

[ FIG. 32]

FIG. 32 shows the predicted frequency of occurrence for H1009/L395 and H1004/L395 ADA and the predicted frequency of occurrence for other pre-existing therapeutic antibodies ADA, as predicted by EpiMatrix.

[ FIG. 33]

Figure 33 shows the change in concentration of individual anti-human IL-8 antibodies 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 comprising an antibody that has a variant Fc region with increased binding to FcRn (H89/L118-F1168m, H89/L118-F1847m, H89/L118-F1848m, H89/L118-F1886m, H89/L118-F1889m, and H89/L118-F1927 m).

[ FIG. 34]

Figure 34 shows binding of antibodies with the variable region of H1009/L395 and whose Fc region is a variant for each Fc γ R (F1886m, F1886s, or F1974 m).

[ FIG. 35]

FIG. 35 shows the change in human IL-8 concentration in mouse plasma when anti-IL-8 antibody is administered to human FcRn transgenic mice in admixture with human IL-8. In this case, the anti-IL-8 antibody is H1009/L395-IgG1(2mg/kg) comprising the variable region of H1009/L395 and the Fc region of native human IgG1, or H1009/L395-F1886s (2, 5 or 10mg/kg) comprising the variable region of H1009/L395 and the modified Fc region.

[ FIG. 36]

FIG. 36 shows the change in antibody concentration in cynomolgus monkey plasma when administered with Hr9-IgG1 or H89/L118-IgG1 (both comprising the Fc region of native human IgG 1), or H1009/L395-F1886s or H1009/L395-F1974m (both comprising the modified Fc region).

[ FIG. 37]

Figure 37 shows IgE plasma concentration time profiles for some anti-IgE antibodies in C57BL6J mice for antibody variable region modification.

[ FIG. 38A ]

Fig. 38 (fig. 38A-38D) shows Octet sensorgrams for selected 25 [ twenty-five ] pH-dependent and/or calcium-dependent antigen-binding clones.

[ FIG. 38B ]

Fig. 38B is a continuation of fig. 38A.

[ FIG. 38C ]

Fig. 38C is a continuation of fig. 38B.

[ FIG. 38D ]

Fig. 38D is a continuation of fig. 38C.

[ FIG. 39]

Figure 39 shows the C5 plasma concentration time profiles of some anti-C5 bispecific antibodies in C57BL6J mice with respect to antibody variable region modification.

[ FIG. 40]

Figure 40 shows IgE plasma concentration time profiles for some anti-IgE antibodies in C57BL6J mice for antibody variable region modification.

Detailed description of the preferred embodiments

Detailed Description

Non-limiting embodiments of the disclosure a, B or C are described below. Description of all embodiments described in the following examples, which are intended to be properly understood in the "detailed description" section, are not limited by any patent practice, ordinance, guidance, etc. that may be sought to narrowly interpret the contents described in the examples in a country where the patent application is desired to be granted.

Disclosure A or disclosure B

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 can be exposed on the surface of the antibody (herein, also referred to as "ion concentration-dependent antibody with increased pI" and antigen-binding domain of the antibody, also referred to as "ion concentration-dependent antigen-binding domain with increased pI" within the scope of disclosure A "). The present invention is based in part on the surprising discovery by the present inventors: removal of antigen from plasma may be facilitated by an ion concentration-dependent antibody whose isoelectric point (pI) is increased by modification of 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 be increased with an ion concentration-dependent antibody with an increased (elevated) pI. The present invention is also based in part on the surprising findings of the inventors: this beneficial effect is brought about by combining two completely different concepts: an ionic concentration-dependent antigen binding domain or an ionic concentration-dependent antibody; and antibodies whose pI is increased by modification of at least one amino acid residue that may be exposed on the surface (herein, also referred to as "antibodies with increased pI" within the scope of disclosure A; and antibodies whose pI is decreased (decreased) by modification of at least one amino acid residue that may be exposed on the surface (also referred to as "antibodies with decreased pI") within the scope of disclosure A). The invention is thus classified as a type of pioneering research that may lead to significant technological innovations in the field (e.g., medical field) to which disclosure a belongs.

In general, for example, an antibody that comprises 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, which is further modified such that the antigen binding activity of the antigen binding domain changes depending on the ion concentration condition, is also included in the scope of the disclosure a described herein (the antibody is also referred to as "ion concentration-dependent antibody with increased pI" in the scope of the disclosure a herein).

Typically, for example, an antibody containing an ion concentration-dependent antigen binding domain, at least one amino acid residue of which may be exposed on the surface of the antibody has a different charge than at least one amino acid residue at a corresponding position in a pre-modified antibody (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody), or a reference or parent antibody (e.g., a pre-modified antibody, or an antibody before or during library construction, etc.), and the net antibody pI thereof is increased) is also included in the disclosure a described herein (said antibody is also referred to as "ion concentration-dependent antibody with increased pI" within the scope of the disclosure a described herein).

Typically, for example, antibodies containing an ion concentration-dependent antigen binding domain whose pI is increased by modification of at least one amino acid residue that may be exposed on the antibody surface in a pre-modified antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody, or a reference or parent antibody (e.g., a pre-modified antibody, or an 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).

Typically, 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 (the antibodies are also referred to as "ion concentration-dependent antibodies with increased pI" within the scope of the disclosure a described herein).

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

When used within the context of disclosures a and B, "modification of amino acids," "modification of amino acid residues," or equivalent terms, are to be understood as not being 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 an antibody amino acid sequence with a molecule 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 an antibody amino acid sequence. Nucleic acids encoding amino acid sequences can be subjected, for example, to site-directed mutagenesis (Kunkel et al, Proc. Natl. Acad. Sci. USA 82: 488-Asan 492(1985)) or overlap extension PCR; via affinity maturation of the antibody, or by using chain shuffling (shuffling) of the antibody heavy or light chains; or by antigen panning-based selection using phage display libraries (Smith et al, Methods enzymol.217: 228-257(1993)) for amino acid additions, deletions, substitutions, or insertions; and these may be performed alone or in an appropriate combination. The amino acid modification is preferably an amino acid addition, deletion, substitution, or insertion by replacing one or more amino acid residues in the amino acid sequence of the antibody with different amino acids (respectively), and the amino acid modification by humanization or chimerization may be performed by a method known in the art. Amino acid (residue) changes or modifications, such as amino acid additions, deletions, substitutions, or insertions, can also be made on the antibody variable region or antibody constant region of the recombinant antibody to be used for producing the antibody of disclosure a or B.

In one embodiment within the scope of the disclosure a and B described herein, substitution of an amino acid (residue) refers to substitution with a different amino acid (residue), and may be designed to modify, for example, the matters as in each of (a) to (c): (a) a polypeptide backbone structure in a folded or helical conformational region; (b) charge or hydrophobicity at the target site; or (c) the size of the side chain.

Amino acid residues are classified, for example, into the following groups 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 that influence chain orientation: gly and Pro; and (6) aromatic: trp, Tyr, and Phe.

Substitutions of amino acid residues within each group are referred to as conservative substitutions, while substitutions of amino acid residues between different groups are referred to as non-conservative substitutions. The substitution of the amino acid residue may be a conservative substitution, a non-conservative substitution, or a combination thereof. A variety of known suitable methods can be used to replace amino acids with those other than the natural amino acids (Wang et al, Annu. Rev. Biophys. Biomol. Structure.35: 225-249 (2006); Forster et al, Proc. Natl. Acad. Sci. USA 100 (11): 6353-6357 (2003)). It is possible to use, for example, a cell-free translation system containing a tRNA to which an unnatural amino acid is linked to an amber suppressor tRNA that is complementary to a UAG codon (amber codon), which is a stop codon (Protein Express).

Within the context of the disclosures a and B described herein, it is to be understood that the structure of an "antigen" is not limited to a particular structure, so long as the antigen includes an epitope that binds an antibody. The antigen may be an inorganic substance or an organic substance. The antigen may be any ligand, including various cytokines, e.g., interleukins, chemokines, and cell growth factors. Alternatively, in general, receptors that exist in soluble form or that are modified to be in soluble form in biological fluids such as plasma, for example, may also be used as antigens. Non-limiting examples of such soluble receptors include Mullberg et al, j.immunol.152 (10): 4958 and 4968 (1994). Furthermore, the antigen may be monovalent (e.g., soluble IL-6 receptor) or multivalent (e.g., IgE).

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

Within the context of the disclosures A and B described herein, "extending the half-life of a target molecule in plasma" or "shortening the half-life of a target molecule in plasma" (the target molecule may be an antigen or an antibody), or equivalent terms thereof, may also be more particularly expressed using any other parameter than the half-life in plasma (t1/2), such as the mean retention time in plasma, the clearance rate in plasma (CL), and the area under the concentration curve (AUC) (pharmacokinetics: Enshuniyoru Rikai (understood by practice) Nanzando). These parameters can be specifically evaluated, for example, by non-compartmental analysis (noncompartmental analysis) according to the protocol attached to in vivo kinetic analysis software winnonlin (pharsight). As known to those skilled in the art, these parameters are typically related to each other.

Within the context of the disclosures a and B described herein, "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, the epitope may be defined by the antigen binding activity of an antibody recognizing the epitope. When the antigen is a peptide or polypeptide, the epitope may be embodied by the amino acid residues constituting the epitope. Alternatively, when the epitope is a sugar chain, the epitope may be specified based on its specific sugar chain structure. The antigen binding domains of disclosures a and B can bind a single epitope or different epitopes on an antigen.

The linear epitope may be a primary amino acid sequence. The linear epitopes typically contain at least three and usually at least five, e.g., 8 to 10 amino acids or 6 to 20 amino acids as unique sequences.

In conformational epitopes, typically the amino acids that make up the epitope do not exist contiguously as primary sequences. Antibodies recognize conformational epitopes in the three-dimensional structure of a peptide or protein. Methods for determining the conformation of an Epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific rotational labeling and electron paramagnetic resonance (Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol.66, Morris (eds)).

Within the scope of the disclosures a and B described herein, "antibody" is not particularly limited and is used in the broadest sense so long as it can bind to a target antigen, non-limiting examples of antibodies broadly include known common antibodies (e.g., natural immunoglobulins (abbreviated as "Ig")), as well as molecules and variants derived therefrom, e.g., Fab ', F (ab')₂Diabodies, ScFv (Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-; 6448 (1993); EP404, 097; WO 93/11161; Peer et al, Nature Nanotechnology 2: 751-760(2007)), low molecular weight antibodies (minibodies) (Orita et al, Blood 105: 562-566(2005)), scaffold proteins, single-armed antibodies (including all embodiments of the single-armed antibodies described in WO 2005/063816), multispecific antibodies (e.g., bispecific antibodies: antibodies specific for two different epitopes, including antibodies recognizing different antigens and antibodies recognizing different epitopes on the same antigen). Within the context of the disclosures a and B described herein, "bispecific antibodies" are not limited to, but can be prepared, for example, as antibody molecules having the common L chain described in WO2005/035756, or by the method described in WO2008/119353, wherein two general types of antibodies having IgG 4-like constant regions are mixed, causing an exchange reaction between the two types of said antibodies (referred to as the "Fab-arm exchange" method for those skilled in the art). In an alternative embodiment, they may be of the type having a structure in which the heavy chain variable region and the light chain variable region are linked together as a single chain (e.g., sc (fv)) ₂) The structural antibody of (1). Alternatively, they may consist of an Fc region (lacking the CH1 structure)Constant region of Domain) with scFv (or sc (fv)₂) (wherein the heavy chain variable region (VH) is linked to the light chain variable region (VL)) linkage results in an antibody-like molecule (e.g., scFv-Fc). Multispecific antibodies consisting of scFv-Fc have (scFv)₂-an Fc structure, wherein the first and second polypeptides are VH 1-linker-VL 1-Fc and VH 2-linker-VL 2-Fc, respectively. Alternatively, they may be antibody-like molecules in which single domain antibodies are linked to an Fc region (Marvin et al, curr. Opin. drug Discov. Dedevel.9 (2): 184- > 193(2006)), Fc fusion proteins (e.g., immunoadhesins) (US2013/0171138), functional fragments thereof, functionally equivalent substances thereto, and sugar chain modified variants thereof. Herein, native IgG (e.g., native IgG1) refers to a polypeptide comprising the same amino acid sequence as a naturally occurring IgG (e.g., native IgG1) and is of the antibody type substantially encoded by immunoglobulin gamma genes. The natural IgG may be a spontaneous mutant thereof, or the like.

Typically, the Y-shaped structure of the four chains (two heavy chain polypeptides and two light chain polypeptides) may be the basic structure when the antibody has a structure that is substantially the same as or similar to a native IgG. Typically, the heavy and light chains may be linked by disulfide bonds (SS bonds) and form heterodimers. The heterodimers may be linked together by disulfide bonds and form a Y-shaped heterotetramer. The two heavy or light chains may be identical to or different from each other.

For example, an IgG antibody can be cleaved by papain to two Fab units (regions) and a single Fc unit (region), which cleaves the hinge region (also referred to as the "hinge" within the scope of the disclosures a and B described herein), wherein the heavy chain Fab region is linked to the Fc region. Typically, the Fab region contains an antigen binding domain. Because phagocytic cells such as lymphocytes and macrophages have receptors capable of binding to the Fc region (Fc receptors), and can recognize antibodies binding to antigens via the Fc receptors and phagocytose the antigens (opsonization). Meanwhile, the Fc region is involved in the mediation of immune responses such as ADCC or CDC, and has an effector function of inducing a response upon binding of an antibody to an antigen. It is known that the effector functions of antibodies vary depending on the type (isotype) of immunoglobulin. An Fc region of the IgG class would indicate, for example, a region spanning cysteine at position 226 or proline at position 230 (EU numbering) to the C-terminus; however, the Fc region is not limited thereto. The Fc region may be suitably obtained by partially digesting monoclonal IgG1, IgG2, IgG3, or IgG4 antibody or the like with a protease such as pepsin, followed by eluting the adsorbed fraction from a protein a or protein G column.

Within the scope of the disclosures A and B described herein, the positions of amino acid residues in the variable region (CDR(s) and/or FR (s)) of an antibody 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).

Within the context of the disclosure a and B described herein, a "library" may refer to a molecule (population) such as a plurality of antibodies having sequence variations, wherein their respective sequences may be the same or different from each other; a plurality of fusion polypeptides comprising said antibody; or nucleic acids or oligonucleotides encoding such amino acid sequences, as detailed in WO2013/125667 (e.g., paragraphs 0121-0125). The library may, for example, contain at least 10⁴An antibody molecule, more preferably, at least 10⁵Individual antibody molecules, even more preferably, at least 10⁶Antibody molecules, particularly preferably at least 10⁷Individual antibody molecules or more. The library may be a phage library. The term "consisting essentially of" means that a certain fraction of many independent clones in a library having different sequences may have antibodies with different antigen binding activity. In one embodiment, an immune library constructed based on antibody genes from lymphocytes from animals immunized with a specific antigen, infected patients, humans with elevated antibody low levels in blood due to immunization, or patients with cancer or autoimmune diseases may be suitably used as the random variable region library. In an alternative embodiment, an unimmunized library containing nonimmune (negative) sequences (antibody sequences that are not biased in the repertoire) constructed from antibody genes derived from lymphocytes from healthy humans may also be suitably used as a randomized variable region library (Gejima et al, human antibodies 11: 121-129 (2002)); cardoso et al, Scand.J. Immuno l.51: 337-344(2000)). Amino acid sequences containing non-immune sequences may refer to those obtained from the non-immune library. In an alternative embodiment, a synthetic library in which a V gene from genomic DNA or a reconstructed functional V gene is replaced with a set of CDR sequences comprising sequence-synthesized oligonucleotides encoding codon sets of appropriate length may also be suitably used as a random variable region library. In this case, it is also possible to replace only the heavy chain CDR3 sequence, since a sequence change was observed in the CDR3 gene. The standard route for creating amino acid diversity in antibody variable regions may be to increase the alteration of amino acid residues at positions that are likely to be exposed on the antibody surface.

In one embodiment, where the antibodies of disclosure a or B, for example, have a structure that is substantially the same as or similar to that of a natural Ig antibody, they typically have variable regions ("V regions") [ heavy chain variable regions ("VH regions") and light chain variable regions ("VL regions") ] as well as constant regions ("C regions") [ "heavy chain constant regions (" CH regions ") and light chain constant regions (" CL regions "). The CH region is further divided into three: CH1 to CH 3. Typically, the Fab region of a heavy chain contains the VH region and CH1, and typically the Fc region of a heavy chain contains CH2 and CH 3. Typically, the hinge region is located between CH1 and CH 2. In addition, variable regions typically have complementarity determining regions ("CDRs") and framework regions ("FRs"). Typically, the VH and VL regions each have three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR 4). Typically, six CDRs in the variable regions of the heavy and light chains interact and form the antigen binding domain of an antibody. On the other hand, in the case where only one single CDR is present, it has the ability to recognize and bind to an antigen while it is known that the antigen binding affinity is lower than in the case where six CDRs are present.

Ig antibodies are classified into various types (isotypes) based on the structural differences in their constant regions. In many mammals, they are classified into five immunoglobulin types based on structural differences in the constant regions: IgG, IgA, IgM, IgD, and IgE. Furthermore, in the human case, IgG is of four types: IgG1, IgG2, IgG3, and IgG 4; and IgA has two subclasses: IgA1 and IgA 2. Heavy chains are classified into γ, μ, α, δ, and ε chains according to differences in 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: lambda chains and kappa chains, and all immunoglobulins have one of these two.

In one embodiment, the antibody of disclosure a or B has a heavy chain, e.g., the heavy chain can be, or can be derived from, any of a gamma chain, a chain, an alpha chain, a delta chain, and an epsilon chain, and wherein the antibody of disclosure a or B has a light chain, e.g., the light chain can be, or can be derived from, a kappa chain or a lambda chain. Further, within the scope of the disclosure a and B described herein, the antibody may be of any isotype (e.g., IgG, IgM, IgA, IgD, or IgE) and of any subclass (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2; mouse IgG1, IgG2a, IgG2B, and IgG3), or may be derived from any of them, but is not limited thereto.

Within the context of the disclosures A and B described herein, an "antigen binding domain" can have any structure so long as it binds the antigen of interest. The domain may include, for example, the variable regions (e.g., 1 to 6 CDRs) of antibody heavy and light chains; a module of about 35 amino acids, called the A domain, contained in Avimer (a cell membrane protein present in vivo) (WO2004/044011 and WO 2005/040229); an Adnectin containing 10Fn3 domain that binds to a protein in the glycoprotein fibronectin (fibronectin) expressed on the cell membrane (WO 2002/032925); affibody, which has a scaffold IgG-binding domain (triple helix bundle of 58 amino acids constituting protein a) (WO 1995/001937); designed Ankyrin (Ankyrin) repeat proteins (DARPins), which are regions exposed on the molecular surface of Ankyrin Repeats (AR), which have the structure: with a repeat stack of subunits containing 33 amino acid residue turns (turn), two antiparallel helices, and loops (WO 2002/020565); anticalins et al, which are one of the central twisted barrel structures supporting the highly conserved eight antiparallel strands in molecules such as neutrophil gelatinase-associated lipocalin (NGAL) The lateral tetracyclic region (WO 2003/029462); and a concave region formed by a parallel-sheet structure inside a horseshoe structure formed by stacked repeats of leucine-rich repeat (LRR) modules of a Variable Lymphocyte Receptor (VLR) which does not have an immunoglobulin structure and is used for a system of acquired immunity in invertebrate animals such as lamprey and congenity eel (WO 2008/016854). Preferred antigen binding domains of disclosure a or B may include those having IgG antibody heavy and light chain variable regions, and more particularly, ScFv, single chain antibody, Fv, ScFv₂(Single chain Fv)₂) Fab, and F (ab')₂。

In one embodiment of disclosure a, "ion concentration" is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. Herein, the "metal ion" may be any of ions of 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 of subgroup a belonging to groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. The metal atom has the property of releasing valence electrons into cations. This is called ionization tendency. Metals with a strong tendency to ionize are considered chemically active.

In one embodiment of disclosure a, the preferred metal ion may be a calcium ion, as detailed in WO2012/073992 and WO 2013/125667.

In one embodiment of disclosure a, the "one or more ion concentration conditions" may be conditions that focus on the difference in biological behavior of the ion concentration-dependent antibody between a low ion concentration and a high ion concentration. Further, "the antigen binding activity varies depending on the ion concentration condition" may mean that the antigen binding activity of the ion concentration-dependent antigen binding domain or the ion concentration-dependent antibody of the disclosure a or B varies between a low ion concentration and a high ion concentration. 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.

In one embodiment of disclosure a, the ionic concentration may be a hydrogen ion concentration (pH) or a calcium ion concentration, and in the case where the ionic concentration is a hydrogen ion concentration (pH), the ionic 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 "calcium ion concentration-dependent antigen-binding domain".

In one embodiment in the context of disclosure a, the ion concentration-dependent antigen binding domain, the ion concentration-dependent antibody, the ion concentration-dependent antigen binding domain with increased pI, and the ion concentration-dependent antibody with increased pI may be obtained from a library consisting essentially of antibodies that differ in sequence (have variability) and whose antigen binding domain contains at least one amino acid residue that causes the antigen binding activity of the antigen binding domain or antibody to change depending on the ion concentration conditions. The antigen binding domain may preferably be located within the light chain variable region (which may be modified) and/or the heavy chain variable region (which may be modified). Furthermore, to construct the library, the light or heavy chain variable regions may be combined with heavy or light chain variable regions constructed as a library of random variable region sequences. In the case where the ion concentration is hydrogen or calcium ion concentration, non-limiting examples of libraries include, for example, those in which the heavy chain variable regions constructed as a library of random variable region sequences are identical to those in which the germline sequence is set forth in SEQ ID NO: 1(Vk1), SEQ ID NO: 2(Vk2), SEQ ID NO: 3(Vk3), or SEQ ID NO: 4(Vk4) with at least one amino acid residue capable of altering antigen binding activity based on ionic concentration. Further, where the ion concentration is calcium ion concentration, the library includes, for example, wherein SEQ ID NO: 5(6RL #9-IgG1) or SEQ ID NO: 6(6KC4-1#85-IgG1) in combination with light chain variable regions constructed as a library of random variable region sequences or light chain variable regions having germline sequences.

In one embodiment, when the ion concentration is a calcium ion concentration, the high calcium ion concentration is not particularly limited to a specific value; however, the concentration may be chosen between 100. mu.M to 10mM, between 200. mu.M to 5mM, between 400. mu.M to 3mM, between 200. mu.M to 2mM, or between 400. mu.M to 1 mM. Concentrations chosen between 500. mu.M and 2.5mM, which are close to the calcium ion plasma (blood) concentration in vivo, may also be preferred. The low calcium ion concentration is not particularly limited to a specific value; however, the concentration may be selected between 0.1. mu.M to 30. mu.M, between 0.2. mu.M to 20. mu.M, between 0.5. mu.M to 10. mu.M, or between 1. mu.M to 5. mu.M, or between 2. mu.M to 4. mu.M. Concentrations chosen between 1 μ M and 5 μ M, which is close to the in vivo calcium ion concentration in early endosomes, may also be preferred.

Whether the antigen binding activity of an antigen binding domain or an antibody containing said domain varies depending on the metal ion concentration (e.g. calcium ion concentration) conditions can be readily determined by known methods, e.g. by the methods described herein in the context of disclosure a, or the methods described in WO 2012/073992. For example, the antigen binding activity of an antigen binding domain or an antibody containing the domain can be measured and compared at low and high calcium ion concentrations. In this case, the conditions other than the calcium ion concentration may preferably be the same. Further, conditions other than the calcium ion concentration in the determination of the antigen-binding activity may be appropriately selected by those skilled in the art. The antigen binding activity can be determined, for example, under the condition of HEPES buffer at 37 ℃ or using BIACORE (GE healthcare) or the like.

In one embodiment in the context of disclosure a, it is preferred that the antigen binding activity of the ionic concentration-dependent antigen binding domain, the ionic concentration-dependent antibody, the ionic concentration-dependent antigen binding domain with increased pI, or the ionic concentration-dependent antibody with 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 the condition of low calcium ion concentration and the antigen-binding activity under the condition of high calcium ion concentration is not limited; however, the ratio of KD (dissociation constant) for an antigen under low calcium ion concentration conditions to KD under high calcium ion concentration conditions, i.e., KD (3 μ M Ca)/KD (2mM Ca), may preferably be 2 or more, more preferably 10 or more, and still more preferably 40 or more. The upper limit of the KD (3. mu.M Ca)/KD (2mM Ca) value is not limited and may be any value such as 400, 1000, or 10000.

In the case where the antigen is a soluble antigen, the dissociation constant (KD) can be used as a value of the antigen-binding activity. Meanwhile, in the case where the antigen is a membrane antigen, an apparent dissociation constant (KD) may 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 curves, or flow cytometry.

Alternatively, for example, the dissociation rate constant (kd) may also be used as another index representing the ratio of binding activities. When the dissociation rate constant (KD) is used instead of the dissociation constant (KD) as an index representing the ratio of antigen-binding activities, the ratio of the low-calcium-ion-concentration-conditional dissociation rate constant (KD) to the high-calcium-ion-concentration-conditional dissociation rate constant (KD), that is, KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition), may be preferably 2 or more, more preferably 5 or more, still more preferably 10 or more, and still more preferably 30 or more. The upper limit of kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) value is not limited and may be any value such as 50, 100, or 200.

In the case where the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as a value of the antigen-binding activity. Meanwhile, in the case where the antigen is a membrane antigen, an apparent dissociation rate constant (kd) may 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.

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

The method may include, for example:

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

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

(c) selecting an antigen binding domain or antibody having an antigen binding activity that is lower at low calcium ion concentrations than at high calcium ion concentrations.

Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen binding domain or antibody, or library thereof, under conditions of high calcium ion concentration;

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

(c) isolating the antigen binding domain or antibody that is dissociated in step (b).

Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen binding domain or antibody, or library thereof, under conditions of low calcium ion concentration;

(b) selecting an antigen binding domain or antibody that does not bind to an antigen or has low antigen binding capacity in step (a);

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

(d) Isolating the antigen binding domain or antibody that binds to the antigen in step (c).

Alternatively, the method may comprise, for example:

(a) contacting the antigen binding domain or antibody, or library thereof, with an antigen immobilized column under conditions of high calcium ion concentration;

(b) eluting the antigen binding domain or antibody bound to the column of step (a) from the column under conditions of low calcium ion concentration; and

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

Alternatively, the method may comprise, for example:

(a) passing the antigen binding domain or antibody, or library thereof, through a column of immobilized antigen under conditions of low calcium ion concentration to collect the antigen binding domain or antibody that elutes without binding to the column;

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

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

Alternatively, the method may comprise, for example:

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

(c) Incubating the antigen binding domain or antibody obtained in step (b) at a low calcium ion concentration; and

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

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

In the case of the disclosure a, in one embodiment, the antigen binding domain or antibody as starting material may be, for example, a modified antigen binding domain or antibody with an increased pI resulting from modifying the charge of at least one amino acid residue capable of being exposed on its surface. In an alternative embodiment, where amino acids that alter the binding activity of the ionic concentration-dependent antigen binding domain are introduced into the sequence, they may be introduced together with a charge modification 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.

Alternatively, in the case of the present disclosure a, for example, it is possible to use a pre-existing antigen binding domain or antibody, a pre-existing library (phage library, etc.); preparing antibodies from hybridomas obtained by immunizing an animal or from B cells of an immunized animal, or a library thereof; or by introducing natural or unnatural amino acid mutations that can sequester calcium therein, an antibody, or a library (described below) (e.g., a library with increased content of calcium-sequestering amino acids, or a library of calcium-sequestering amino acids introduced at specific sites).

In one embodiment in the case of the disclosure a, in the case where the ion concentration is a calcium ion concentration, there is no limitation on the types of amino acids that change the binding activity of the ion concentration-dependent antigen-binding domain or the ion concentration-dependent antigen-binding domain having an increased pI, as long as they are capable of forming a calcium-binding motif. For example, calcium-binding motifs are known to those of skill 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. ol. 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)), onou et al (EMBO J. 9: 349-354 (1990)); Wzburg et al (structure. 14: 6)) 1049 (1058)); calcium-binding motifs such as those having a calcium binding motif that can be altered in a concentration range as found in GPR-23), such as the binding motif can be altered under conditions for binding to a calcium binding motif such as those described in the GPR Protein binding motif Other than those described above) e.g., SEQ ID NO: 7 (which corresponds to "Vk 5-2").

In one embodiment in the case of the disclosure a, in the case where the ion concentration is a calcium ion concentration, an amino acid having a metal chelating activity may be used as an amino acid that changes the binding activity of the ion concentration-dependent antigen binding domain or the ion concentration-dependent antigen binding domain having an increased pI. For example, any amino acids may be suitably used as the amino acids having metal chelating activity as long as they are capable of forming a calcium-binding motif. In particular, the amino acids include those having electron donating properties. Preferred amino acids include, but are not limited to, Ser (S), Thr (T), Asn (N), Gln (Q), Asp (D), and Glu (E).

The position of the amino acid having 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 heavy chain variable region and/or the light chain variable region that may form an antigen binding domain. The at least one amino acid residue that causes a calcium ion concentration-dependent change in the antigen binding activity of the antibody may be contained in, for example, a CDR (one or more of CDR1, CDR2 and CDR 3) and/or an FR (one or more of FR1, FR2, FR3, and FR 4) of a heavy chain and/or a 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 alone or in combination.

Troponin C, calmodulin, parvalbumin, myosin light chain, and the like are known to have multiple calcium-binding sites and are suspected of originating from common sources in molecular evolution, and in one embodiment, one or more of the light chain CDR1, CDR2, and CDR3 may be designed to contain its binding motif. For the above purpose, for example, a cadherin domain; EF hands contained in calmodulin; the C2 domain contained in protein kinase C; the G1a domain contained in factor IX; c-type lectins of asialoglycoprotein receptors or mannose-binding receptors; an A domain contained in LDL receptors; annexin (Annexin); a thrombospondin type 3 domain; and EGF-like domains.

In one embodiment, in the ionWhen the concentration is a hydrogen ion concentration (pH), the proton, that is, the concentration condition of the nucleus of the hydrogen atom is used synonymously with the condition of the hydrogen index (pH). Active amount of hydrogen ion in aqueous solution using aH⁺In the case shown, the pH was defined as-log 10aH⁺. The ionic strength in aqueous solution is low (e.g., less than 10)^-3) In the case of (a), aH⁺Almost equal to the hydrogen ion strength. For example, the ionic product of water at 25 ℃ and 1 atmosphere is Kw ═ aH ⁺*aOH＝10^-14(ii) a Thus, for pure water, aH⁺＝aOH＝10^-7. In this case, pH 7 is neutral, and aqueous solutions with pH less than 7 are acidic, and aqueous solutions with pH greater than 7 are basic. Thus, the hydrogen ion concentration condition may be a condition focused on a difference in biological behavior of the pH-dependent antibody at a high hydrogen ion concentration (acidic pH range) and at a low hydrogen ion concentration (neutral pH range) with respect to the hydrogen ion concentration condition or the pH condition. For example, in the case of the disclosure a, "the antigen binding activity under the condition of high hydrogen ion concentration (acidic pH range) is lower than the antigen binding activity under the condition of low hydrogen ion concentration (neutral pH range)" may mean that the antigen binding activity of the ionic concentration-dependent antigen binding domain, the ionic concentration-dependent antibody, the ionic concentration-dependent antigen binding domain having an increased pI, or the ionic concentration-dependent antibody having 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 may mean that the antigen binding activity in early endosome pH is weaker than the antigen binding activity in plasma pH; and specifically means that the antigen binding activity of the antibody, e.g., at pH 5.8, is weaker than the antigen binding activity at, e.g., pH 7.4.

Whether the antigen binding activity of an antigen binding domain or an antibody containing said domain varies depending on the hydrogen ion concentration conditions can be readily assessed by known methods, e.g., by the assay methods described in the context of disclosure a herein, or in WO 2009/125825. For example, the antigen binding activity of an antigen binding domain or an antibody containing said domain on an antigen of interest can be measured and compared at low and high hydrogen ion concentrations. In this case, it is preferable that the conditions other than the hydrogen ion concentration are the same. In the case of determining the antigen binding activity, the skilled person can appropriately select conditions other than the hydrogen ion concentration, and for example, measurement can be performed under the condition of HEPES buffer at 37 ℃, or using biacore (ge healthcare), or the like.

Within the scope of disclosure a described herein, unless the context specifically indicates otherwise, "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 convenient measurement, for example, pH 7.0 may be used.

Within the scope of disclosure a described herein, 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 unless the context specifically indicates otherwise; 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 of the early endosomes, but for convenience, pH 6.0 may be used, for example.

In one embodiment in the case of the disclosure a, in the case where the ion concentration is a hydrogen ion concentration, it is preferable that the antigen binding activity of the ion concentration-dependent antigen binding domain, the ion concentration-dependent antibody, the ion concentration-dependent antigen binding domain having an increased pI, or the ion concentration-dependent antibody having an increased pI is higher 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 an antigen, the ratio of dissociation constant (KD) under acidic pH conditions to KD under neutral pH conditions, i.e., KD (acidic pH range)/KD (neutral pH range), (e.g., KD (pH 5.8)/KD (pH 7.4)) may be 2 or more; 10 or more; or 40 or more. The upper limit of KD (acidic pH range)/KD (neutral pH range) value is not limited and may be any value such as 400, 1000, or 10000.

In an alternative embodiment, it is also possible to express the above binding activity ratio using, for example, the dissociation rate constant (kd) as an index. In the case of expressing the binding activity ratio using the dissociation rate constant (KD) instead of the dissociation constant (KD) as an index, the ratio of the dissociation rate constant (KD) under the high hydrogen ion concentration condition to that under the low hydrogen ion concentration condition for the antigen, that is, KD (acidic pH range)/KD (neutral pH range) may be 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of kd (acidic pH range)/kd (neutral pH range) value is not limited and can be any value such as 50, 100, or 200.

In the case where the antigen is a soluble antigen, the value of the antigen-binding activity may be represented by an off-rate constant (kd), and in the case where the antigen is a membrane antigen, the value may be represented by an apparent off-rate constant (apparent kd). The dissociation rate constant (kd) and the apparent dissociation rate constant (apparent kd) can be determined by known methods, for example, by using biacore (ge healthcare) or flow cytometry.

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

The method may include, for example:

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

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

(c) selecting an antigen binding domain or antibody having an antigen binding activity at acidic pH conditions that is lower than at neutral pH conditions.

Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen binding domain or an antibody, or a library thereof, under neutral pH conditions;

(b) incubating the antigen binding domain or antibody that binds to the antigen in step (a) under acidic pH conditions; and

Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen binding domain or an antibody, or a library thereof, under acidic pH conditions;

(c) allowing the antigen to bind to the antigen binding domain or antibody selected in step (b) under neutral pH conditions; and

Alternatively, the method may comprise, for example:

(a) contacting the antigen binding domain or antibody, or library thereof, with an antigen immobilized column under neutral pH conditions;

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

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

Alternatively, the method may comprise, for example:

(a) passing the antigen binding domain or antibody, or library thereof, through a column of immobilized antigen under acidic pH conditions to collect eluted antigen binding domain or antibody that does not bind to the column;

(b) allowing the antigen binding domain or antibody collected in step (a) to bind to antigen under neutral pH conditions; and

(c) isolating the antigen binding domain or antibody bound to the antigen in step (b).

Alternatively, the method may comprise, for example:

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

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

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

Each of these different screening methods may be repeated several times, or the steps may be combined. The above conditions can be appropriately selected for acidic and neutral pH conditions. The desired pH-dependent antigen-binding domain or pH-dependent antibody can thus be obtained.

In the case of disclosure a, in one embodiment, the antigen binding domain or antibody as starting material may be, for example, a modified antigen binding domain or antibody having an increased pI due to modification of the charge of at least one amino acid residue capable of being exposed on its surface. In an alternative embodiment, where amino acids that alter the binding activity of the ionic concentration-dependent antigen binding domain are introduced into the sequence, they may be introduced together with at least one charge modification of amino acid residues that are capable of being exposed on the surface of the antigen binding domain or antibody to increase the pI.

Alternatively, in the case of the present disclosure a, for example, it is possible to use a pre-existing antigen binding domain or antibody, a pre-existing library (phage library, etc.); antibodies prepared from hybridomas obtained by immunizing an animal or from B cells of an immunized animal, or a library thereof; or by introducing into it natural or unnatural amino acid mutations having a side chain pKa of 4.0-8.0 (described below), antibodies, or libraries (e.g., libraries of natural or unnatural amino acid mutations having an increased side chain pKa of 4.0-8.0, or libraries of natural or unnatural amino acid mutations having a side chain pKa of 4.0-8.0 at a particular site). The preferred antigen binding domain may have, for example, an amino acid sequence in which at least one amino acid residue is replaced by an amino acid having a side chain pKa of 4.0-8.0 and/or which is inserted with an amino acid having a side chain of 4.0-8.0, as described in WO 2009/125825.

In one embodiment in the case of the disclosure a, the site of introducing the amino acid mutation having a side chain pKa of 4.0 to 8.0 is not limited, and the mutation may be introduced to any site as long as the antigen binding activity is weaker in an acidic pH range than in a neutral pH range (KD (acidic pH range)/KD (neutral pH range) value is increased or KD (acidic pH range)/KD (neutral pH range) value is increased, as compared to before the substitution or insertion. Where the antibody has a variable region or one or more CDRs, the site may be within the variable region or one or more CDRs. The number of substituted or inserted amino acids can be appropriately determined by those skilled in the art; and the number may be one or more. In addition, other amino acids may be deleted, added, inserted, and/or substituted, or modified (in addition to the above substitutions or insertions). Substitution or insertion of an amino acid with a side chain pKa of 4.0-8.0 with an amino acid with a side chain pKa of 4.0-8.0 can be performed in a random manner by scanning methods, such as histidine scanning, wherein alanine is replaced with histidine in an alanine scan known to the skilled person, and/or antibodies with an increased KD (acidic pH range)/KD (neutral pH range) value or KD (acidic pH range)/KD (neutral pH range) value compared to before mutation can be selected from antigen binding domains or antibodies obtained by random substitution or random insertion mutation of these amino acids, or libraries thereof.

Furthermore, antigen binding domains or antibodies may preferably be those whose antigen binding activity is not significantly reduced, not substantially the same, or increased at neutral pH ranges before and after these mutations; and in other words, those whose activity can be maintained at least 10% or more, preferably 50% or more, still more preferably 80% or more, and still more preferably 90% or more, or even more. In the case where the binding activity of the antigen-binding domain or the antibody is decreased due to the substitution or insertion of an amino acid having a pKa of 4.0 to 8.0 with an amino acid having a pKa of 4.0 to 8.0, the binding activity may 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.

In an alternative embodiment, the amino acid with a side chain pKa of 4.0 to 8.0 may be placed anywhere within the heavy and/or light chain variable region that may form the antigen binding domain. At least one amino acid residue having a side chain pKa of 4.0 to 8.0 may be located, for example, in a CDR (one or more of CDR1, CDR2 and CDR 3) and/or FR (one or more of FR1, FR2, FR3 and FR 4) 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 the light chain variable region CDR1 according to Kabat numbering; an amino acid residue according to Kabat numbering at one or more of

positions

50, 51, 52, 53, 54, 55 and 56 of the light chain variable region CDR 2; and/or an amino acid residue according to Kabat numbering at one or more of positions 89, 90, 91, 92, 93, 94 and 95A of the light chain variable region CDR 3. Those amino acid residues may be contained therein alone or in combination as long as the antigen-binding activity of the antibody is changed depending on the hydrogen ion concentration condition.

In one embodiment within the scope of disclosure a, any amino acid residue may be suitably used as an amino acid residue of the antigen binding domain or the antigen binding activity of the antibody which changes depending on the hydrogen ion concentration condition. Specifically, the amino acid residues may include those having a side chain pKa of 4.0 to 8.0. The amino acids having electron donating properties may 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 residue may preferably include, for example, an amino acid having a side chain pKa of 6.0 to 7.0, particularly His (H).

Within the scope of disclosure a described herein, unless otherwise indicated and unless there is a discrepancy in context, it is understood that the isoelectric point (pI) may be a theoretically or experimentally determined isoelectric point, and it is also referred to as "pI".

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.).

In one embodiment, whether the pI of an antibody having an increased pI, or an antibody of disclosure a, is increased compared to the antibody prior to modification (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (e.g., an antibody prior to antibody modification, or an antibody prior to or during library construction) can be determined by performing (in addition to or in place of the methods described above) antibody pharmacokinetic testing using plasma (e.g., from a mouse, rat, rabbit, dog, monkey, or human), in combination with methods such as BIACORE, cell proliferation assay, ELISA, Enzyme Immunoassay (EIA), Radioimmunoassay (RIA), or fluorescence immunoassay.

In the context of the disclosure a described herein, "amino acid residues capable of being exposed on a surface" may generally refer to amino acid residues located on the surface of a polypeptide constituting an antibody. "amino acid residue located on the surface of a polypeptide" may refer to an amino acid residue whose side chain may be in contact with a solvent molecule (which may typically be predominantly a water molecule). However, the side chain does not have to be completely in contact with the solvent molecule, and even when a part of the side chain is in contact with the solvent molecule, the amino acid residue is defined as "amino acid located on the surface". Amino acid residues located on the surface of a polypeptide may also include amino acid residues adjacent to the surface of an antibody and thus may have shared charge effects from its side chains, even other amino acid residue or residues that are partially in contact with a solvent molecule. One skilled in the art can make homology models for polypeptides or antibodies by, for example, homology modeling using commercially available software. Alternatively, it is possible to use methods such as X-ray crystallography. Amino acid residues that may be exposed on the surface can be determined from the three-dimensional model integration of the antibody, for example, using computer software such as the insight ii program (Accelrys). The site of surface-exposure can be determined using algorithms known in the art (e.g., Lee and Richards (J.mol.biol.55: 379-400 (1971)); connolly (J.appl.Crystal.16: 548-558 (1983)). the sites that can be exposed at the surface can be determined using software suitable for protein modeling and three-dimensional structural information obtained from antibodies. For example, SYBYL Biopolymer Module software (Tripos Associates.) when the algorithm requires the user to enter size parameters, the "size" of the probe used in the calculation may be set to a radius of about 1.4 angstroms or less, and further, a method for determining surface-exposed areas and areas using software for personal computers is described by Pacios (Pacios, comput.chem18 (4): 377-386 (1994); j.mol.model.1: 46-53(1995)) based on the above information, an appropriate amino acid residue located on the surface of a polypeptide constituting an antibody can be selected.

Methods of increasing the pI of a protein are, for example, decreasing the number of amino acids having negatively charged side chains (e.g., aspartic acid and glutamic acid) and/or increasing the number of amino acids having positively charged side chains (e.g., arginine, lysine and histidine) under neutral pH conditions. An amino acid residue having a negatively charged side chain has a negative charge denoted-1 at a pH sufficiently above the pKa of its side chain, a theory well known to those skilled in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the side chain has a negative charge expressed as-1 at neutral pH conditions (e.g., in solution at pH 7.0). In contrast, an amino acid residue having a positively charged side chain has a positive charge denoted as +1 at a pH 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 expressed as +1 under neutral pH conditions (e.g., in solution at pH 7.0). Amino acid residues whose side chains are known to be uncharged under neutral pH conditions (e.g., in solution at pH 7.0) include 15 types of natural amino acids, i.e., alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. In general, it is understood that the amino acids used to alter the pI may be unnatural amino acids.

In conclusion, 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 amino acid sequence of a protein, the side chain of which has a negative charge of-1, the charge of protein +1 can be intentionally investigated by replacing the amino acid (residue) with an uncharged side chain. Furthermore, for example, for amino acids (residues) with uncharged side chains, the charge imparted to the protein +1 can be altered by substituting 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 imparted to the protein +2 at a time can be changed by substituting arginine or lysine (whose side chain has a positive charge of + 1). Alternatively, in order to increase the pI of the protein, an amino acid having an uncharged side chain and/or an amino acid having a positively charged side chain is added or inserted into the amino acid sequence of the protein, or the amino acid having an uncharged side chain and/or the amino acid having a negatively charged side chain present in the amino acid sequence of the protein may be deleted. It is understood, for example, that the N-terminal and C-terminal amino acid residues of a protein have charges derived from the backbone (NH of the amino group at the N-terminus) in addition to charges derived from their side chains ³⁺And COO of C-terminal carboxyl group^-). Thus, the pI of the protein may also be increased by making some additions, deletions, substitutions, or insertions to the functional groups derived from the backbone.

It will be appreciated by those skilled in the art that the effect of altering the net charge or pI of a protein, obtained (in terms of the presence or scale of the charge of amino acids (residues)) by modifying one or more amino acids (residues) in the amino acid sequence, does not only (or substantially) depend on the type of amino acid sequence itself or the target antigen from which the antibody is made, but also on the type and number of amino acid residues that are added, deleted, substituted or inserted.

Antibodies modified to have an increased pI by a modification on at least one amino acid residue that may be exposed on the surface of the antibody ("antibodies with increased pI" or "pI-increased antibodies") may be taken into cells more rapidly or are capable of facilitating the removal of antigen from plasma, as described or suggested in, for example, WO2007/114319, WO2009/041643, WO2014/145159, or WO 2012/016227.

Among the various antibody isotypes, for example, IgG antibodies have a sufficiently large molecular weight and their major metabolic pathways are not excreted through the kidneys. IgG antibodies (which have an Fc region as part of the molecule) are known to be recycled through rescue pathways via FcRn, and thus have a long half-life in vivo. IgG antibodies are thought to be metabolized primarily via metabolic pathways in endothelial cells (He et al, J.Immunol.160 (2): 1029-1035 (1998)). Specifically, it is believed that IgG antibodies are recycled by binding to FcRn when taken up non-specifically into endothelial cells, while IgG antibodies that cannot be bound are metabolized. The plasma half-life of IgG antibodies can be shortened when their Fc region is modified such that their FcRn-binding activity is reduced. On the other hand, it has been shown that the plasma half-life of antibodies with increased pI depends in a highly relevant manner on pI, as described, for example, in WO2007/114319 and WO 2009/041643. In particular, the plasma half-life of the pI-enhanced antibodies described in the above 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 techniques for increasing pI can be widely applied, even for any type of antibody molecule whose primary metabolic pathway is renal excretion, such as scFv, Fab, or Fc fusion proteins.

The pH concentration in biological fluids (e.g., plasma) is in the neutral pH range. Without being bound by a particular theory, it is believed that in biological fluids, the net positive charge of pI-raised antibodies increases due to the increased pI, and thus the antibodies adsorb more strongly to the surface of endothelial cells whose net charge is negative through physicochemical Coulomb interactions than do antibodies whose pI is not raised; through non-specific binding, the antibody binds to it and is taken up into the cell, which results in a reduction of the half-life of the antibody in the plasma or an enhancement of the removal of antigen from the plasma. Furthermore, increasing the pI of an antibody enhances the uptake of the antibody (or antigen/antibody complex) into cells and/or intracellular permeability, which is thought to result in a decrease in antibody concentration in plasma, a decrease in antibody bioavailability, and/or a decrease in antibody half-life in plasma; and these phenomena are expected to occur frequently in vivo regardless of cell type, tissue type, organ type, and the like. Furthermore, in the case where an antibody forms a complex with an antigen and is taken into a cell, not only the pI of the antibody but also the pI of the antigen may have an effect on a decrease or increase in the uptake into the cell.

In one embodiment, methods of generating or screening antibodies with increased pI may include, for example, those described in WO2007/114319 (e.g., paragraphs 0060-0087), WO2009/041643 (e.g., paragraphs 0115), WO2014/145159, and WO 2012/016227. The method may include, for example:

(a) Modifying a nucleic acid encoding an antibody comprising 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, thereby increasing the pI of the antibody;

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

(c) collecting the antibody from the host cell culture.

Alternatively, the method may comprise, for example:

(a') modifying a nucleic acid encoding an antibody comprising at least one amino acid residue that may be exposed on the surface of the antibody such that one or more amino acid residue charges are modified;

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

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

(d') (optionally confirmed or measured and) selecting an antibody with an increased pI compared to the antibody before modification. Here, the antibody as a 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 ionic concentration-dependent antigen-binding domain may also be included in the sequence.

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

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

(A) modifying nucleic acid encoding the first and/or second polypeptide, and optionally the third and/or fourth polypeptide, any one or more of which 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;

(B) culturing the host cell to express the nucleic acid; and

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

Alternatively, the method may comprise, for example:

(a') modifying a nucleic acid encoding the first and/or second polypeptide, and optionally the third and/or fourth polypeptide, any one or more of which 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 altered;

(B') culturing the host cell to express the nucleic acid;

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

(D') (optionally identified and) selecting an antibody with an increased pI compared to the antibody before modification.

Herein, the antibody as a 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, amino acids that alter the binding activity of the ionic concentration-dependent antigen-binding domain may also be included in the sequence.

Alternatively, the method may simply comprise a method of culturing the host cell obtained in step (B) or (B') and collecting the antibody from the cell culture. In this case, the polypeptide whose nucleic acid is modified may preferably be a homo-polymer of the first polypeptide, a homo-polymer of the second polypeptide, or a hetero-polymer of the first and second polypeptides (and optionally a homo-polymer of the third polypeptide, a homo-polymer of the fourth polypeptide, or a hetero-polymer of the third and fourth polypeptides).

In an alternative embodiment, the method may, for example, be a method of producing a humanized or human antibody with a reduced half-life in plasma comprising: comprising one or more CDRs selected from the group consisting of one or more CDRs from a human source, one or more CDRs from an animal other than a human source, and one or more synthetic CDRs; one or more FR of human origin; and a human constant region, (I) 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 a constant region is modified to one or more amino acid residues having a different charge from one or more amino acid residues present at the corresponding position before the modification, whereby the pI of the antibody is increased.

Alternatively, the method may comprise, for example, a method comprising selecting a CDR from the group consisting of one or more CDRs of human origin, one or more CDRs of an animal other than human origin, and one or more synthetic CDRs; one or more FR of human origin; 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 a 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 the modification; and

(II') (optional confirmation) selecting antibodies with an increased pI compared to the antibody before modification.

Here, the antibody as a 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 ionic concentration-dependent antigen binding domain may also be included in the sequence.

Alternatively, for example, it is possible to use pre-existing antigen binding domains or antibodies, pre-existing libraries (phage libraries, etc.); an antibody prepared from a hybridoma obtained by immunizing an animal or from a B cell of an immunized animal, or a library thereof; or an antigen binding domain or antibody or library thereof with an increased pI prepared by modifying at least one amino acid residue in the antigen binding domain, antibody, or library thereof that is likely to be exposed on the surface according to, for example, any of the embodiments described above.

In one embodiment of the antibody of disclosure a, the pI value may preferably be increased, e.g., 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 significantly shorten the antibody half-life in plasma, compared to the antibody prior to modification or alteration (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody), or a reference or parent antibody (e.g., an antibody prior to antibody modification, or an antibody prior to or during library construction)), the pI value may be increased, e.g., 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, the optimal pI value of the antibody of the disclosure a can be appropriately and routinely determined by those skilled in the art in consideration of the balance between pharmacological effects and toxicity, and, for example, the number of antigen-binding domains of the antibody or the pI of the antigen. Without being bound by a particular theory, it is believed that the antibody of disclosure a is beneficial in one embodiment because, in addition to shuttling between plasma and endosomes and the feature of repeated binding of a single antibody molecule to multiple antigens due to the presence of the ionic concentration-dependent antigen binding domain, the net positive charge of the antibody is increased due to the increase in pI, and this enables rapid cellular uptake of the antibody. These properties may shorten the half-life of the antibody in plasma, increase the extracellular matrix binding activity of the antibody, or enhance antigen removal from plasma. The skilled person can decide the optimal pI value to take advantage of these properties.

In one embodiment in the context of disclosure a, an ion concentration-dependent antibody of disclosure a having an increased pI may preferably enhance antigen removal from plasma, e.g., by 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, when compared to an antibody that modifies or changes at least one amino acid residue to increase pI (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody), or a reference or parent antibody (e.g., an antibody that is modified or an antibody prior to or during library construction), which may be an ion concentration-dependent antibody), 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 may preferably be increased, for example, by 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.

In one embodiment in the context of disclosure a, an ion concentration-dependent antibody of disclosure a having an increased pI can preferably enhance antigen removal from plasma, e.g., by 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, when compared to the antibody prior to introduction of the ion concentration-dependent antigen binding domain (a native antibody (e.g., a native Ig antibody, preferably a native IgG antibody), or a reference or parent antibody (e.g., an antibody modified, or an antibody prior to or during library construction), 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 may preferably be increased, for example, by 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.

In one embodiment, the assay method used to assess whether the extracellular matrix binding activity of the antibody of disclosure a is increased compared to the antibody prior to modification or alteration (a native antibody (e.g., a native Ig antibody, which may be a native IgG antibody), or a reference or parent antibody (e.g., an antibody modification, or an antibody prior to or during library construction), which may be an ion concentration-dependent antibody or antibody with an increased pI) is not limited. For example, the assay may be performed using an ELISA system that detects binding between an antibody and extracellular matrix, wherein the antibody is added to a plate on which the extracellular matrix is immobilized, and a labeled antibody against the antibody is added thereto. Alternatively, as described in examples 1 to 4 and WO2012/093704 herein, it is also possible to use Electrochemiluminescence (ECL), which enables the detection of extracellular matrix binding capacity with high sensitivity. The method may be performed, for example, using an ECL system in which a mixture of an antibody and a ruthenium antibody is added to a plate on which an extracellular matrix is immobilized, and the binding between the antibody and the extracellular matrix is measured based on electrochemiluminescence of ruthenium. The concentration of the antibody to be added may be set as appropriate; the added concentration can be high, thereby increasing the sensitivity of detecting extracellular matrix binding. The extracellular matrix may be derived from animals or plants as long as they contain glycoproteins such as collagen, proteoglycans, fibronectin, laminin, entactin, fibrin, and perlecan; and extracellular matrices of animal origin may be preferred. For example, it is possible to use extracellular matrices derived from animals such as humans, mice, rats, monkeys, rabbits, or dogs. For example, a natural extracellular matrix of human origin can be used as an indicator of the pharmacokinetics of antibodies in human plasma. The condition under which extracellular matrix binding of the antibody is evaluated may preferably be a neutral pH range of about pH 7.4, which is a physiological condition; however, the conditions need not be in the neutral range, and binding can also be assessed in the acidic pH range (e.g., about pH 6.0). Alternatively, when assessing extracellular matrix binding of an antibody, the assay may be performed in the co-presence of antigen molecules to which the antibody binds, and by assessing the binding activity of the antigen-antibody complex to the extracellular matrix.

In one embodiment, the antibody of disclosure a may retain antigen binding activity (substantially) compared to an antibody prior to modifying or altering at least one amino acid residue to increase pI (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (e.g., an antibody modification, or an antibody prior to or during library construction)). In this case, "to (substantially) retain antigen binding activity" may mean to have 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 of activity as compared to the binding activity of the antibody before modification or alteration. Alternatively, the antibodies of disclosure a need only retain binding activity to an extent that allows them to retain their function when bound to an antigen; thus, the affinity determined under physiological conditions at 37 ℃ may be, for example, 100nM or less, preferably 50nM or less, more preferably 10nM or less, and still more preferably 1nM or less.

In one embodiment of disclosure a, the phrase "modifying at least one amino acid residue that may be exposed on the surface of an antibody" or an equivalent phrase may mean making one or more of an addition, a deletion, a substitution, and an insertion to the at least one amino acid residue that may be exposed on the surface of an antibody. The modification may preferably comprise the substitution of at least one amino acid residue.

The substitution of an amino acid residue may include, for example, substitution of an amino acid residue whose side chain is uncharged with an amino acid residue having a negatively charged side chain, substitution of an amino acid residue having a positively charged side chain with an amino acid residue whose side chain is uncharged, and substitution of an amino acid residue having a positively charged side chain with an amino acid residue having a negatively charged side chain in the amino acid sequence of the target antibody, which may be performed alone or in an appropriate combination. The insertion or addition of amino acid residues may include, for example, insertion or addition of an amino acid whose side chain is uncharged and/or insertion or addition of an amino acid having a positively charged side chain, in the amino acid sequence of the target antibody, which may be performed alone or in an appropriate combination. Deletion of amino acid residues may include, for example, deletion of amino acid residues whose side chains are uncharged and/or deletion of amino acid residues having negatively charged side chains in the amino acid sequence of the target antibody, which may be performed alone or in an appropriate combination.

One skilled in the art can appropriately combine one or more of these additions, deletions, substitutions, and insertions in the amino acid sequence of the target antibody. Modifications that result in a reduction of the local charge of the amino acid residues are also acceptable, as only the net pI of the antibody of disclosure a must be increased. For example, if desired, antibodies whose pI is increased (too much) may be modified to decrease the pI (slightly). It is also acceptable that the modification of at least one amino acid residue is carried out simultaneously or non-simultaneously for other purposes (e.g. increasing antibody stability or decreasing immunogenicity) resulting in a local charge reduction of the amino acid residue. Such antibodies include antibodies from libraries constructed for specific purposes.

In one embodiment, among the amino acids (residues) used for modifying at least one amino acid residue that may be exposed on the surface of the antibody, natural amino acids are as follows: the amino acid having a negatively charged side chain may be glu (e) or asp (d); the amino acid having no charge in its side chain may 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 the amino acid having a positively charged side chain may be His (H), Lys (K), or Arg (R).

As detailed in examples 1-4, in neutral pH (e.g., pH 7.0) solutions, lysine and arginine have almost 100% positive charge when present as residues in antibodies, whereas histidine has only about 9% positive charge when present as residues in antibodies, with the remaining major portion being considered to have no charge. Thus, Lys (K) or Arg (R) is preferably selected as the amino acid having a positively charged side chain.

In one embodiment, the antibody of disclosure a preferably has a variable region and/or a constant region. Furthermore, the variable region may preferably have a heavy chain variable region and/or a light chain variable region, and/or may preferably have one or more CDRs (e.g., one or more of CDR1, CDR2 and CDR 3) and/or one or more FRs (e.g., one or more of FR1, FR2, FR3 and FR 4). The constant region may preferably have a heavy chain constant region and/or a light chain constant region, and may be, for example, an IgG-type constant region (preferably, a human IgG1, a human IgG2, a human IgG3, or a human IgG 4-type constant region, a human kappa chain constant region, and a human lambda chain constant region) in terms of sequence and type. It is possible to use modified variants of these constant regions.

In one embodiment, the modification of at least one amino acid residue that may be exposed on the surface of the antibody may be a single amino acid modification or a combination of multiple amino acid modifications. A preferred method may be to introduce a combination of multiple amino acid substitutions at positions where amino acids are likely to be exposed on the surface of the antibody. Further, without limitation, the plurality of amino acid substitutions are preferably introduced at positions close to each other in three dimensions. When an amino acid having a positively charged side chain (e.g., Lys (K) or Arg (R)) is substituted with an amino acid that may be exposed on the surface of an antibody molecule (which is preferably, but not limited to, an amino acid having a negatively charged side chain (e.g., Glu (E) or Asp (D)), or when a positively charged pre-existing amino acid (e.g., Lys (K) or Arg (R)) is used, for example, one or more amino acids that are three-dimensionally close to the amino acid (which may include amino acids embedded within the antibody molecule as the case may be) may also be substituted with a positively charged amino acid, thereby correspondingly creating a dense state of local positive charges at three-dimensionally adjacent positions Whether a 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.

In addition to those described above, methods of imparting multiple positive charges at sites that are close to each other in three dimensions may include those using 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 proximal positions by substituting positively charged amino acids at sites three-dimensionally proximal to these positively charged amino acids.

In the case of antibodies of disclosure a having variable regions (which may be modified), amino acid residues that are not covered by antigen binding (i.e., may still be exposed on the surface) may be modified, and/or amino acid modifications may not be introduced at sites covered by antigen binding or amino acid modifications may be made that do not (substantially) inhibit antigen binding. In the case where amino acid residues present in the ion concentration-dependent binding domain that may be exposed on the surface of the antibody molecule are modified, the amino acids of the antigen binding domain may be modified in this manner: the modification does not (substantially) reduce the binding activity of amino acid residues (e.g., calcium-binding motifs, or those in histidine insertion sites and/or histidine substitution sites) that may alter the antigen-binding activity of the antibody depending on the ion concentration conditions, or the amino acid residues may be modified at sites other than the amino acid residues that alter the antigen-binding activity of the antibody depending on the ion concentration conditions. On the other hand, when amino acid residues that may be exposed on the surface of an antibody molecule present in the ion concentration-dependent binding domain have been modified, the type or position of the amino acid residues that may change the antigen binding activity of the antibody depending on the ion concentration conditions may be selected such that the pI of the antibody is not reduced below an acceptable level. In the case where the pI of the antibody is reduced below an acceptable level, the pI of the whole antibody may be increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody molecule.

Without limitation, the FR sequence with high pI may preferably be selected from the sequences of human germline FR sequences or regions equivalent thereto, whose amino acids may be modified in some cases.

In the case where the antibody of disclosure a has a constant region (which may be modified) comprising an fcyr-binding domain (which may be a binding domain for any of the fcyr isoforms and allotypes described below) and/or an FcRn-binding domain, the site for modifying at least one amino acid residue that may be exposed on the surface of the constant region may be an amino acid residue other than those in the fcyr-binding domain and/or those in the FcRn-binding domain, if desired. Alternatively, where the modification site is selected from amino acid residues in an Fc γ R-binding domain and/or an FcRn-binding domain, it may be preferable to select a site that does not (substantially) affect the binding activity or binding affinity for Fc γ R and/or FcRn, or if they do, select a biologically or pharmacologically acceptable site.

In one embodiment, the site of the at least one amino acid residue modified to produce 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 (which may be modified) is not limited; however, the position may be selected from the group consisting of: (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDRs of the light chain variable region, wherein the amino acid at each position may be selected from any of the amino acids described above with respect to side chain charge, such as lys (k), arg (r), gin (q), gly (g), ser(s), or asn (n), but is not limited thereto, after modification. 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.

In one embodiment, among the positions to be modified, the following positions may be used in combination with other positions that may themselves have sufficient effect to increase the pI of the antibody to aid in the increase of the pI of the antibody of disclosure a. The position for assisting in the increase of pI may be, for example, for the light chain variable region, selected from the group consisting of positions 27, 52, 56, 65, and 69 (numbering according to Kabat).

Furthermore, the position of at least one amino acid residue modified in the CDR and/or FR is not limited; however, the site may 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 CDRs of the heavy chain variable region; (c) positions 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDRs 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.

In the case where the modification site of at least one amino acid residue is selected from, for example, the group comprising the above groups, the types of modified amino acids in the heavy chain variable region are, for example:

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

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

any two or more positions selected from the group consisting of 16, 43, 64, and 105; any two or more positions selected from the group consisting of (numbering according to Kabat): positions 77, 82a, and 82 b; positions 77 and 85; positions 41 and 44; positions 82a and 82 b; positions 82 and 82 b; positions 82b and 83; or positions 63 and 64, wherein the amino acid at each position after modification may be selected from any of the above with respect to side chain charge, such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N), but is not limited thereto.

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

Similarly, the types of modified amino acids 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.

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 locations at a location selected from the group consisting of 16, 24, and 27; any two or more locations at a location selected from the group consisting of 24, 27, and 37; any two or more locations at a location selected from the group consisting of 25, 26, and 37; any two or more locations at a location selected from the group consisting of 27, 76, and 79; any two or more locations at a location selected from the group consisting of 41, 74, and 77; any two or more locations at a location selected from the group consisting of 41, 76, and 79; any two or more locations at a location selected from the group consisting of 24, 27, 41, and 42; any two or more locations at a location selected from the group consisting of 24, 27, 52, and 56; any two or more locations at a location selected from the group consisting of 24, 27, 65, and 69; any two or more locations at a location selected from the group consisting of 24, 27, 74, and 77; any two or more locations at a location selected from the group consisting of 24, 27, 76, and 79; any two or more locations at a location selected from the group consisting of 25, 26, 52, and 56; any two or more locations at a location selected from the group consisting of 25, 26, 65, and 69; any two or more locations at a location selected from the group consisting of 25, 26, 76, and 79; any two or more locations at a location selected from the group consisting of 27, 41, 74, and 77; any two or more locations at a location selected from the group consisting of 27, 41, 76, and 79; any two or more locations at a location selected from the group consisting of 52, 56, 74, and 77; any two or more locations at a location selected from the group consisting of 52, 56, 76, and 79; any two or more locations at a location selected from the group consisting of 65, 69, 76, and 79; any two or more locations at a location selected from the group consisting of 65, 69, 74, and 77; any two or more locations at a location selected from the group consisting of 18, 24, 45, 79, and 107; any two or more locations at a location selected from the group consisting of 27, 52, 56, 74, and 77; any two or more locations at a location selected from the group consisting of 27, 52, 56, 76, and 79; any two or more locations at a location selected from the group consisting of 27, 65, 69, 74, and 77; any two or more locations at a location selected from the group consisting of 27, 65, 69, 76, and 79; any two or more locations at a location selected from the group consisting of 41, 52, 56, 74, and 77; any two or more locations selected from the group consisting of 41, 52, 56, 76, and 79; any two or more locations at a location selected from the group consisting of 41, 65, 69, 74, and 77; any two or more locations at a location selected from the group consisting of 41, 65, 69, 76, and 79; any two or more locations at a location selected from the group consisting of 24, 27, 41, 42, 65, and 69; any two or more locations at a location selected from the group consisting of 24, 27, 52, 56, 65, and 69; any two or more locations at a location selected from the group consisting of 24, 27, 65, 69, 74, and 77; any two or more locations at a location selected from the group consisting of 24, 27, 65, 69, 76, and 79; any two or more locations at a location selected from the group consisting of 24, 27, 41, 42, 74, and 77; any two or more locations at a location selected from the group consisting of 24, 27, 52, 56, 74, and 77; any two or more locations at a location selected from the group consisting of 24, 27, 41, 42, 76, and 79; any two or more locations at a location selected from the group consisting of 24, 27, 52, 56, 76, and 79; any two or more locations at a location selected from the group consisting of 24, 27, 74, 76, 77, and 79; any two or more locations at a location selected from the group consisting of 52, 56, 65, 69, 74, and 77; or any two or more positions (numbering according to Kabat) at positions selected from the group consisting of 52, 56, 65, 69, 76, and 79, wherein the amino acid at each position after modification may be selected from any of the above amino acids in terms of side chain charge, such as lys (k), arg (r), gin (q), gly (g), ser(s), or asn (n), but is not limited thereto.

Specific combinations may 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.

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 only (or substantially) on the amino acid sequence itself constituting the antibody or the type of target antigen, but rather it depends on the type and number of amino acid residues that are substituted. It has also been demonstrated that the antigen binding activity for several types of antigens is (substantially) maintained, or at least can be expected to be maintained with high probability by the person skilled in the art, even after modification of some amino acids.

For example, WO2009/041643 shows, inter alia, that in the context of SEQ ID NO: 8, the preferred modification sites of amino acid residues that may be exposed on the surface in the heavy chain FR of the humanized glypican 3(glypican 3) antibody shown in fig. 8 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 the Kabat numbering. It is also reported 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 amino acid residues at positions 52, 54, 62, 63, 65, and 66 in the heavy chain CDRs of an antibody are preferred. It also shows, as shown in SEQ ID NO: 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 in the FR of the light chain of the humanized glypican 3 antibody shown in fig. 9 according to the Kabat numbering are preferred. It also indicates that amino acid residues at positions 24, 27, 33, 55, 59 in the light chain CDRs of the antibody are preferred. Furthermore, WO2009/041643 specifically shows that SEQ ID NO: the amino acid residues at positions 31, 64, and 65 according to Kabat numbering in the heavy chain CDRs of the anti-human IL-6 receptor antibody shown in 10 are preferred sites that allow modification of amino acid residues that may be exposed on the surface while retaining antigen binding activity. It also shows, as shown in SEQ ID NO: 11, the amino acid residues at positions 24, 27, 53, and 55, according to Kabat numbering, in the light chain CDRs of the anti-human IL-6 receptor antibody are preferred. It also specifically shows that SEQ ID NO: 12 is a preferred position according to Kabat numbering of the amino acid residue at position 31 in the heavy chain CDR of the anti-human IL-6 receptor antibody, which allows modification of amino acid residues that may be exposed on the surface while retaining antigen binding activity. It also shows that SEQ ID NO: the amino acid residues at positions 24, 53, 54, and 55 according to Kabat numbering in the light chain CDRs of the anti-human IL-6 receptor antibody set forth in 13 are preferred. WO2009/041643 also shows that SEQ ID NO: the amino acid residues at positions 61, 62, 64, and 65 according to Kabat numbering in the heavy chain CDRs of the anti-human glypican 3 antibody shown in 14 are preferred sites that allow modification of amino acid residues that may be exposed on the surface while retaining antigen binding activity. It is also shown that SEQ ID NO: 15 the amino acid residues at positions 24 and 27 according to Kabat numbering in the light chain CDRs of the anti-human glypican 3 antibody are preferred. It is also shown that SEQ ID NO: the amino acid residues at positions 61, 62, 64, and 65 according to Kabat numbering in the heavy chain CDRs of the anti-human IL-31 receptor antibody shown in 16 are preferred sites that allow modification of amino acid residues that may be exposed on the surface while retaining antigen binding activity. WO2009/041643 also shows that SEQ ID NO: 17, the amino acid residues at positions 24 and 54, according to Kabat numbering, in the light chain CDRs of the anti-human IL-31 receptor antibody are preferred. Similarly, WO2007/114319 reports antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and hB26-PF, which are generated by modifying the charge of one or more amino acid residues that may be exposed on the surface, show an alteration in pI (as demonstrated by isoelectric focusing), and have comparable binding activity to factor IXa or factor X (which is their antigen) compared to the antibody before the modification or alteration. It was also reported that when these antibodies were administered to mice, the pI of each antibody showed a high correlation with its clearance rate in plasma (CL), retention in plasma, and half-life in plasma (T1/2). WO2007/114319 also shows that as sites for modification of amino acid residues that may be exposed on the surface, amino acid residues at positions 10, 12, 23, 39, 43, 97, and 105 in the variable region are preferred.

In an alternative or another embodiment, amino acid residues that may be exposed on the surface of the antibody constant region can be identified to determine the modification site for at least one amino acid residue of the antibody whose pI is increased for use in generating disclosure a, for example, using known methods such as X-ray crystallography or by homology models constructed from homology modeling of antibody constant regions (which are preferably human constant regions, more preferably human Ig-type constant regions, and still more preferably human IgG-type constant regions, but not limited thereto). The modification site of at least one amino acid residue that may be exposed on the surface of the constant region is not limited; however, the site may preferably be selected from the group consisting of: positions 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and position 443, 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, wherein the amino acid at each position may be selected from the above amino acids in terms of side chain charge after modification, such as, but not limited to, Lys (K), Arg (R), Gln (Q), or Asn (N). When the modification site of at least one amino acid residue is selected, for example, from the group comprising the above-mentioned groups, for example, the type of amino acid at each site after modification may be as follows:

254K, 254R, 254Q, or 254N at position 254; 258K, 258R, 258Q, or 258N at position 258;

281K, 281R, 281Q, or 281N at position 281; 282K, 282R, 282Q, or 282N at position 282;

285K, 285R, 285Q, or 285N at position 285; 309K, 309R, 309Q, or 309N at position 309;

311K, 311R, 311Q, or 311N at position 311; 315K, 315R, 315Q, or 315N at location 315;

327K, 327R, 327Q, or 327N at position 327; 330K, 330R, 330Q, or 330N at location 330;

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

345K, 345R, 345Q, or 345N at position 345; 356K, 356R, 356Q, or 356N at position 356;

358K, 358R, 358Q, or 358N at position 358; 359K, 359R, 359Q, or 359N at position 359;

361K, 361R, 361Q, or 361N at position 361; 362K, 362R, 362Q, or 362N at position 362;

384K, 384R, 384Q, or 384N at position 384; 385K, 385R, 385Q, or 385N at position 385;

386K, 386R, 386Q, or 386N at location 386; 387K, 387R, 387Q, or 387N at position 387;

389K, 389R, 389Q, or 389N at position 389; 399K, 399R, 399Q, or 399N at position 399;

400K, 400R, 400Q, or 400N at position 400; 401K, 401R, 401Q, or 401N at position 401;

402K, 402R, 402Q, or 402N of location 402; 413K, 413R, 413Q, or 413N at position 413;

418K, 418R, 418Q, or 418N at position 418; 419K, 419R, 419Q, or 419N at position 419;

421K, 421R, 421Q, or 421N at position 421; 433K, 433R, 433Q, or 433N at position 433;

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

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

In one embodiment of the antibody of disclosure a, the net pI of the antibody may be increased by modifying at least one amino acid residue as described above that may be exposed on the surface of the variable region (which may be modified) and at least one amino acid residue as described above that may be exposed on the surface of the constant region (which may be modified).

Within the scope of the disclosures a and B described herein, where the antibody of disclosure a or B is an IgG-type antibody or a molecule derived therefrom, the antibody heavy chain constant region may comprise a constant region of IgG1 type, IgG2 type, IgG3 type, or IgG4 type. In the disclosure a or B, the heavy chain constant region may be a human heavy chain constant region, but is not limited thereto. It is known that there are many allotypes for human IgG. In particular, some differences in the amino acid sequence of the human IgG constant region between 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).

Among these, for example, for human IgG1, allotypes called G1m1, 17, and G1m3 are known. Allotypes differ in their amino acid sequence: g1m1, 17 has an aspartic acid at position 356 and a leucine at position 358 (numbering according to EU), whereas G1m3 has a glutamic acid at position 356 and a methionine at position 358 (numbering according to EU). However, no significant differences in basic antibody function and properties were reported among the reported allotypes. Therefore, 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 embodiments and have the same effect with any allotype prediction. Within the scope of the disclosures a and B described herein, when referred to as "human IgG 1", "human IgG 2", "human IgG 3", or "human IgG 4", the allotype is not limited to a particular allotype and may include all reported allotypes (allotype).

In an alternative or another embodiment of the disclosure a or B, the light chain constant region of the antibody may comprise any constant region of the kappa chain (IgK) type or the lambda chain (IgL1, IgL2, IgL3, IgL6, or IgL7) type. The light chain constant region may preferably be a human light chain constant region, but is not limited thereto. As in Sequences of proteins of immunological interest, NIH publication No.91-3242, there are reports on various allotypic Sequences due to gene polymorphisms of the human kappa chain constant region and the human lambda chain constant region. Such allotypes include, for example, the human kappa chain constant region (SEQ ID NO: 22) and the human lambda chain constant region (SEQ ID NO: 23). However, no report is available suggesting significant differences in basic antibody function and properties among the reported allotypes. Thus, one skilled in the art can readily appreciate that the same effect is expected for any allotype (hereinafter also collectively referred to as the natural (human) IgG (type) constant region) when reference is made to a specific allotype within the scope of the disclosure a and B described herein.

Furthermore, since the Fc region of a natural IgG antibody constitutes a part of the constant region of a 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 may have an Fc region contained in the constant region of a natural IgG (IgG1, IgG2, IgG3, or IgG4 type) (hereinafter also collectively referred to as a natural (human) IgG (type) Fc region). The Fc region of a native IgG may relate to an Fc region consisting of the same amino acid sequence as an Fc region derived from a naturally occurring IgG. Specific examples of the Fc region of natural human IgG may 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), or the Fc region contained in the human IgG4 constant region (SEQ ID NO: 21) as described above (the Fc region of the IgG type may refer, for example, to the C-terminus from cysteine at position 226 according to EU numbering, or to the C-terminus from proline at position 230 according to EU numbering).

In one embodiment, the antibodies of disclosure a and B may comprise variants in which one or more modifications selected from amino acid substitutions, additions, deletions, or insertions are made to the constant region of a native (preferably human) IgG (heavy chain constant region and/or light chain constant region) or to the Fc region of a native (preferably human) IgG.

Within the scope of the disclosure a described herein, WO2013/081143 reports that, for example, due to avidity (sum of binding strengths between various 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 multimeric antigen and a multispecific ion concentration-dependent antibody or a multiple paratope (multivalent) 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 are able to bind Fc γ R, FcRn, complement receptor more strongly and thus the antibody is taken into cells more rapidly. Therefore, the above-mentioned ion concentration-dependent antibody (which is capable of forming a multivalent immunocomplex with a multimeric antigen or a monomeric antigen) can also be used as the antibody of the disclosure a (an ion concentration-dependent antibody having an increased pI) when modified to have an increased pI by modifying at least one amino acid residue that is likely to be exposed on the surface of the antibody. One skilled in the art will appreciate that an ion concentration-dependent antibody with increased pI capable of forming a multivalent immune complex with a multimeric or monomeric antigen may be taken up into a cell more rapidly than an ion concentration-dependent antibody with increased pI not capable of forming a multivalent immune complex. One skilled in the art will also appreciate that in one embodiment, the activity of the antibody of disclosure a in binding to FcRn and/or fcyr may be increased under neutral pH conditions and in that case, ion concentration-dependent antibodies with increased pI capable of forming multivalent immune complexes with multimeric or monomeric antigens may be taken up into cells even more rapidly.

In one embodiment, the antibody of disclosure a may be a one-armed antibody (including all embodiments of the one-armed antibody described in WO 2005/063816). Typically, a one-armed antibody is an antibody lacking one of the two Fab regions possessed by a normal IgG antibody, and may be (without limitation) produced, for example, by the method described in WO 2005/063816. Without limitation, in an IgG-type antibody having a heavy chain whose structure is, for example, VH-CH 1-hinge-CH 2-CH3, when one of the Fab regions is cleaved at a site more N-terminal with respect to the hinge (e.g., VH or CH1), the antibody will be expressed in a form containing excess sequence, and when one of the Fab regions is cleaved at a site more C-terminal with respect to the hinge (e.g., CH2), the Fc region will have an incomplete form. Therefore, without limitation, from the viewpoint of stability of the antibody molecule, it is preferable that the one-armed 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 after cleavage by an intramolecular disulfide bond. WO2005/063816 reports that such single-armed antibodies have increased stability compared to Fab molecules. Antibodies with increased or decreased pI can also be produced by making such single-arm antibodies. Furthermore, when the ion concentration-dependent antigen binding domain is introduced into an antibody having an increased pI, which is a one-armed antibody, the half-life of the antibody in plasma may be further shortened, cellular uptake of the antibody may be further enhanced, removal of antigen from plasma may be further enhanced, or affinity of the antibody for extracellular matrix may be further increased, as compared to an antibody having an increased pI, which does not have the ion concentration-dependent antigen binding domain.

Without being bound by a particular theory, embodiments can be envisaged where the cellular uptake-accelerating effect of the one-armed antibody is expected, but not limited to, a situation where the pI of the soluble antigen is lower than that of the antibody. The net pI of a complex consisting of an antibody and an antigen can be calculated by known methods, taking into account that the complex is a single molecule. In this case, the lower the pI of the soluble antigen, the lower the net pI of the complex; and the higher the pI of the soluble antigen, the higher the net pI of the complex. When a normal type IgG antibody molecule (with two Fabs) binds to a single low-pI soluble antigen, the net pI of the complex in the latter case is lower compared to binding to two low-pI soluble antigens. When such a general type antibody is converted into a one-armed antibody, only one antigen can bind to a single molecule of the antibody; the reduction in pI of the complex due to binding of the second antigen can thereby be inhibited. In other words, it is believed that when the pI of the soluble antigen is lower than that of the antibody, the conversion to the one-armed antibody increases the pI of the complex compared to the ordinary antibody, and the uptake into cells is accelerated.

Furthermore, without limitation, when the Fab of a normal IgG-type antibody molecule (with two Fabs) has a lower pI than Fc, conversion to a one-armed antibody increases the net pI of the complex consisting of the one-armed antibody and the antigen. Further, when such a transition to a one-armed antibody is performed, it is preferable from the viewpoint of stability of the one-armed antibody that one of the Fabs is cleaved in the hinge region located at the junction between the Fab and the Fc. In this case, it can be expected that the pI can be effectively increased by selecting a site that will increase the pI of the single-arm antibody to a desired degree.

Thus, it will be appreciated by those skilled in the art that, not only (or substantially) depending on the antibody amino acid sequence itself and the type of soluble antigen, the pI of the antibody may be increased and cellular uptake of the accompanying antigen may be accelerated by converting the antibody to a one-armed antibody, by calculating the relationship between the theoretical pI of the antibody (theoretical pI for Fc and Fab) and the theoretical pI of the soluble antigen and predicting the difference in their theoretical pI values.

In one embodiment, the antibody of disclosure a or B may be a multispecific antibody, which may be, but is not limited to, a bispecific antibody. The multispecific antibody may be a multispecific antibody comprising a first polypeptide and a second polypeptide. Herein, "multispecific antibody comprising a first polypeptide and a second polypeptide" refers to an antibody that binds to at least two or more types of different antigens or at least two or more types of epitopes in the same antigen. The first and second polypeptides may preferably comprise a heavy chain variable region, and more preferably the variable region comprises one or more CDRs and/or one or more FRs. In another embodiment, the first polypeptide and the second polypeptide may preferably each comprise a heavy chain constant region. In another embodiment, the multispecific antibody may comprise a third polypeptide and a fourth polypeptide, each comprising a light chain variable region and preferably also a light chain constant region. In this case, the first to fourth polypeptides may be assembled together to form a multispecific antibody.

In one embodiment, where the antibody of disclosure a is a multispecific antibody and the multispecific antibody contains a heavy chain constant region, in order to reduce its pI, for example, the following sequences may be used: an IgG2 or IgG4 sequence at position 137; an IgG1, IgG2, or IgG4 sequence at position 196; an IgG2 or IgG4 sequence at position 203; an IgG2 sequence at position 214; an IgG1, IgG3, or IgG4 sequence at position 217; an IgG1, IgG3, or IgG4 sequence at position 233; an IgG4 sequence at position 268; an IgG2, IgG3, or IgG4 sequence at position 274; an IgG1, IgG2, or IgG4 sequence at position 276; an IgG4 sequence at position 355; an IgG3 sequence at position 392; an IgG4 sequence at position 419; or an IgG1, IgG2, or IgG4 sequence at position 435. Meanwhile, in order to increase the pI thereof, for example, the following sequence may be used: an IgG1 or IgG3 sequence at position 137; an IgG3 sequence at position 196; an IgG1 or IgG3 sequence at position 203; an IgG1, IgG3, or IgG4 sequence at position 214; an IgG2 sequence at position 217; an IgG2 sequence at position 233; an IgG1, IgG2, or IgG3 sequence at position 268; an IgG1 sequence at position 274; an IgG3 sequence at position 276; an IgG1, IgG2, or IgG3 sequence at position 355; an IgG1, IgG2, or IgG4 sequence at position 392; an IgG1, IgG2, or IgG3 sequence at position 419; or an IgG3 sequence at position 435.

In one embodiment, where the antibody of disclosure a has two heavy chain constant regions, the pI of the two heavy chain constant regions may be the same or different from each other. The heavy chain constant region may be an IgG1, IgG2, IgG3 and IgG4 heavy chain constant region initially having different pis. Alternatively, it is possible to introduce a pI difference between the two heavy chain constant regions. The modification site of at least one amino acid residue used to introduce such a pI difference in the constant region may be one or more positions as described above or one or more positions selected from, for example, the group consisting of: position 137, position 196, position 203, position 214, position 217, position 233, position 268, position 274, position 276, position 297, position 355, position 392, position 419, and position 435 (according to EU numbering) in the heavy chain constant region described in WO 2009/041643. Alternatively, the amino acid residue at position 297, which is a glycosylation site, may be modified to remove the sugar chain, since removal of the sugar chain from the heavy chain constant region results in a difference in pI.

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

In one embodiment, the antibodies of disclosure a or B may be derived from, without limitation, any animal species (e.g., human; or non-human animals such as mice, rats, hamsters, rabbits, monkeys, cynomolgus monkeys, macaques, baboons (hamadras baboon), chimpanzees, goats, sheep, dogs, pigs, or camels), or any bird; and the antibody is preferably derived from human, monkey or mouse.

In one embodiment, the antibody of disclosure a or B may be an Ig-type antibody, and may preferably be an IgG-type antibody.

Within the context of the disclosures a and B described herein, an Fc receptor (also referred to as "FcR") refers to a receptor protein that can bind to the Fc region, or Fc region variants, of an immunoglobulin (antibody) or molecule derived therefrom. For example, in the context of the disclosure a described herein, Fc receptors for IgG, IgA, IgE and IgM are known as Fc γ R, Fc α R, Fc ∈ R and Fc μ R, respectively. Within the scope of the disclosures a and B described herein, the Fc receptor may also be, for example, FcRn (also referred to as "neonatal Fc receptor").

Within the scope of disclosure a described herein, "fcyr" may refer to a receptor protein that may bind to the Fc region, or Fc region variant, of an IgG1, IgG2, IgG3, or IgG4 antibody or molecule derived therefrom, and may include any one or more or all of the members of a family of proteins substantially encoded by the fcyr gene. In humans, the family includes, but is not limited to, Fc γ RI (CD64) (including isoforms Fc γ RIa, Fc γ RIb, and Fc γ RIc); fc γ RII (CD32) (including isoforms Fc γ RIIa ((including allotype H131 (type H) and R131 (type R))), Fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc); and Fc γ RIII (CD16) (including isoforms Fc γ RIIIa (including allotype V158 and F158) and Fc γ RIIIb (including allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2)), as well as all unidentified human Fc γ Rs and Fc γ R isoforms and allotypes. In addition, Fc γ RIIb1 and Fc γ RIIb2 have been reported as splice variants of human Fc γ RIIb (hFc γ RIIb). There is also a report of a splice variant called Fc γ RIIb3 (Brooks et al, J.Exp.Med.170: 1369-1385 (1989)). In addition to those described above, hfcyriib includes all splice variants, such as those recorded under NP _001002273.1, NP _001002274.1, NP _001002275.1, NP _001177757.1, and NP _003992.3 in NCBI. The hFc γ RIIb also includes all the genetic polymorphisms that have been reported, for example, Fc γ RIIb (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)), and all the genetic polymorphisms that will be reported in the future.

The Fc γ R may be derived from any organism, and may include those derived from human, mouse, rat, rabbit, or monkey, but is not limited thereto. Mouse Fc γ Rs include, but are not limited to, Fc γ RI (CD64), Fc γ RII (CD32), Fc γ RIII (CD16) and Fc γ RIII-2(CD16-2), as well as all unidentified mouse Fc γ Rs, and Fc γ R isoforms and allotypes. Such preferred Fc γ rs include, for example, human Fc γ RI (CD64), Fc γ RIIA (CD32), Fc γ RIIB (CD32), Fc γ RIIIA (CD16), or Fc γ RIIIB (CD 16). Since Fc γ R exists in vivo in a membrane form, it can be used in experimental systems after artificial conversion to an appropriate soluble form.

For example, as shown in WO2014/163101, the oligonucleotide and amino acid sequences of Fc γ RI may be the sequences shown in NM _000566.3 and NP _000557.1, respectively; the oligonucleotide sequence and amino acid sequence of Fc γ RIIA may be the sequences shown in BC020823.1 and AAH20823.1, respectively. The oligonucleotide sequence and amino acid sequence of Fc γ RIIB can be the sequences shown in BC146678.1 and AAI46679.1, respectively; the oligonucleotide sequence and amino acid sequence of Fc γ RIIIA may be the sequences shown in BC033678.1 and AAH33678.1, respectively; the oligonucleotide sequences and amino acid sequences of Fc γ RIIIB may be the sequences shown in BC128562.1 and AAI28563.1, respectively (showing RefSeq accession numbers).

Fc γ RIIa has two genetic polymorphisms in which the amino acid at position 131 of Fc γ RIIa is replaced with histidine (type H) or arginine (type R) (j.exp.med.172: 19-25, 1990).

In Fc γ RI (CD64), which includes Fc γ RIa, Fc γ RIb, and Fc γ RIc, and Fc γ RIII (CD16), which includes Fc γ RIIIa (including allotypes V158 and F158), the α chain of the Fc region that binds IgG is associated with a common γ chain that has ITAMs that transmit intracellular activation signals. Fc γ RIIIb (including allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2) is a GPI-anchor protein. Meanwhile, the cytoplasmic domain of Fc γ RII (CD32), which includes Fc γ RIIa (including allotypes H131 and R131) and Fc γ RIIc isoforms, contains ITAMs. These receptors are expressed on many immune cells such as macrophages, mast cells, and antigen presenting cells. Activation signals transduced upon binding of these receptors to the Fc region of IgG promote phagocytic capacity of macrophages, production of inflammatory cytokines, degranulation of mast cells, and increased function of antigen-presenting cells. Fc γ rs having the above-described ability to transduce an activation signal are also referred to as activating Fc γ rs within the scope of the disclosures a and B described herein.

Meanwhile, the cytoplasmic domain of Fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2) contains ITIM, which transmits inhibitory signals. In B cells, cross-linking between Fc γ RIIb and B Cell Receptor (BCR) inhibits activation signals from the BCR, which results in inhibition of antibody production by the BCR. In macrophages, crosslinking of Fc γ RIII and Fc γ RIIb inhibits phagocytic ability and the ability to produce inflammatory cytokines. Within the scope of the disclosures a and B described herein, an Fc γ R having the ability to transduce the inhibitory signals described above is also referred to as an inhibitory Fc γ receptor.

Within the scope of disclosure a described herein, the increased, or (substantially) maintained, or decreased binding activity of an antibody or Fc region (variant) to various fcyr compared to the antibody or Fc region (variant) prior to modification can be assessed by methods known to those skilled in the art. The method is not particularly limited and those described in the present embodiment may be used, and for example, BIACORE (proc.natl.acad.sci.usa (2006)103(11), 4005-. Alternatively, for example, ELISA and Fluorescence Activated Cell Sorting (FACS) and ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay). In these assays, the extracellular domain of human Fc γ R can be used as a soluble antigen (e.g., WO 2013/047752).

As the pH condition for measuring the binding activity between the Fc γ R-binding domain and the Fc γ R contained in the antibody or Fc region (variant), acidic or neutral pH conditions may be suitably used. For the temperature used in the measurement conditions, the binding activity (binding affinity) between the Fc γ R-binding domain and the Fc γ R can be arbitrarily evaluated, for example, between 10 ℃ and 50 ℃. Preferred temperatures for determining the binding activity (binding affinity) of the human Fc γ R-binding domain to Fc γ R are, for example, 15 ℃ to 40 ℃. More preferably, to determine the binding activity (binding affinity) between the Fc γ R-binding domain and the Fc γ R, any temperature from 20 ℃ to 35 ℃ may be used, such as, for example, any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 ℃. A non-limiting example of such a temperature is 25 ℃.

In one embodiment, where the antibody of disclosure a or B has a constant region (which may be modified), the constant region may have an Fc region or Fc region variant (preferably, a human Fc region or human Fc region variant), and preferably has an fcyr-binding domain within the scope of disclosure a and an FcRn-binding domain within the scope of disclosures a and B described herein.

In one embodiment, where the antibody of disclosure a has fcyr-binding activity, it may have an fcyr-binding domain, preferably a human fcyr-binding domain. The Fc γ R-binding domain is not particularly limited as long as the antibody has binding activity or affinity for Fc γ R at acidic pH and/or neutral pH, and it may be a domain having activity of directly or indirectly binding Fc γ R.

In one embodiment, where the antibody of disclosure a has fcyr-binding activity, it is preferred that the fcyr-binding activity of the antibody is increased under neutral pH conditions as compared to a reference antibody containing a native IgG constant region. From the viewpoint of comparing Fc γ R-binding activities of both, it is preferable, without limitation, that the antibody of disclosure a and the reference antibody containing a natural IgG constant region have the same amino acid sequence in a region (e.g., variable region) other than the constant region of the antibody modified at one or more amino acid residues of disclosure a.

In one embodiment, where the antibody of disclosure a has fcyr-binding activity or increased fcyr-binding activity under neutral pH conditions (e.g., pH 7.4), without being limited by theory, the antibody is considered to have a combination of the following properties: the property of shuttling between plasma and endosomes and repeatedly binding multiple antigens by having an ionic concentration-dependent antigen binding domain as a single antibody molecule; the property of rapid uptake into cells by having an increased pI and increased positive charge in the whole antibody; and a property of being rapidly taken into cells by having an increased Fc γ R-binding activity under neutral pH conditions. Thus, the half-life of the antibody in plasma may be further shortened, or the binding activity of the antibody to the extracellular matrix may be further increased, or the removal of the antigen from plasma may be further facilitated; the antibodies of disclosure a are therefore beneficial. One skilled in the art can routinely determine the optimal pI value for an antibody to take advantage of these properties.

In one embodiment, an Fc γ R-binding domain having a higher Fc γ R-binding activity than the Fc region or constant region of a native human IgG in which the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar chain may be produced by modifying amino acid residues in the Fc region or constant region of a native human IgG (see WO 2013/047752). In addition, any structural domain that binds to Fc γ R can be used as an Fc γ R-binding domain. In this case, the Fc γ R-binding domain may be produced without the need to introduce amino acid modifications, and alternatively its affinity for Fc γ R may be increased by introducing additional modifications. The Fc γ R-binding domain may include Schlapschly et al (Protein Eng. Des. Sel.22 (3): 175-.

In one embodiment of disclosure a, the initial Fc γ R-binding domain preferably comprises, for example, a (human) IgG Fc region or a (human) IgG constant region. Any Fc region or constant region can be used as the initial Fc region or initial constant region, as long as the initial Fc region or variant of the initial constant region is capable of binding to human fcyr at neutral pH ranges. The Fc region or constant region further obtained by further modifying the starting Fc region or starting constant region whose one or more amino acid residues have been modified from the Fc region or constant region may also be suitably used as the Fc region or constant region of disclosure a. The initial Fc region or initial constant region may refer to the polypeptide itself, a composition comprising the initial Fc region or initial constant region, or an amino acid sequence encoding the initial Fc region or initial constant region. The starting Fc region or starting constant region may comprise a known Fc region or known constant region produced by recombinant techniques. The origin of the initial Fc region or initial constant region is not limited, and it may be obtained from any organism other than a human animal or a human. Furthermore, the initial Fc γ R-binding domain may be obtained from a cynomolgus monkey, marmoset, macaque, chimpanzee, or human. The starting Fc region or starting constant region may preferably be obtained from human IgG 1; however, it is not limited to a specific IgG class. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as an appropriate starting Fc γ R-binding domain, and it also means that, within the scope of the disclosure a described herein, an Fc region or constant region of IgG type or subclass derived from any organism can be preferably used as the starting Fc region or starting constant region. Examples of natural IgG variants or modified forms are described in well known literature such as Strohl, curr, opin, biotechnol.20 (6): 685-; presta, curr, opin, immunol.20 (4): 460-470 (2008); davis et al, Protein eng.des.sel.23 (4): 195- > 202 (2010); WO2009/086320, WO 2008/092117; WO 2007/041635; and WO2006/105338, but is not limited thereto.

In one embodiment, the amino acid residues of the initial Fc γ R-binding domain, the initial Fc region, or the initial constant region may 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; inserting one or more amino acid residues into an amino acid residue in the initial Fc region or the initial constant region; or one or more amino acid residues are deleted from those of the starting Fc region or the starting constant region. The amino acid sequence of the modified Fc region or constant region is preferably an amino acid sequence that contains at least a portion of an Fc region or constant region that may not be naturally occurring. The variant must have less than 100% sequence identity or similarity to the starting Fc region or starting constant region. For example, a variant has from about 75% to less than 100%, more preferably from about 80% to less than 100%, even more preferably from about 85% to less than 100%, yet more preferably from about 90% to less than 100%, and yet more preferably from 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 and the starting Fc region or starting constant region of disclosure a.

In one embodiment, the Fc region or constant region (which may be comprised in the antibody of disclosure a) having fcyr-binding activity at acidic pH ranges and/or at neutral pH ranges may be obtained by any method. In particular, Fc region or constant region variants having Fc γ R-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. The IgG antibody Fc region or IgG antibody constant region suitable for modification may include, for example, an Fc region or constant region of human IgG (IgG1, IgG2, IgG3, or IgG4, or variants thereof), and a mutant spontaneously produced therefrom. As for the Fc region or constant region of human IgG1, human IgG2, human IgG3, or human IgG4 antibody, 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 in publication A. In particular, for the human IgG1 sequence, the amino acid sequence according to EU numbering positions 356 to 358 may be DEL or EEM.

In another embodiment within the scope of disclosure a, modifications of other amino acids are not limited as long as the variant has Fc γ R-binding activity in the neutral pH range. One or more amino acid positions of the modification are reported in, for example, 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 WO 2014/030750.

The sites of amino acid modification in the constant region or Fc region to increase Fc γ R-binding activity in the neutral pH range may include, for example, one or more positions selected from the group consisting of: 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, 324, 325, 326, 327, 328, 329, 330, 335, 332, 333, 334, 427, 337, 378, 339, 385, 376, 377, 382, 35, 428, 434, 047752, and numbering according to EU, 2, 142, or EU, 2, and EU, as shown in WO2, 48, 377, 21, 48, and 21. The modification of the amino acid residues can increase binding of the Fc region or constant region of the IgG antibody to Fc γ R under neutral pH conditions. WO2013/047752 describes, as preferred modifications in an IgG-type constant region or Fc region, for example, modifications of one or more amino acid residues selected from the group consisting of: the amino acid modification at position 221 is Lys or Tyr; the amino acid modification at position 222 is any one of Phe, Trp, Glu, and Tyr; a modification of the amino acid at position 223 to any one of Phe, Trp, Glu, and Lys; the amino acid modification at position 224 to any one of Phe, Trp, Glu, and Tyr; the amino acid modification at position 225 is any one of Glu, Lys, and Trp; the amino acid modification at position 227 is any one of Glu, Gly, Lys, and Tyr; the amino acid modification at position 228 is any one of Glu, Gly, Lys, and Tyr; the amino acid modification at position 230 is any one of Ala, Glu, Gly, and Tyr; the amino acid modification at position 231 is any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid modification at position 232 is any one of Glu, Gly, Lys, and Tyr; the amino acid modification at position 233 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 234 is 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 modification at position 235 is 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 modification at position 236 is any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 237 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 238 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 239 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, gin, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 240 to any one of Ala, Ile, Met, and Thr; the amino acid modification at position 241 is any one of Asp, Glu, Leu, Arg, Trp, and Tyr; the amino acid modification at position 243 is any one of Glu, Leu, Gln, Arg, Trp, and Tyr; the amino acid modification at position 244 is His; the amino acid modification at position 245 is Ala; the amino acid modification at position 246 is any one of Asp, Glu, His, and Tyr; the amino acid modification at position 247 is any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; the amino acid modification at position 249 is any one of Glu, His, Gln, and Tyr; the amino acid modification at position 250 is Glu or Gln; the amino acid at position 251 is modified to Phe; a modification of the amino acid at position 254 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 modification at position 256 is any one of Ala, Met, and Pro; the amino acid modification at position 258 is any one of Asp, Glu, His, Ser, and Tyr; the amino acid modification at position 260 is any one of Asp, Glu, His, and Tyr; a modification of the amino acid at position 262 to any one of Ala, Glu, Phe, Ile, and Thr; the amino acid modification at position 263 is any one of Ala, Ile, Met, and Thr; the amino acid modification at position 264 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, gin, Arg, Ser, Thr, Trp, and Tyr; the amino acid modification at position 265 is 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 modification at position 266 is any one of Ala, Ile, Met, and Thr; the amino acid modification at position 267 is any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, gin, Arg, Thr, Val, Trp, and Tyr; an amino acid modification at position 268 to any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp; the amino acid modification at position 269 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 270 is any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid modification at position 271 is 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 modification at position 272 is any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 273 is Phe or Ile; the amino acid modification at position 274 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 275 is Leu or Trp; the amino acid modification at position 276 is any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 278 is any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, gin, Arg, Ser, Thr, Val, and Trp; the amino acid modification at position 279 is Ala; the amino acid modification at position 280 is any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid modification at position 281 is any one of Asp, Lys, Pro, and Tyr; the amino acid modification at position 282 to any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid modification at position 283 is any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid modification at position 284 is any one of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid modification at position 285 is any one of Asp, Glu, Lys, gin, Trp, and Tyr; the amino acid modification at position 286 to any one of Glu, Gly, Pro, and Tyr; the amino acid modification at position 288 is any one of Asn, Asp, Glu, and Tyr; the amino acid modification at position 290 is any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr; the amino acid modification at position 291 is any one of Asp, Glu, Gly, His, Ile, Gln, and Thr; the amino acid modification at position 292 is any one of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid modification at position 293 is any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 294 is any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 295 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 296 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val; the amino acid modification at position 297 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; an amino acid modification at position 298 to any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid modification at position 299 is 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 modification at position 300 is any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, gin, Arg, Ser, Thr, Val, and Trp; the amino acid modification at position 301 is any one of Asp, Glu, His, and Tyr; the amino acid modification at position 302 is Ile; the amino acid modification at position 303 is any one of Asp, Gly, and Tyr; the amino acid modification at position 304 is any one of Asp, His, Leu, Asn, and Thr; the amino acid modification at position 305 is any one of Glu, Ile, Thr, and Tyr; the amino acid modification at position 311 is any one of Ala, Asp, Asn, Thr, Val, and Tyr; the amino acid at position 313 is modified to Phe; the amino acid modification at position 315 is Leu; the amino acid modification at position 317 is Glu or Gln; the amino acid modification at position 318 is any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid modification at position 320 is any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 322 is any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 323 is Ile; the amino acid modification at position 324 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid modification at position 325 is 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 modification at position 326 is any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 327 is any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid modification at position 328 is 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 modification at position 329 is any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 330 is any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid modification at position 331 is any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid modification at position 332 is 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 modification at position 333 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid modification at position 334 is any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; the amino acid modification at position 335 is any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid modification at position 336 to any one of Glu, Lys, and Tyr; the amino acid modification at position 337 is any one of Glu, His, and Asn; the amino acid modification at position 339 to any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr; the amino acid modification at position 376 to Ala or Val; the amino acid modification at position 377 is Gly or Lys; an amino acid modification at position 378 to Asp; the amino acid modification at position 379 is Asn; an amino acid modification at position 380 to any one of Ala, Asn, and Ser; the amino acid modification at position 382 is Ala or Ile; the amino acid modification at position 385 to Glu; the amino acid modification at position 392 is Thr; the amino acid modification at position 396 is Leu; the amino acid at position 421 is modified to Lys; the amino acid modification at position 427 is Asn; the amino acid modification at position 428 is Phe or Leu; an amino acid modification at position 429 to Met; the amino acid modification at position 434 to Trp; the amino acid modification at position 436 is Ile; and amino acid modification at position 440 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 only an amino acid at one position or an amino acid at two or more positions. Combinations of amino acid modifications at two or more positions are shown in table 5 of WO 2013/047752. Modifications of these amino acid residues may also be appropriately introduced into the antibody of disclosure a.

In one embodiment, the binding activity of (the Fc γ R-binding domain of) an antibody of disclosure a to (a human) Fc γ R, such as any one or more of Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa and Fc γ RIIIb, may be higher than the binding activity of (the Fc region or constant region of) a native IgG or a reference antibody comprising the starting Fc region or starting constant region. For example, the Fc γ R-binding activity of (the Fc γ R-binding domain of) the antibody of disclosure a may 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 of the Fc γ R-binding activity of a reference antibody, 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.

In another embodiment, the increased level of binding activity (in the neutral pH range) for inhibitory Fc γ R (Fc γ RIIb-1 and/or Fc γ RIIb-2) may be greater than the increased level of binding activity (in the neutral pH range) for activating Fc γ R (Fc γ RIA: Fc γ RIb; Fc γ RIc; Fc γ RIIIa (including allotype V158); Fc γ RIIIa (including allotype F158); Fc γ RIIIb (including allotype Fc γ RIIIb-NA 1); Fc γ RIIIb (including allotype Fc γ RIIIb-NA 2); Fc γ RIIa (including allotype H131); or Fc γ RIIa (including allotype R131)).

In one embodiment, the antibodies of disclosure a can have binding activity to Fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2).

In one embodiment, preferred Fc γ R-binding domains of disclosure a also include, for example, Fc γ R-binding domains (Fc γ R-binding domains with selective Fc γ R-binding activity) that have greater binding activity for a particular Fc γ R than for other Fc γ rs. In the case of using an antibody (or Fc region as an fcyr-binding domain), a single antibody molecule may bind only a single fcyr molecule. Thus, a single antibody molecule in a state of binding to an inhibitory Fc γ R cannot bind to other activating Fc γ rs, and a single antibody molecule in a state of binding to an activating Fc γ R cannot bind to other activating Fc γ rs or inhibitory Fc γ rs.

As described above, activating Fc γ R preferably includes, for example, Fc γ RI (CD64) such as Fc γ RIa, Fc γ RIb, or Fc γ RIc; and Fc γ RIII (CD16) such as Fc γ RIIIa (e.g. allotype V158 or F158) or Fc γ RIIIb (e.g. allotype Fc γ RIIIb-NA1 or Fc γ RIIIb-NA 2). Meanwhile, the inhibitory Fc γ R preferably includes, for example, Fc γ RIIb (such as Fc γ RIIb-1 or Fc γ RIIb-2).

In one embodiment, an Fc γ R-binding domain with higher binding activity for inhibitory Fc γ R than for activating Fc γ R can be used as the selective Fc γ R-binding domain comprised in the antibody of disclosure a. The selective Fc γ R-binding domain may include, for example, an Fc γ R-binding domain having greater binding activity to Fc γ RIIb (such as Fc γ RIIb-1 and/or Fc γ RIIb-2) than to any one or more of the activating Fc γ rs selected from the group consisting of: fc γ RI (CD64) such as, for example, Fc γ RIa, Fc γ RIb, or Fc γ RIc; fc γ RIII (CD16) such as Fc γ RIIIa (e.g., allotype V158 or F158) or Fc γ RIIIb (e.g., Fc γ RIIIb-NA1 or Fc γ RIIIb-NA 2); fc γ RII (CD32) such as Fc γ RIIa (including allotype H131 or R131); and Fc γ RIIc.

Further, whether an Fc γ R-binding domain has selective binding activity can be evaluated by comparing the binding activity to each Fc γ R determined by the above-described method, for example, by comparing a value (ratio) obtained by dividing a KD value for an activating Fc γ R by a KD value for an inhibiting Fc γ R, more specifically by comparing Fc γ R selectivity indexes shown in the following equation 1:

Equation 1 Fc γ R selectivity index (KD value for activating Fc γ R/KD value for inhibiting Fc γ R)

In equation 1, the KD value for an activating Fc γ R refers to the KD value for one or more of: fc γ RIa; fc γ RIb; fc γ RIc; fc γ RIIIa (including allotypes V158 and/or F158); fc γ RIIIb (including Fc γ RIIIb-NA1 and/or Fc γ RIIIb-NA 2); fc γ RIIa (including allotypes H131 and/or R131); and Fc γ RIIc; and the KD value for inhibitory Fc γ R refers to the KD value for Fc γ RIIb-1 and/or Fc γ RIIb-2. The activating Fc γ R and the inhibiting Fc γ R used to determine the KD values may be selected in any combination. For example, a value (ratio) determined by dividing the KD value for Fc γ RIIa (including allotype H131) by the KD value for Fc γ RIIb-1 and/or Fc γ RIIb-2 may be used, without being limited thereto.

The Fc γ R selectivity index may 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, 280 or higher, 270 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 more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, 8000 or more, 8500 or more, 9000 or more, 9500 or more, 10000 or more, or 100000 or more; it is not limited thereto.

In one embodiment, a human IgG (IgG1, IgG2, IgG3, or IgG4) which is an Fc region variant or constant region variant (antibody comprising the same) having Asp or Glu as the amino acid at position 238 or 328, respectively, according to EU numbering, may be preferably used as the antibody of publication A comprising the Fc region variant or constant region variant, because the optical properties of the optical fibers are improved as described in particular in WO2013/125667, WO2012/115241, and WO2013/047752, it has greater binding activity to Fc γ RIIb-1 and/or Fc γ RIIb-2 than to Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa (including allotype V158), Fc γ RIIIa (including allotype F158), Fc γ RIIIb (including allotype Fc γ RIIIb-NA1), Fc γ RIIIb (including allotype Fc γ RIIIb-NA2), Fc γ RIIa (including allotype H131), Fc γ RIIa (including allotype R131), and/or Fc γ RIIc. In this embodiment, the antibodies of disclosure A have binding activity to all activating Fc γ Rs (herein, selected from the group consisting of Fc γ RIA, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb, Fc γ RIIa) and Fc γ RIIb, and their Fc γ RIIb-binding activity is maintained or increased and/or their binding activity to all activating Fc γ Rs is decreased as compared to a reference antibody containing a native IgG constant region or a native IgG Fc region.

In one embodiment, for antibodies of disclosure a containing a variant Fc region or a variant constant region, their binding activity to Fc γ RIIb may be maintained or increased and their binding activity to Fc γ RIIa (type H) and Fc γ RIIa (type R) may be decreased compared to a reference antibody having a constant region or Fc region of a native IgG. The antibodies can have increased binding selectivity for Fc γ RIIb over Fc γ RIIa.

Within the scope of disclosure a described herein, the degree of "reduced binding activity to all activating Fc γ rs" 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.

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

Within the scope of disclosure a described herein, the "reduced binding activity to Fc γ RIIa (type H) and Fc γ RIIa (type R)" or "reduced binding activity to Fc γ RIIa (type H) and Fc γ RIIa (type R)" degree 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.

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

The above modifications may be in a single position alone or in combination in two or more positions. Alternatively, the preferred modifications may include, for example, those shown in tables 14 to 15, 17 to 24, and 26 to 28 of WO2013/047752, e.g., variants of a human constant region or 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; further, one or more of positions 233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396 according to EU numbering may be replaced. In this case, variants may include, but are not limited to, variants of a human constant region or human Fc region comprising one or more of:

asp at position 233, Tyr at position 234, Asp at position 237, Ile at position 264, Glu at position 265, Phe, Met, and Leu at position 266, any of Ala, Glu, Gly, and gin at position 267, Asp or Glu at position 268, Asp at position 269, Asp at position 272, Phe, Ile, Met, Asn, and gin, 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 at position 333, any of Lys, and Arg at position 296, and any of Asp, Glu, Phe, Ile, Lys, Leu, Met, gin, Arg, and Tyr at position 396 (according to EU numbering).

In an alternative embodiment, an antibody of disclosure a containing an Fc region variant or constant region variant may have retained or increased binding activity to Fc γ RIIb and reduced binding activity to Fc γ RIIa (type H) and Fc γ RIIa (type R) compared to a reference antibody containing a constant region or Fc region of a native IgG. Preferred sites for amino acid substitutions of such variants may be as reported in WO2014/030728, e.g., an amino acid at position 238 according to EU numbering and at least one amino acid at a position selected from the group consisting of: positions 233, 234, 235, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409, and 419 (according to EU numbering).

More preferably, the variant may have Asp at position 238 according to EU numbering and at least one amino acid selected from the group of amino acids 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 at position 266, Leu, or Met, Ala, Glu, Gly at position 267, or Gln, Asp at position 268, Gln, or Glu, Asp at position 269, Gly at position 271, Asp at position 272, Phe, Ile, Met, Asn, Pro, or Gln, Gln at position 274, Asp or Phe at position 296, Ala or Asp at position 326, Gly at position 327, Lys at position 330, Arg, or Ser, Ser at position 331, Lys at position 332, Arg, Ser, or Thr, Lys, Arg, Ser, or Thr at position 333, Arg, Ser at position 334, or Thr, 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, Arg at position 409, and Glu at position 419 (according to EU numbering).

In an alternative embodiment, an antibody of disclosure a comprising an Fc region variant or constant region variant may have retained binding activity to Fc γ RIIb and reduced binding activity to all activating Fc γ Rs, Fc γ RIIa (type R) compared to a reference antibody comprising a constant region or Fc region of a native IgG. Preferred sites for amino acid substitutions for such variants may be at least one amino acid at a position selected from positions 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 according to EU numbering, in addition to the amino acid at position 238, as reported in WO2014/163101, for example according to EU numbering. More preferably, the variant may have Asp at position 238 according to EU numbering, and at least one amino acid selected from the group of amino acids 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).

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

Within the scope of disclosure a described herein, the aforementioned "reduced level of binding activity to all activating Fc γ Rs, particularly Fc γ RIIa (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.

WO2014/030750 also reports variants of mouse constant and Fc regions. In one embodiment, an antibody of disclosure a or B may comprise such a variant.

Within the scope of the disclosures A and B described herein, unlike Fc γ R, FcRn, and in particular human FcRn, belonging to the immunoglobulin superfamily, are structurally similar to polypeptides of the major histocompatibility complex type I (MHC) and exhibit sequence identity of 22% to 29% with MHC class I molecules (Ghetie et al, Immunol. today 18(12), 592. 598 (1997)). FcRn is expressed as a heterodimer consisting of a soluble β or light chain (β 2 microglobulin) complexed with transmembrane α or heavy chain. Like MHC, the α chain of FcRn contains three extracellular domains (α 1, α 2, and α 3), and its short cytoplasmic domain links the protein to the cell surface. The α 1 and α 2 domains interact with the FcRn-binding domain of the Fc region of an antibody (Raghavan et al, Immunity 1: 303-315 (1994)).

FcRn is expressed in the maternal placenta and yolk sac of mammals and is involved in maternal to fetal IgG transfer. Furthermore, in the small intestine of neonatal rodents expressing FcRn, FcRn is involved in the transfer of mature IgG from ingested colostrum or milk across the brush border epithelium. FcRn is expressed in a variety of other tissues and endothelial cell systems of various species. FcRn is also expressed in adult vascular endothelium, muscle vasculature, and hepatic sinusoid capillaries. FcRn is thought to play a role in the maintenance of plasma IgG concentrations by binding to IgG and recycling IgG to serum. In general, FcRn binding to IgG molecules is strictly pH dependent. The best binding was observed in the acidic pH range below 7.0.

The oligonucleotide and amino acid sequences of human FcRn can be derived, for example, from the precursors (containing signal sequences) shown in NM _004107.4 and NP _004098.1, respectively, (RefSeq accession numbers shown in parentheses).

The precursor forms a complex with human beta 2-microglobulin in vivo. Thus, by using known recombinant expression techniques, soluble human FcRn capable of forming a complex with human β 2-microglobulin can be prepared for appropriate use in various experimental systems. The soluble human FcRn can be used to assess FcRn-binding activity of an antibody or Fc region variant. In the disclosure a or B, FcRn is not particularly limited as long as it is a form capable of binding to FcRn-binding domain; however, a preferred FcRn may be a human FcRn.

Within the scope of the disclosures a and B described herein, where the 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 with activity to bind FcRn directly or indirectly. Such domains include, but are not limited to, the Fc region of an IgG-type immunoglobulin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides, and anti-FcRn scaffold molecules (which have activity for directly binding to FcRn), and molecules that bind to IgG or albumin (which have activity for indirectly binding to FcRn. in disclosures A or B, it is also possible to use domains that have FcRn-binding activity in the acidic pH range and/or in the neutral pH range The neutral pH range has FcRn-binding activity. Alternatively, the amino acids of the domain that initially have FcRn-binding activity in the acidic pH range and/or in the neutral pH range may be modified to further increase its FcRn-binding activity. FcRn-binding activity in the acidic pH range and/or in the neutral pH range can be compared to before and after amino acid modifications to find amino acid modifications of interest to the FcRn-binding domain.

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

Within the scope of the disclosures a and B described herein, whether the FcRn-binding activity of an antibody or Fc region (variant) is increased, (substantially) retained, or decreased, as compared to the antibody or Fc region (variant) before modification can be assessed by known methods, such as those described in the examples herein, and for example, BIACORE, Scatchard curves and flow cytometers (see WO 2013/046722). The extracellular domain of human FcRn can be used as a soluble antigen in these assays. In the measurement of FcRn-binding activity of an antibody or Fc region (variant), the conditions (other than pH) may be appropriately selected by those skilled in the art. The assay may, for example, be performed in MES buffer and at 37 ℃, as described in WO 2009/125825. The FcRn-binding activity of an antibody or Fc region (variant) can be assessed, for example, by loading FcRn as an analyte on a chip on which the antibody is immobilized.

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

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

In one embodiment, where the antibodies of disclosure a or B have FcRn-binding activity, they 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 a binding activity or affinity for FcRn at acidic pH and/or neutral pH, and it may be a domain having an activity of directly or indirectly binding FcRn. In a specific embodiment, it may be preferred that the antibody of disclosure a or B has, e.g., increased FcRn-binding activity under neutral pH conditions compared to a reference antibody containing the constant region of a native IgG (see WO 2013/046722). From the viewpoint of comparing FcRn-binding activities of both, it may be preferable, without limitation, that the antibody of disclosure a or B and a reference antibody containing a constant region of a natural IgG have the same amino acid sequence in a region (e.g., a variable region) other than the constant region modified at one or more amino acid residues of the antibody of disclosure a or B.

In one embodiment, within the scope of disclosure a described herein, where the antibody of disclosure a has increased FcRn-binding activity under neutral pH conditions, without being limited by a particular theory, the antibody of disclosure a may have a combination of any two or more of the following properties: the property of shuttling between plasma and endosomes and repeatedly binding multiple antigens as a single antibody molecule by having an ionic concentration-dependent antigen binding domain; the property of being taken up rapidly into cells by having an increased pI and an increased positive charge in the whole antibody; and the property of being rapidly taken up into cells by having an increased FcRn-binding activity under neutral pH conditions. Thus, the antibody half-life in plasma may be further shortened, or the binding activity of the antibody to the extracellular matrix may be further increased, or the removal of antigen from plasma may be further facilitated. One skilled in the art can determine the optimal pI values for the antibodies of disclosure a to take advantage of these properties.

Within the scope of the disclosures A and B described herein, the activity of native human IgG1 binding to human FcRn is KD 1.7. mu.M in the acidic pH range (pH 6.0) and the activity is barely detectable in the neutral pH range, according to Yeung et al (J.Immunol.182: 7663-7671 (2009)). Therefore, in order to increase FcRn-binding activity in the neutral pH range, as the antibody of the 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 μ M or greater in the acidic pH range and whose human FcRn-binding activity is comparable to or greater than native human IgG in the neutral pH range; preferably an antibody or constant region variant or Fc region variant whose human FcRn-binding activity is KD 2.0 μ M or greater in the acidic pH range and whose human FcRn-binding activity is KD40 μ M or greater in 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. mu.M or more in the acidic pH range and whose human FcRn-binding activity is KD 15. mu.M or more in the neutral pH range. The KD value was determined by the method described in Yeung et al (J.Immunol.182: 7663-7671(2009) (by immobilizing the antibody on the chip and loading human FcRn as analyte)).

Within the scope of the disclosures a and B described herein, a domain of any structure that binds FcRn can be used as an FcRn-binding domain. In this case, the FcRn-binding domain may be generated without the need to introduce amino acid modifications, or the affinity for FcRn may be increased by introducing additional modifications.

Within the scope of the disclosures a and B described herein, the initial FcRn-binding domain may comprise, for example, the Fc region or constant region of a (human) IgG. Any Fc region or constant region may be used as an initiating Fc region or initiating constant region, as long as the variant of the initiating Fc region or initiating constant region is capable of binding FcRn at an acidic pH range and/or at a neutral pH range. Alternatively, an Fc region or constant region obtained by further modifying the starting Fc region or starting constant region, the amino acid residues of which have been modified from the Fc region or constant region, may also be suitably used as the Fc region or constant region. The initial Fc region or initial constant region may include an Fc region known to be produced by recombination. The initial Fc region or initial constant region may refer to the polypeptide itself, a composition containing the initial Fc region or initial constant region, or an amino acid sequence encoding the initial Fc region or initial constant region, depending on the context. The origin of the starting Fc region or starting constant region is not limited, and it may be obtained from any organism other than a human or animal. Furthermore, the starting FcRn-binding domain may be obtained from cynomolgus monkey, velvet, rhesus monkey, chimpanzee and human. The starting Fc region or starting constant region may be obtained from human IgG1, but is not limited to any particular IgG class. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be used as an appropriate starting FcRn-binding domain, and the Fc region or constant region of an IgG class or subclass derived from any organism can be used as a starting Fc region or as a starting constant region. Examples of natural IgG variants or modified forms are described in, e.g., Strohl, curr, opin, biotechnol.20 (6): 685-; presta, curr, opin, immunol.20 (4): 460-470 (2008); davis et al, Protein eng.des.sel.23 (4): 195-; WO 2007/041635; and WO 2006/105338).

Within the scope of the disclosure a and B described herein, the amino acid residues of the initial FcRn-binding domain, initial Fc region, or initial constant region may contain, for example, one or more mutations: for example, substitution mutations using amino acid residues that differ from the amino acid residues in the starting Fc region or the starting constant region; inserting one or more amino acid residues into an amino acid residue in the initial Fc region or the initial constant region; or one or more amino acid residues are deleted from the amino acid residues of the starting Fc region or the starting constant region. The amino acid sequence of the modified Fc region or constant region may preferably be an amino acid sequence containing at least a portion of an Fc region or constant region that does not naturally occur. 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 from about 75% to less than 100%, more preferably from about 80% to less than 100%, even more preferably from about 85% to less than 100%, yet more preferably from about 90% to less than 100%, and still more preferably from 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 differs between the modified Fc region or constant region and the starting Fc region or starting constant region of disclosure a or B.

The Fc region or constant region having FcRn-binding activity in the acidic pH range and/or in the neutral pH range within the scope of the disclosures a and B described herein may be obtained by any method. In particular, variants of the Fc region or constant region having FcRn-binding activity in the acidic pH range and/or in the neutral pH range may be obtained by modifying the amino acids of a human IgG-type antibody which may be used as the starting Fc region or starting constant region. Suitable IgG-type antibody Fc regions or constant regions for modification include, for example, the Fc region or constant region of human IgG (IgG1, IgG2, IgG3, and IgG4, and variants thereof), and mutants spontaneously produced therefrom are also included in the IgG Fc region or constant region. As for the Fc region or constant region 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 publication A or B. In particular, for the human IgG1 sequence, the amino acid sequence according to EU numbering positions 356 to 358 may be DEL or EEM.

In one embodiment of the disclosure a or B, the modification to other amino acids is not particularly limited as long as the resulting variant has FcRn-binding activity in the acidic pH range and/or in the neutral pH range, and preferably in the neutral pH range. Sites for modification of amino acids to increase FcRn-binding activity under neutral pH conditions are described, for example, in WO 2013/046722. The modification site includes, 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 WO 2013/046722. WO2013/046722 also describes, as part of preferred modifications in the Fc region or constant region, for example, modifications of one or more amino acids selected from the group consisting of: the amino acid modification at position 256 is Pro, the amino acid modification at position 280 is Lys, the amino acid modification at position 339 is Thr, the amino acid modification at position 385 is His, the amino acid modification at position 428 is Leu, and the amino acid modification at position 434 is Trp, Tyr, Phe, Ala, or His (numbering according to EU). The number of amino acids to be modified is not particularly limited, and modification may be performed at a single position alone or at two or more positions. Modification of these amino acid residues may enhance FcRn binding of the Fc region or constant region of IgG-type antibodies under neutral pH conditions. Modifications of these amino acid residues may also be suitably introduced into the antibodies of disclosure a or B.

In further or alternative embodiments, it is also possible to use appropriate amino acid modification sites for increasing FcRn-binding activity under acidic pH conditions. Among such modification sites, one or more modification sites that allow for an increase in FcRn binding may also be suitable for use in disclosure a or B in the neutral pH range. Such modification sites include, for example, those reported in WO2011/122011, WO2013/046722, WO2013/046704, and WO 2013/046722. The amino acid positions and modified amino acid types allowing for such modification of the constant or Fc region of human IgG-type antibodies are reported in table 1 of WO 2013/046722. WO2013/046722 also describes, particularly preferred, modification sites in the constant region or Fc region, e.g. 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 in the neutral pH range. WO2013/046722 also describes, as part of preferred modifications in an IgG-type constant region or Fc region, for example, modifications of one or more amino acid residues selected from the group consisting of: (a) the amino acid modification at position 237 to Met; (b) the amino acid modification at position 238 to Ala; (c) the amino acid modification at position 239 to Lys; (d) the amino acid modification at position 248 to Ile; (e) an amino acid modification at position 250 to any one of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, and Tyr; (f) the amino acid modification at position 252 to any one of Phe, Trp, and Tyr; (g) the amino acid modification at position 254 is Thr; (h) the amino acid modification at position 255 to Glu; (i) the amino acid modification at position 256 is any one of Asp, Glu, and Gln; (j) the amino acid modification at position 257 is any one of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, and Val; (k) the amino acid modification at position 258 is His; (l) The amino acid modification at position 265 to Ala; (m) the amino acid at position 270 is modified to Phe; (n) the amino acid modification at position 286 to Ala or Glu; (o) the amino acid modification at position 289 is His; (p) modification of the amino acid at position 297 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 modification at position 305 is Ala; (t) the amino acid modification at position 307 is any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; (u) modification of the amino acid at position 308 to any one of Ala, Phe, Ile, Leu, Met, Pro, Gln, and Thr; (v) the amino acid modification at position 309 is any one of Ala, Asp, Glu, Pro, and Arg; (w) the amino acid modification at position 311 is any one of Ala, His, and Ile; (x) The amino acid modification at position 312 is Ala or His; (y) the amino acid at position 314 is modified to Lys or Arg; (z) the amino acid modification at position 315 is Ala or His; (aa) modification of the amino acid at position 317 to Ala; (ab) the amino acid modification at position 325 to Gly; (ac) the amino acid modification at position 332 to Val; (ad) the amino acid modification at position 334 is Leu; (ae) the amino acid modification at position 360 is His; (af) the amino acid modification at position 376 to Ala; (ag) an amino acid modification at position 380 to Ala; (ah) the amino acid modification at position 382 is Ala; (ai) the amino acid at position 384 is modified to Ala; (aj) the amino acid modification at position 385 is 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) an amino acid modification at position 389 to Ala or Ser; (an) the amino acid modification at position 424 is Ala; (ao) modification of the amino acid at position 428 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 modified to Lys; (aq) amino acid modifications at position 434 to Ala, Phe, His, Ser, Trp, and Tyr; and (ar) the amino acid modification at position 436 is His (according to EU numbering). The number of amino acids to be modified is not particularly limited, and the modification may be performed at a single position alone 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 WO 2013/046722. Modifications of these amino acid residues may also be appropriately introduced into the antibodies of publications a and B.

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

In one embodiment, the amino acid sequence to be modified in the antibody of disclosure a or B may preferably comprise a human sequence (a sequence present in a naturally human antibody) so as not to increase the immunogenicity of the antibody when the antibody is administered in vivo (preferably, into a human). Alternatively, after modification, mutations may be introduced at positions other than the amino acid modification site in such a manner 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, Ono et al, mol. 387-395 (1999). Humanization Methods are known in the art and include, but are not limited to, Methods 36 (1): 43-60 (2005).

In one embodiment, the framework region sequences (also referred to as "FR sequences") of the heavy and/or light chain variable regions of the antibodies of disclosure a or B may comprise human germline framework region sequences. When the framework region sequences are entirely human germline sequences, the antibody is expected to elicit little or no immunogenic response when administered to a human (e.g., to treat or prevent a disease).

The FR sequences may 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 used as appropriate in the disclosure A or B. 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-.

Fully human VH sequences may 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 VH 1-69); subgroup VH2 (e.g., VH2-5, VH2-26, and VH 2-70); 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 VH 3-74); subgroup VH4(VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, and VH 4-61); subgroup VH5(VH 5-51); subgroup VH6(VH 6-1); or subgroup VH7(VH7-4 and VH 7-81). These are also described, for example, in Matsuda et al (J.Exp.Med.188: 1973-1975(1998)), and can be designed by those skilled in the art appropriately based on the information of these sequences. It is also preferable to use other completely human FR sequences or sequences of regions corresponding thereto.

Fully human vk sequences may 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 as subgroup Vk 1; a1, a2, A3, a5, a7, a17, a18, a19, a23, O1, and O11, which are classified as subgroups Vk 2; a11, a27, L2, L6, L10, L16, L20, and L25, which are classified as subgroups Vk 3; b3, subgroup Vk 4; b2 (also referred to as "Vk 5-2"), classified as subgroup Vk 5; or A10, A14, and A26, which are classified as subgroups Vk6(Kawasaki et al (Eur. J. Immunol.31: 1017-1028 (2001)); hopper Seyler biol. chem.374: 1001-1022 (1993)); Brensing-Kuppers et al (Gene 191: 173-181 (1997)).

Fully human V λ sequences may 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 as subgroups VL 1; v2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19, which are classified as subgroups VL 2; v3-2, V3-3, and V3-4, which are classified as subgroups VL 3; v4-1, V4-2, V4-3, V4-4, and V4-6, which are classified as subgroups VL 4; or V5-1, V5-2, V5-4, and V5-6, which are classified as subgroup VL5(Kawasaki et al Genome Res.7: 250-261 (1997)).

Typically, these FR sequences differ from each other at one or more amino acid residues. These FR sequences can be used to modify the amino acid residues of antibodies. Fully human FR sequences that can be used for modification also include, for example, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (see, for example, Kabat et al (1991) supra; Wu et al (J.exp. Med.132: 211-.

Within the context of the disclosure a and B described herein, a "flexible residue" may refer to an alteration of an amino acid residue present at a position that exhibits high amino acid diversity (where the light or heavy chain variable region has a plurality of different amino acids when compared to the amino acid sequence of a known and/or native antibody or antigen binding domain). Locations that show high diversity are usually located in the CDRs. The data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991), can effectively determine locations with high diversity in known and/or natural antibodies. In addition, multiple databases on the network (vbase. mrc-cpe. cam. ac. uk/, bioif. org. uk/abs/index. html) provide a collection of many human light and heavy chain sequences and their locations. These sequence and position information are used to determine the position of the flexible residues. Without limitation, for example, a position can be judged to exhibit (high) diversity when the amino acid residue at that position has a variability of, preferably, 2 to 20, 3 to 19, 4 to 18, 5 to 17, 6 to 16, 7 to 15, 8 to 14, 9 to 13, or 10 to 12 amino acid residues.

In one embodiment, it is understood that where the antibody of disclosure a or B contains all or a portion of a light chain variable region and/or a heavy chain variable region, the antibody may contain one or more suitable flexible residues, if desired. For example, the heavy chain and/or light chain variable region sequences selected to have FR sequences that originally contain amino acid residues that alter the antigen-binding activity of the antibody according to the conditions of ion concentration (hydrogen ion concentration or calcium ion concentration) may be designed to contain amino acid residues other than these amino acid residues. In this case, for example, the number and position of the flexible residues can also be determined without being limited to a specific embodiment as long as the antigen-binding activity of the antibody of the disclosure a or B is changed depending on the ion concentration condition. In particular, the CDR sequences and/or FR sequences of the heavy and/or light chains may contain at least one flexible residue. For example, where the ionic concentration is calcium, flexible residues that can be introduced into the light chain variable region sequence (Vk 5-2, supra) include, but are not limited to, one or more of the amino acid residue positions shown in Table 1 or Table 2. Likewise, suitable flexible residues may be introduced, for example, into an ion concentration-dependent or ion concentration-independent antibody containing all or a portion of the light chain variable region and/or the heavy chain variable region, wherein at least one amino acid residue that may be exposed on the surface of the antibody is modified in order to increase the pI.

[ Table 1]

(positions are shown according to Kabat numbering.)

[ Table 2]

(positions are shown according to Kabat numbering.)

In one embodiment, in humanizing the chimeric antibody, the pI of the chimeric antibody is increased by modifying one or more amino acid residues that may be exposed on the surface of the antibody, thereby producing a humanized antibody of either disclosure a or B having a reduced plasma half-life as compared to the chimeric antibody lacking such modification. Modification of amino acid residues that may be exposed on the surface of the humanized antibody may be performed prior to or simultaneously with the humanized antibody. Alternatively, by using a humanized antibody as a starting material, amino acid residues that may be exposed on the surface may be modified to further alter the pI of the humanized antibody.

Adams et al (Cancer Immunol. Immunother.55 (6): 717-727(2006)) reported that humanized antibodies, trastuzumab (trastuzumab) (antigen: HER2), bevacizumab (bevacizumab) (antigen: VEGF), and pertuzumab (pertuzumab) (antigen: HER2), which were humanized using the same human antibody FR sequence, were nearly equivalent in plasma pharmacokinetics. Specifically, it is understood that plasma pharmacokinetics are almost comparable when humanization is performed using the same FR sequences. According to one embodiment of disclosure a, in addition to the humanization step, the antigen concentration in plasma is reduced by increasing the pI of the antibody by modifying amino acid residues that may be exposed on the surface of the antibody. In alternative embodiments of disclosure a or B, human antibodies may be used. The ability of the originally produced human antibody to remove antigen from plasma can be increased by modifying amino acid residues that can be exposed on the surface of the human antibody (produced from a human antibody library, a human antibody-producing mouse, a recombinant cell, etc.) and increasing the pI of the human antibody.

In one embodiment, the antibody of disclosure a may contain a modified sugar chain. Antibodies having modified sugar chains include, for example, antibodies having modified glycosylation (WO99/54342), antibodies lacking fucose (WO 00/61739; WO02/31140, WO 2006/067847; WO2006/067913), and antibodies having bisected GlcNAc sugar chains (WO 02/79255).

In one embodiment, the antibodies of disclosure a or B may be used, for example, in techniques that exhibit increased anti-tumor activity against cancer cells or techniques that facilitate removal of biologically harmful antigens from plasma.

In an alternative embodiment, the disclosure a or B relates to a library of ion concentration-dependent antigen binding domains with increased pI or ion concentration-dependent antibodies with increased pI, as described above.

In an alternative embodiment, the disclosure a or B relates to a nucleic acid (oligonucleotide) encoding the above-described ion concentration-dependent antigen binding domain with increased pI or ion concentration-dependent antibody with increased pI. In one embodiment, the nucleic acid may be obtained using an appropriate known method. For specific embodiments, reference may be made to, for example, WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241, WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, and WO2012/093704, each of which is incorporated herein by reference in its entirety.

In one embodiment, the nucleic acid of disclosure a or B may be an isolated or purified nucleic acid. The nucleic acid encoding the antibody of disclosure a or B may be any gene, and may be DNA or RNA, or other nucleic acid analogs.

Within the disclosures a and B described herein, after modifying the amino acid sequence of an antibody, the amino acid sequence of the antibody before modification may be a known sequence or the amino acid sequence of a newly obtained antibody. For example, antibodies can be obtained from a library of antibodies, or by cloning nucleic acids encoding the antibodies from hybridomas or B cells that produce monoclonal antibodies. Methods for obtaining nucleic acids encoding antibodies from hybridomas can use the following techniques: immunizing by a conventional immunization method using the antigen of interest or cells expressing the antigen of interest as a sensitizing antigen; fusing the obtained immune cells with known parent cells by a conventional cell fusion method; screening cells producing monoclonal antibodies (hybridomas) by conventional screening methods; synthesizing cDNA of a variable region (V region) of the antibody from the mRNA of the obtained hybridoma using reverse transcriptase; and the cDNA is ligated with DNA encoding the constant region (C region) of the target antibody.

Sensitizing antigens useful for obtaining nucleic acids encoding the above-described heavy and light chains include, but are not limited to, intact antigens and incomplete antigens (including haptens that do not exhibit immunogenicity) that are immunogenic. For example, it is possible to use the entire protein under investigation or a partial peptide of said protein. Furthermore, substances consisting of polysaccharides, nucleic acids, lipids and other compositions are known to be possible antigens. Thus, in some embodiments, the antigen of the antibody of disclosure a or B is not particularly limited. Antigens can be prepared, for example, by baculovirus-based methods (see, e.g., WO 98/46777). Hybridomas may be obtained, for example, according to the Methods of g.kohler and c.milstein, Methods enzymol.73: 3-46 (1981)). When the immunogenicity of the antigen is low, immunization can be carried out by linking the antigen to a macromolecule having immunogenicity, such as albumin. Alternatively, if desired, soluble antigens may be prepared by linking the antigen to other molecules. When a transmembrane molecule such as a membrane antigen (e.g., receptor) is used as the antigen, a portion of the extracellular region of the membrane antigen may be used as a fragment, or a cell expressing the transmembrane molecule on its surface may be used as the immunogen.

In some embodiments, antibody-producing cells can be obtained by immunizing an animal with an appropriate sensitizing antigen as described above. Alternatively, antibody-producing cells may be prepared by in vitro immunization of antibody-producing lymphocytes. Various animals can be used for immunization and other conventional antibody production procedures. Commonly used animals include rodents, lagomorphs and primates. Animals may include, for example, rodents such as mice, rats and hamsters; rabbits such as rabbits; and primates (including monkeys such as cynomolgus monkeys, macaques, baboons, and chimpanzees. furthermore, transgenic animals carrying a genome bank of human antibodies (repotoreire) are also known, and these animals can be used to obtain human antibodies (see, for example, WO 96/34096; Mendez et al, nat. Genet.15: 146-156 (1997); WO93/12227, WO92/03918, WO94/02602, WO96/34096, and WO 96/33735). instead of using the transgenic animals, it is also possible to obtain desired human antibodies with antigen binding activity by, for example, sensitizing human lymphocytes in vitro with the desired antigen or cells expressing the desired antigen and then fusing the sensitized lymphocytes with human myeloma cells such as U266 (JP pat. Publ.No. H01-59878).

Animal immunization can be carried out, for example, by appropriately diluting and suspending the sensitizing antigen in Phosphate Buffered Saline (PBS), physiological saline, or the like, and mixing it with an adjuvant to emulsify (if necessary); and then injected intraperitoneally or subcutaneously into the animal. The sensitizing antigen mixed with Freund's incomplete adjuvant may then preferably be administered several times every four to 21 days. Antibody production can be confirmed, for example, by measuring the titer of the antibody of interest in the serum of the animal.

Antibody-producing cells obtained from lymphocytes or animals immunized with the desired antigen can be fused with myeloma cells using conventional fusing agents (e.g., polyethylene glycol) to produce hybridomas (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). If desired, the hybridomas are cultured and expanded, and the binding specificity of the antibodies produced by the hybridomas is assessed by, for example, immunoprecipitation, Radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA). Then, if desired, antibody-producing hybridomas (for which specificity, affinity, or activity has been determined) can also be subcloned by methods such as limiting dilution.

Nucleic acids encoding the selected antibodies can be cloned from hybridomas or antibody-producing cells (sensitized lymphocytes, etc.) using probes that can specifically bind to the antibodies (e.g., oligonucleotides complementary to sequences encoding antibody constant regions). Alternatively, the nucleic acid may be cloned from mRNA using RT-PCR. The heavy and light chains used to produce the antibodies of disclosure a or B may be derived, for example, from antibodies belonging to any of the Ig antibody classes and subclasses, and IgG may be preferred.

In one embodiment, the nucleic acid encoding the amino acid sequence of the heavy chain (whole or part thereof) and/or light chain (whole or part thereof) that make up the antibody of disclosure a or B is modified, e.g., by genetic engineering techniques. Recombinant antibodies, such as chimeric or humanized antibodies, having artificial sequence modifications to, e.g., reduce heterologous antigenicity to humans, can be generated, as appropriate, by modifying, e.g., the nucleotide residues encoding the amino acid sequences associated with components of an antibody, e.g., a mouse, rat, rabbit, hamster, sheep, or camel antibody. The chimeric antibody can be obtained, for example, by ligating a DNA encoding a variable region of a murine antibody with a DNA encoding a constant region of a human antibody and incorporating the ligated DNA coding sequence into an expression vector, and then introducing the resulting recombinant vector into a host to express the gene. A humanized antibody, also known as a reshaped human antibody, is an antibody in which one or more FRs of a human antibody are joined to one or more CDR frames of an antibody isolated from a non-human mammal, such as a mouse, to form a coding sequence. The DNA sequence encoding the humanized antibody can be synthesized by overlap extension PCR using a plurality of oligonucleotides as templates. Materials and experimental methods for overlap extension PCR are described in WO98/13388 and the like. For example, DNA encoding the amino acid sequence of an antibody variable region of, for example, publication a or B, can be obtained by overlap extension PCR using a plurality of oligonucleotides designed to have overlapping nucleotide sequences. The overlapping DNA is then ligated in-frame (in frame) with DNA encoding the constant region to form the coding sequence. The DNA ligated as described above may then be inserted into an expression vector so that the DNA may be expressed, and the resulting vector may be introduced into a host or host cell. The antibodies encoded by the DNA may be expressed by the culture host or by the cultured host cells. The expressed antibody can be appropriately purified from the culture medium of a host or the like (EP 239400; WO 96/02576). In addition, one or more FRs of a humanized antibody linked via one or more CDRs can be selected, for example, to allow the CDRs to form an antigen binding site suitable for an antigen. If desired, the amino acid residues of one or more FRs that make up the variable region of the selected antibody can be modified, for example, by appropriate substitution.

In one embodiment, the nucleic acid cassette can be cloned into an appropriate vector for expression of an antibody of disclosure a or B or a fragment thereof. For this purpose, various types of vectors, such as phagemid vectors, are available. In general, a phagemid vector may contain a variety of elements, including regulatory sequences such as promoters or signal sequences, phenotype selection genes, origins of replication, and other necessary elements.

Methods for introducing desired amino acid modifications into antibodies have been established in the art. For example, a library may be constructed by introducing at least one modified amino acid residue that may be exposed on the surface of an antibody of disclosure a or B and/or at least one amino acid that may alter the antigen binding activity of the antibody depending on the ionic concentration conditions. Furthermore, if desired, flexible residues can be added using the method of Kunkel et al (Methods enzymol.154: 367-382 (1987)).

In an alternative embodiment, disclosure a relates to a vector comprising a nucleic acid encoding the above-described ion concentration-dependent antigen binding domain with increased pI or the above-described ion concentration-dependent antibody with increased pI. In a specific embodiment, the vector may be obtained by, for example, the vectors described in WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241, WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, or WO2012/093704, each of which is incorporated herein by reference in its entirety.

In one embodiment, a nucleic acid encoding an embodiment of disclosure a or B may be operably cloned (inserted) into an appropriate vector and introduced into a host cell. For example, when Escherichia coli is used as the host, the vector includes a cloning vector, pBluescript vector (Stratagene) or any of various other commercially available vectors.

In one embodiment, the expression vector is used as a vector containing the nucleic acid of disclosure a or B. Expression vectors can be used to allow expression of the polypeptide in vitro, in E.coli, in cultured cells, or in vivo. For example, it is possible to use the pBEST vector (Promega) for in vitro expression; pET vector (Invitrogen) for E.coli expression; pME18S-FL3 vector (GenBank accession AB009864) for cultured cell expression; and the pME18S vector (Takebe et al, mol. cell biol. 8: 466-472(1988)) for in vivo expression. The DNA can be inserted into the vector using restriction enzyme sites by conventional methods, for example, by ligase reaction (see, Current protocols in Molecular Biology edge. Ausubel et al (1987) Publish. John Wiley & sons. section 11.4-11.11).

In an alternative embodiment, disclosure a relates to a host or host cell comprising a vector containing a nucleic acid encoding the above-described ion concentration-dependent antigen binding domain with increased pI or the above-described ion concentration-dependent antibody with increased pI. In a particular embodiment, the host or host cell can be prepared by, for example, the methods described in WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241, WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, or WO2012/093704, each of which is incorporated herein by reference in its entirety.

The type of the host cell of the disclosure a or B is not particularly limited, and the host cell includes, for example, bacterial cells such as escherichia coli, and various animal cells. Host cells may be suitably used as production systems for the production and expression of antibodies. Both eukaryotic and prokaryotic cells may be used.

Eukaryotic cells useful as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of the animal cells include mammalian cells, for example, CHO (Puck et al, J.Exp.Med.108: 945-956(1995)), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero; amphibian cells, e.g., Xenopus oocytes (Valle et al, Nature 291: 338-340 (1981)); and insect cells, for example, Sf9, Sf21, and Tn 5. The recombinant vector and the like can be introduced into the host cell, for example, using a calcium phosphate method, a DEAE-dextran method, a method using cationic liposome DOTAP (Boehringer-Mannheim), electroporation, and lipofection.

Plant cells known for use as protein production systems include, for example, tobacco-derived cells and duckweed (Lemna minor) -derived cells. Callus may be cultured from these cells to produce antibodies of disclosure a or B. Fungal cell-based protein production systems include those using yeast cells, for example, cells of the genus Saccharomyces such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Schizosaccharomyces pombe (Schizosaccharomyces pombe); and filamentous fungal cells, e.g., Aspergillus such as Aspergillus niger. When prokaryotic cells are used, bacterial cell-based production systems may be used. Production systems based on bacterial cells include, for example, those using Bacillus subtilis and Escherichia coli.

To produce an antibody of disclosure a or B using a host cell, the host cell is transformed with an expression vector containing a nucleic acid encoding the antibody of disclosure a or B and cultured to express the nucleic acid. For example, when animal cells are used as a host, the culture medium may include, for example, DMEM, MEM, RPMI1640, and IMDM, which may be used in appropriate combination with serum supplements such as FBS or Fetal Calf Serum (FCS). Alternatively, the cells may be cultured in serum-free medium.

Alternatively, an animal or plant can be used in an in vivo production system to produce an antibody of disclosure a or B, e.g., one or more nucleic acids encoding an antibody of disclosure a or B can be introduced into the animal or plant, the antibody produced in vivo, and the antibody can be subsequently collected from the animal or plant.

When an animal is used as the host, a production system using mammals or insects is available. Preferred mammals include, but are not limited to, goats, pigs, sheep, mice, and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Transgenic animals may also be used.

In one example, a nucleic acid encoding an antibody of disclosure a or B is prepared as a fusion gene with a gene encoding a polypeptide specifically included in milk, such as goat β -casein. Then, the goat embryo is injected with an oligonucleotide fragment containing the fusion gene and transplanted into a female goat. The antibody of interest may be obtained from milk produced by a transgenic goat (which is produced by a goat receiving embryos) or its offspring. Hormones may be suitably administered to the transgenic goat to increase the amount of antibody-containing milk produced by the goat (Ebert et al, Bio/Technology 12: 699-.

Insects useful for producing the antibodies of disclosure a or B include, for example, silkworm. When silkworms are used, baculovirus in which an oligonucleotide encoding an antibody of interest is inserted into the viral genome is used to infect silkworms. The antibody of interest may be obtained from the body fluid of the silkworm under investigation (Susumu et al, Nature 315: 592-594 (1985)).

When plants are used to produce antibodies of disclosure a or B, tobacco can be used. When tobacco is used, a recombinant vector obtained by inserting an oligonucleotide encoding an antibody of interest into a plant expression vector, for example, pMON530, can be introduced into bacteria such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). The resulting bacteria can be used to infect tobacco, for example, tobacco (Nicotiana tabacum) (Ma et al, Eur. J. Immunol.24: 131-138(1994)) and obtain the desired antibody from the leaves of the infected tobacco. The modified bacteria can also be used to infect duckweed (Lemna minor) and obtain the desired antibody from the cloned cells of the infected duckweed (Cox et al, nat. Biotechnol.24 (12): 1591-1597 (2006)).

For secretion of antibodies expressed in host cells into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the polypeptide of interest. The signal may be endogenous to the antibody of interest or may be an exogenous signal known in the art.

Antibodies of disclosure a or B produced as described above can be isolated from the host cell or from the interior or exterior of the host (e.g., culture medium and milk) and purified as substantially pure and homogeneous antibodies. The antibody can be appropriately separated and purified, for example, by appropriately selecting and combining chromatography columns, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and the like. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. The chromatography may be performed, for example, by using liquid chromatography such as HPLC and FPLC. The column used for affinity chromatography may be a protein a column or a protein G column. Protein A columns include, for example, Hyper D, POROS, Sepharose F.F (Pharmacia).

If desired, the modified antibody or peptide may be partially deleted by treating the antibody with an appropriate protein modifying enzyme before or after purification of the antibody. For such protein-modifying enzymes, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase and glucosidase can be used.

In an alternative embodiment, disclosure a relates to a method of producing an antibody containing an antigen binding domain whose antigen binding activity changes depending on the ionic concentration conditions, which may include culturing host cells or breeding hosts and collecting the antibody from cultures of these cells, secreted substances from the hosts, or by other means known in the art.

In one embodiment, disclosure a relates to a method of production comprising one or more steps selected from the group consisting of: (a) selecting antibodies capable of facilitating removal of antigen from plasma; (b) selecting an antibody having enhanced binding activity to the extracellular matrix; (c) selecting an antibody having enhanced Fc γ R-binding activity under neutral pH conditions; (d) selecting an antibody having enhanced Fc γ RIIb-binding activity under neutral pH conditions; (e) selecting an antibody having maintained or enhanced Fc γ RIIb-binding activity and reduced binding activity to one or more activating Fc γ rs selected from the group consisting of Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb, and Fc γ RIIa; (f) selecting an antibody having enhanced FcRn-binding activity under neutral pH conditions; (g) selecting an antibody with an increased pI; (h) confirming the pI of the collected antibodies, and then selecting antibodies with increased pI; and (i) selecting an antibody whose antigen binding activity is altered or increased depending on the ion concentration condition as compared to the reference antibody.

Herein, reference antibodies include, but are not limited to, natural antibodies (e.g., natural Ig antibodies, preferably natural IgG antibodies) and antibodies prior to modification (antibodies prior to or during library construction, e.g., ion concentration-dependent antibodies that increase their pI, or antibodies with increased pI prior to conferring an ion concentration-dependent antigen binding domain).

After production of the antibody of disclosure a, the resulting antibody can be evaluated by antibody pharmacokinetic assays using plasma of e.g. mice, rats, rabbits, dogs, monkeys, humans, to select antibodies with enhanced antigen removal from plasma compared to a reference antibody.

Alternatively, after the antibody of disclosure a is produced, the resulting antibody may be compared with a reference antibody in terms of extracellular matrix binding capacity by electrochemiluminescence or the like to select an antibody having increased binding to extracellular matrix.

Alternatively, after the antibody of the disclosure a is produced, the resultant antibody may be compared with a reference antibody with respect to binding activity to various Fc γ rs under neutral pH conditions using BIACORE (registered trademark) or the like to select an antibody having increased binding activity to various Fc γ rs under neutral pH conditions. In this case, the various Fc γ Rs may be one of the Fc γ Rs studied, e.g., Fc γ RIIb. Similarly, it is also possible to select antibodies whose Fc γ RIIb-binding activity (under neutral pH conditions) is maintained or increased and whose binding activity to one or more activating Fc γ rs selected from the group consisting of Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb and Fc γ RIIa, etc., is reduced. In this case, the Fc γ R may be a human Fc γ R.

Alternatively, after production of the antibody of disclosure a, the resulting antibody can be compared to a reference antibody for FcRn-binding activity under neutral pH conditions using known techniques such as BIACORE to select an antibody having increased FcRn-binding activity under neutral pH conditions. In this case, the FcRn may be a human FcRn.

Alternatively, after the antibody of the disclosure a is produced, the pI of the resulting antibody is evaluated by isoelectric focusing or the like, and an antibody having an increased pI as compared to the reference antibody is selected. In this case, it is possible to select antibodies whose pI values are increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 or more, or at least 0.6, 0.7, 0.8, or 0.9 or more; or an antibody whose pI value is increased by at least 1.0, 1.1, 1.2, 1.3, 1.4, or 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, or 2.5 or more, or 3.0 or more.

Alternatively, after the antibody of the disclosure a is produced, the resulting antibody can be compared with a reference antibody with respect to the binding activity to a desired antigen under low and high ion concentration conditions using BIACORE or the like to select an antibody whose antigen binding activity is changed or increased depending on the ion concentration conditions. The ion concentration may be, for example, a hydrogen ion concentration or a metal ion concentration. When the ion concentration is a metal ion concentration, it may be, for example, a calcium ion concentration. Whether the binding activity is altered or increased can be assessed based on the presence of, for example: (a) altered or enhanced uptake of antigen by cells; (b) altered or increased ability to bind different antigenic molecules multiple times; (c) altered or enhanced reduction of antigen concentration in plasma; or (d) plasma retention of the altered antibody. Alternatively, any two or more of these selection methods may be appropriately combined, if necessary.

In an alternative embodiment, disclosure a relates to a method for producing or screening an antibody containing an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and whose pI is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody ("ion concentration-dependent antibody with increased pI"). The production method can be carried out, for example, by appropriately combining, as necessary, related embodiments described in the scope of the disclosure a herein, for example, an embodiment of a method for producing or screening the above-mentioned antibody having an increased pI, and an embodiment of a method for producing or screening the above-mentioned calcium ion concentration-dependent antigen binding domain or calcium ion concentration-dependent antibody whose antigen binding activity is higher under a high calcium ion concentration condition than under a low calcium ion concentration condition, or a library thereof, and/or an embodiment of a method for producing or screening the above-mentioned pH-dependent antigen binding domain or pH-dependent antibody whose antigen binding activity is higher under a neutral pH condition than under an acidic pH condition, or a library thereof.

In an alternative embodiment, disclosure a provides a method for producing or screening an antibody containing an antigen-binding domain whose extracellular matrix-binding activity is increased, wherein its antigen-binding activity is changed according to ion concentration conditions and its pI is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody ("ion concentration-dependent antibody with increased pI"). An increase in the pI of the ion concentration-dependent antibody can be considered in this method. The method can be performed, for example, by appropriately combining, as needed, related embodiments described within the scope of the disclosure a, for example, an embodiment of a method for producing or screening the above-described antibody having an increased pI, and an embodiment of a method for producing or screening the above-described calcium ion concentration-dependent antigen binding domain or calcium ion concentration-dependent antibody whose antigen binding activity is higher under a high calcium ion concentration condition than under a low calcium ion concentration condition, or a library thereof, and/or an embodiment of a method for producing or screening the above-described pH-dependent antigen binding domain or pH-dependent antibody whose antigen binding activity is higher under a neutral pH condition than under an acidic pH condition, or a library thereof. For example, the resulting antibody can be compared to a reference antibody in terms of extracellular matrix binding ability, by known techniques such as electrochemiluminescence, to select an antibody with increased extracellular matrix binding.

Herein, the reference antibody may include, but is not limited to, a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) and an antibody before modification (an antibody before or during library construction, e.g., an ion concentration-dependent antibody before its pI is increased or an antibody having an increased pI before imparting an ion concentration-dependent antigen-binding domain thereto).

In an alternative embodiment, disclosure a relates to a method of 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 likely to be exposed on the surface of the antibody so as to increase the isoelectric point (pI). In some embodiments, the amino acid residue modification comprises a modification selected from the group consisting of: (a) replacing a negatively charged amino acid residue with an uncharged amino acid residue; (b) replacing a negatively charged amino acid residue with a positively charged amino acid residue; and (c) replacing the uncharged amino acid residue with a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is substituted with histidine. In another embodiment, the antibody comprises a variable region and/or a constant region, and amino acid residues in the variable region and/or the constant region are modified. In another embodiment, at least one amino acid residue modified according to the method is at a position in a CDR or FR selected from the group consisting of: (a) positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in the FR of the heavy chain variable region; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 (numbering according to Kabat) in the CDRs of the light chain variable region. In another embodiment, at least one amino acid residue modified according to the method is at a position in a CDR or FR 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 CDRs of the heavy chain variable region; (c) positions 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in the FR of the light chain variable region; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDRs of the light chain variable region. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody produced according to the method for its corresponding antigen at acidic pH (e.g., pH 5.8) and neutral pH (e.g., pH 7.4). In another embodiment, the method comprises selecting an antibody having a KD (acidic pH range (e.g., pH 5.8))/KD (neutral pH range (e.g., pH 7.4)) for the antigen of 2 or greater. In some embodiments, the method further comprises comparing the antigen binding activity of an antibody produced according to the method under conditions of high ion concentration (e.g., hydrogen ion or calcium ion concentration) and low ion concentration. In another embodiment, the method further comprises selecting an antibody that has a higher (e.g., 2-fold) antigen binding activity at high ion concentrations than at low ion concentrations. In some embodiments, where the ion concentration is calcium ion concentration, a high calcium ion concentration may be selected between 100 μ M to 10mM, between 200 μ M to 5mM, between 400 μ M to 3mM, between 200 μ M to 2mM, or between 400 μ M to 1 mM. It may be preferred to select a concentration between 500 μ M and 2.5mM, which is close to the plasma (blood) concentration of calcium ions in vivo. In some embodiments, the low calcium ion concentration may be selected between 0.1 μ M to 30 μ M, between 0.2 μ M to 20 μ M, between 0.5 μ M to 10 μ M, or between 1 μ M to 5 μ M, or between 2 μ M to 4 μ M. It may also be preferred to select a concentration between 1. mu.M and 5. mu.M, which is close to the concentration of calcium ions in the early endosome in vivo. In some embodiments, the lower limit of the KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 μ M Ca)/KD (2mM Ca)) value is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less. In alternative embodiments, the lower limit of the kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) (e.g., kd (3 μ M Ca)/kd (2mM Ca)) value is 2 or more, 5 or more, 10 or more, or 30 or more, and the upper limit thereof is 50 or less, 100 or less, or 200 or less. In some embodiments, where the ionic concentration is a hydrogen ion concentration, the low hydrogen ion concentration (neutral pH range) may 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 low hydrogen ion concentration may preferably be pH 7.4, which is close to the in vivo plasma (blood) pH, but for convenience of measurement, for example, pH 7.0 may be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) may 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 the early endosome, but for convenient measurement, for example, pH 6.0 may be used. In some embodiments, the lower limit of KD (acidic pH range)/KD (neutral pH range) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less. In some embodiments, the method further comprises comparing the removal of antigen from plasma following administration of an antibody produced according to the method compared to when administered only in that it does not comprise one or more modified reference antibodies introduced according to the method. In another embodiment, the method further comprises selecting an antibody produced according to the method that promotes removal of antigen from plasma (e.g., 2-fold) compared to an antibody that does not contain the modification introduced according to the method. In some embodiments, the method further comprises comparing extracellular matrix binding of an antibody produced according to the method as compared to an antibody that differs only in that it does not comprise one or more modifications introduced according to the method. In another embodiment, the method further comprises selecting an antibody produced according to the method that has increased extracellular matrix binding (e.g., 2-fold when bound to an antigen) compared to an antibody that differs only in that it does not comprise one or more modifications introduced according to the method. In another embodiment, an antibody produced according to the present method retains (substantially) antigen binding activity when compared to an antibody that has been modified or altered at least one amino acid residue to increase pI pre-pI (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (e.g., an antibody modification, or an antibody before or during library construction)). In this case, "to (substantially) retain antigen binding activity" may mean to have 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 of activity as compared to the binding activity of the antibody before modification or alteration.

In a further embodiment, disclosure a relates to 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 a constant region of the antibody so as to increase the isoelectric point (pI). In some embodiments, the amino acid residue modification comprises a modification selected from the group consisting of: (a) replacing a negatively charged amino acid residue with an uncharged amino acid residue; (b) replacing a negatively charged amino acid residue with a positively charged amino acid residue; and (c) replacing the uncharged amino acid residue with a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is substituted with histidine. In another embodiment, the antibody comprises a variable region and/or a constant region, and amino acid residues in the variable region and/or the constant region are modified. In another embodiment, at least one amino acid residue modified according to the method is in the constant region at a position selected from the group consisting of: positions 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 (according to EU numbering). In another embodiment, at least one amino acid residue modified according to the method is in the constant region 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 (according to EU numbering). In another embodiment, at least one amino acid residue modified according to the method is in the constant region 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). In some embodiments, the method further comprises comparing the antigen binding activity of an antibody produced according to the method under conditions of high ion concentration (e.g., hydrogen ion or calcium ion concentration) and low ion concentration. In another embodiment, the method further comprises selecting an antibody that has a higher antigen binding activity at high ion concentrations than at low ion concentrations. In some embodiments, the method comprises comparing the removal of antigen from plasma following administration of an antibody produced according to the method compared to when the administration differs only in that it does not comprise one or more modifications introduced according to the invention. In another embodiment, the method further comprises selecting an antibody produced according to the method that promotes removal of antigen from plasma (e.g., 2-fold) compared to an antibody that does not contain the modification introduced according to the invention. In some embodiments, the method comprises comparing extracellular matrix binding of an antibody produced according to the method as compared to an antibody that differs only in that it does not comprise one or more modifications introduced according to the invention. In another embodiment, the method further comprises selecting an antibody produced according to the method that has increased extracellular matrix binding (e.g., 2-fold when bound to an antigen) compared to an antibody that differs only in that it does not comprise one or more modifications introduced according to the invention. In some embodiments, the method comprises comparing the Fc γ receptor (Fc γ R) -binding activity of an antibody produced according to the method at neutral pH (e.g., pH 7.4) to a reference antibody comprising a constant region of native IgG. In another embodiment, the method comprises selecting an antibody produced according to the method that has enhanced fcyr-binding activity at neutral pH (e.g., pH 7.4) as compared to a reference antibody comprising a constant region of native IgG. In some embodiments, the selected antibodies produced according to the methods have enhanced Fc γ RIIb binding activity at neutral pH. In some embodiments, the selected antibody produced according to said method has binding activity to one or more activating Fc γ rs, preferably selected from the group consisting of Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb and Fc γ RIIa, and to Fc γ RIIb, and optionally said Fc γ RIIb-binding activity is maintained or enhanced and the binding activity to activating Fc γ rs is reduced compared to a reference antibody differing only in that its constant region is a constant region of a native IgG. In some embodiments, the method further comprises comparing the FcRn-binding activity of an antibody produced according to the present method at neutral pH conditions as compared to a reference antibody differing only in that its constant region is that of a native IgG. In another embodiment, the method further comprises selecting an antibody produced according to the method that has increased FcRn-binding activity (e.g., 2-fold) under neutral pH conditions as compared to a reference antibody that differs only in that its constant region is that of a native IgG. In some embodiments, an antibody produced according to the present methods retains antigen binding activity (substantially) when compared to an antibody that modifies or changes at least one amino acid residue to increase pI pre-pI (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (e.g., an antibody modification, or an antibody before or during library construction)). In this case, "to (substantially) retain antigen binding activity" may mean to have 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 of activity as compared to the binding activity of the antibody before modification or alteration.

In further embodiments, the method comprises modifying at least one amino acid residue that may expose the surface of the variable and constant regions of the antibody so as to increase the isoelectric point (pI). In another embodiment, at least one amino acid residue modified according to the present method is in a position in the constant region disclosed above. In another embodiment, at least one amino acid residue modified according to the present methods is in a position in the variable region disclosed above. In another embodiment, at least one amino acid residue modified according to the present method is in a position in the constant region disclosed above and at least one amino acid residue modified according to the present method is in a position in the variable region disclosed above. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody produced according to the method for its corresponding antigen in acidic pH (e.g., pH 5.8) and neutral pH (e.g., pH 7.4). In another embodiment, the method comprises selecting an antibody having a KD (acidic pH range)/KD (neutral pH range) for the antigen of 2 or greater. In some embodiments, the method further comprises comparing the antigen binding activity of an antibody produced according to the method under conditions of high ion concentration (e.g., hydrogen ion or calcium ion concentration) and low ion concentration. In another embodiment, the method further comprises selecting an antibody that has a higher antigen binding activity at high ion concentrations than at low ion concentrations. In some embodiments, where the ion concentration is calcium ion concentration, a high calcium ion concentration may be selected between 100 μ M to 10mM, between 200 μ M to 5mM, between 400 μ M to 3mM, between 200 μ M to 2mM, or between 400 μ M to 1 mM. Concentrations chosen between 500. mu.M and 2.5mM may also be preferred. In some embodiments, the low calcium ion concentration may be selected between 0.1 μ M to 30 μ M, between 0.2 μ M to 20 μ M, between 0.5 μ M to 10 μ M, or between 1 μ M to 5 μ M or between 2 μ M to 4 μ M. Concentrations chosen between 1. mu.M and 5. mu.M may also be preferred. In some embodiments, the lower limit of the KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 μ M Ca)/KD (2mM Ca)) value is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less. In alternative embodiments, the lower limit of the kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) (e.g., kd (3 μ M Ca)/kd (2mM Ca)) value is 2 or more, 5 or more, 10 or more, or 30 or more, and the upper limit thereof is 50 or less, 100 or less, or 200 or less. In some embodiments, where the ionic concentration is a hydrogen ion concentration, the low hydrogen ion concentration (neutral pH range) may 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 low hydrogen ion concentration may preferably be pH 7.4, which is close to the pH in plasma (blood) in vivo, but for convenience of measurement, for example, pH 7.0 may be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) may 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. For example, the acidic pH range may be pH 5.8 or pH 6.0. In some embodiments, the lower limit of KD (acidic pH range)/KD (neutral pH range) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less.

In some embodiments, the method further comprises comparing the removal of antigen from plasma following administration of an antibody produced according to the method compared to when administered only in that it does not comprise one or more modified reference antibodies introduced according to the invention. In another embodiment, the method further comprises selecting an antibody produced according to the method that promotes removal (e.g., 2-fold) of antigen from plasma as compared to an antibody that does not contain the modification introduced according to the invention. In some embodiments, the method further comprises comparing extracellular matrix binding of an antibody produced according to the method as compared to an antibody differing only in that it does not comprise one or more modifications introduced according to the invention. In another embodiment, the method further comprises selecting an antibody produced according to the method that has increased extracellular matrix binding (e.g., 5-fold when complexed with an antigen) compared to an antibody that differs only in that it does not comprise one or more modifications introduced according to the invention. In some embodiments, the method further comprises comparing the Fc γ receptor (Fc γ R) -binding activity of an antibody produced according to the method to a reference antibody comprising a constant region of native IgG at neutral pH (e.g., pH 7.4). In another embodiment, the method comprises selecting an antibody produced according to the method that has enhanced fcyr-binding activity at neutral pH (e.g., pH 7.4) as compared to a reference antibody comprising a constant region of native IgG. In some embodiments, the antibodies selected for production according to the present methods have enhanced Fc γ RIIb binding activity at neutral pH. In some embodiments, the selected antibodies produced according to the present method have binding activity to one or more activating Fc γ rs, preferably selected from the group consisting of Fc γ RIa, Fc γ RIb, Fc γ RIc, Fc γ RIIIa, Fc γ RIIIb and Fc γ RIIa, and to Fc γ RIIb, and optionally Fc γ RIIb-binding activity is maintained or enhanced and binding activity to the activating Fc γ rs is reduced as compared to a reference antibody differing only in that its constant region is a constant region of native IgG. In some embodiments, the method further comprises comparing the FcRn-binding activity of an antibody produced according to the present method at neutral pH conditions as compared to a reference antibody differing only in that its constant region is that of a native IgG. In another embodiment, the method further comprises selecting an antibody produced according to the method that has increased FcRn-binding activity (e.g., 2-fold) under neutral pH conditions as compared to a reference antibody that differs only in that its constant region is that of a native IgG. In another embodiment, an antibody produced according to the present method (substantially) retains antigen binding activity when compared to an antibody that has been modified or altered at least one amino acid residue to increase pI pre-pI (a natural antibody (e.g., a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (e.g., an antibody modification, or an antibody before or during library construction)). In this case, "to (substantially) retain antigen binding activity" may mean to have 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 of activity as compared to the binding activity of the antibody before modification or alteration.

In an alternative embodiment, disclosure a relates to an antibody obtained by the method of disclosure a above for use in generating or screening antibodies.

In an alternative embodiment, disclosure a relates to a composition or pharmaceutical composition comprising the antibody of disclosure a above. In one embodiment, the pharmaceutical composition of disclosure a may be a pharmaceutical composition for accelerating antigen removal from a biological fluid (preferably, plasma, etc.) of a subject and/or for increasing extracellular matrix binding when an antibody of disclosure a is administered to (applied to) a subject (preferably, in vivo). The pharmaceutical composition of disclosure a may optionally contain a pharmaceutically acceptable carrier. Herein, a pharmaceutical composition may generally refer to an agent for treating, preventing, diagnosing or examining a disease.

The disclosure a composition or pharmaceutical composition may be suitably formulated. In some embodiments, they may be administered parenterally, for example, in the form of a sterile solution or suspension in water or any other pharmaceutically acceptable liquid. The compositions may be suitably formulated in unit dosage forms as required by generally accepted pharmaceutical practice, by appropriate combination with a pharmaceutically acceptable carrier or vehicle. The pharmaceutically acceptable carriers or media include, but are not limited to, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives and binders. The amount of active ingredient in the composition may be adjusted in such a way that the dosage falls within a suitable predetermined range.

In some embodiments, the composition or pharmaceutical composition of disclosure a may be administered parenterally. The composition or pharmaceutical composition may be suitably prepared, for example, as an injectable, nasal, pulmonary or transdermal composition. The composition or pharmaceutical composition may be administered systemically or locally, e.g., by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

In some embodiments, the present disclosure provides antibodies whose pI is increased by modifying at least one amino acid residue that may be exposed at the surface (antibodies with increased pI); methods of producing these antibodies; or the use of these antibodies to enhance removal of antigen from plasma when the antibodies are administered to a subject in vivo. It will be understood that the scope of disclosure a described herein and that described in the corresponding examples herein may be suitably employed in this embodiment. In other embodiments, the disclosure provides antibodies whose pI is reduced by modifying at least one amino acid residue that may be exposed at the surface ("antibodies with reduced pI"); methods of producing these antibodies; or the use of these antibodies to improve plasma retention when the antibodies are administered to a subject in vivo. The present inventors revealed that the internalization of antibodies can be enhanced by introducing specific amino acid mutations into specific sites in the amino acid sequence of the constant region to increase its pI. It will be appreciated by those skilled in the art that antibody plasma retention can be prolonged due to a reduction in pI by introducing amino acids with different side chain charge properties into the above-mentioned sites, thereby inhibiting cellular internalization of the antibody. It will be understood that the scope of disclosure a described herein and that described in the corresponding examples herein may be suitably applied to the described embodiments.

In one embodiment, the present disclosure provides a method for producing a modified antibody (with an increased or decreased half-life in plasma compared to the antibody prior to modification), wherein the method comprises: (a) modifying a nucleic acid encoding the pre-modified antibody to alter the charge of at least one amino acid residue located at a position selected from the group consisting of: positions 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 (according to EU numbering); (b) culturing the host cell to express the modified nucleic acid and produce the antibody; and (c) collecting the produced antibody from the host cell culture.

Further embodiments provide a method of increasing or decreasing the half-life of an antibody in plasma, wherein the method comprises modifying at least one amino acid residue located at a position selected from the group consisting of: positions 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 (according to EU numbering).

The methods may further comprise determining that the half-life of the collected and/or modified antibody is increased or decreased in plasma compared to before the antibody modification.

The change in charge can be achieved by one or more amino acid substitutions. In some embodiments, the one or more substituted amino acid residues may be selected from the group consisting of amino acid residues of the following groups (a) and (b), but are not limited thereto: (a) glu (E) and Asp (D); and (b) Lys (K), Arg (R) and His (H).

In some embodiments, the antibody may be an Ig-type antibody such as an IgG antibody. In some embodiments, the antibody can be a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody can be a multispecific antibody such as a bispecific antibody.

Disclosure B

In non-limiting embodiments, disclosure B relates to Fc region variants, uses thereof, and methods of producing the same.

Within the context of the disclosure a and B described herein, "Fc region variant" may refer to an Fc region modified from the Fc region of a native IgG antibody, for example, by modifying at least one amino acid with another amino acid, or may refer to an Fc region modified from the Fc region variant by further modifying at least one amino acid with another amino acid. Herein, the Fc region variant includes not only an Fc region into which an amino acid modification is introduced, but also an Fc region containing the same amino acid sequence as the aforementioned Fc region.

In an alternative embodiment, disclosure B relates to an FcRn-binding domain-containing Fc region variant comprising Ala at position 434; contains at position 438 any of Glu, Arg, Ser, and Lys; and contains any one of Glu, Asp, and Gln at position 440 (according to EU numbering) (within the scope of disclosure B described herein, the Fc region variant is also referred to as a "novel Fc region variant" for descriptive purposes).

In practice, the Fc region variants of disclosure B can be incorporated in virtually any antibody (e.g., multispecific antibodies such as bispecific antibodies) regardless of the type of target antigen. For example, anti-factor IXa/factor X bispecific antibodies can be generated using the Fc region variants shown in example 20 (e.g., F8M-F1847mv [ F8M-F1847mv1(SEQ ID NO: 323) and F8M-F1847mv2(SEQ ID NO: 324) as heavy chains and F8ML (SEQ ID NO: 325) as light chains ], F8M-F1868mv [ F8M-F1868mv1(SEQ ID NO: 326) and F8M-F1868mv2(SEQ ID NO: 327) as heavy chains and F8ML (SEQ ID NO: 325) as light chains ], and F8M-F7 1927mv [ F8M-F1927mv1(SEQ ID NO: 1 8) and F8M-F7 mv 1927 (SEQ ID NO: 2) as heavy chains and F8 67325) as light chains) (SEQ ID NO: 67325) and F8 ML.

As described above, WO2013/046704 reports that Fc region variants incorporating mutations to increase their FcRn binding under acidic conditions (a combination with specific mutations (a representative example is the double-residue mutation Q438R/S440E (according to EU numbering))) exhibit significantly reduced binding to rheumatoid factors. However, WO2013/046704 does not describe that the variant Fc region with reduced binding of rheumatoid factor due to modification of Q438R/S440E is superior in plasma retention compared to antibodies with native Fc region. Thus, there is a need for safe and more advantageous Fc region variants that allow for improved plasma retention, but do not bind pre-existing ADA. The inventors herein disclose safe and more advantageous Fc region variants that allow improved plasma retention, but do not bind anti-drug antibodies (pre-existing ADA, etc.). In particular, it was first disclosed herein that, surprisingly, Fc region variants containing a combination of amino acid residue mutations, which are substitutions of the amino acid at position 434 according to EU numbering with ala (a) and a specific double-residue mutation (a representative example is Q438R/S440E), are preferred for prolonged retention of antibody in plasma while maintaining significantly reduced binding to rheumatoid factor.

Thus, the novel Fc region variants of disclosure B disclosed herein provide advantageous and unexpected improvements over the Fc region variants described in WO2013/046704 (which is incorporated herein by reference in its entirety).

In one embodiment, disclosure B provides a novel combination of amino acid substitutions of the FcRn-binding domain that increases FcRn-binding activity of the antibody in the acidic pH range and in the neutral pH range, particularly, in the acidic pH range.

In one embodiment, the Fc region variant of disclosure B contains an Ala at position 434; any one of Glu, Arg, Ser, and Lys at position 438; and any one of Glu, Asp, and Gln at position 440 (according to EU numbering); and more preferably Ala at position 434; arg or Lys at position 438; and Glu or Asp at position 440 (according to EU numbering). Preferably, the Fc-region variant of publication B additionally contains any one of he or Leu at position 428, and/or any one of he, Leu, Val, Thr, and Phe at position 436 (numbering according to EU). More preferably, the variant Fc-region comprises Leu at position 428, and/or Val or Thr at position 436 (according to EU numbering).

In one embodiment, the Fc region variant of disclosure B may be an Fc region variant of a native Ig antibody, and more preferably is an Fc region variant of a native IgG (IgG1, IgG2, IgG3, or IgG4 type) antibody. Native Fc regions are described herein in part within the scope of disclosures a and B. More specifically, in disclosure B, a native Fc region may refer to an unmodified or naturally occurring Fc region, and preferably, an unmodified or naturally occurring Fc region of a native Ig antibody (whose Fc region amino acid residues remain unmodified). The antibody source for the Fc region may be an Ig such as IgM or IgG, for example, human IgG1, IgG2, IgG3, or IgG 4. In one embodiment, it may be human IgG 1. Meanwhile, a (reference) antibody comprising a native Fc region may refer to an antibody comprising an unmodified or naturally occurring Fc region.

Positions 428, 434, 438, and 440 are common to the Fc region of all native human IgG1, IgG2, IgG3, and IgG4 antibodies. However, at position 436 of the Fc region, native human IgG1, IgG2, and IgG4 antibodies share tyr (y), while native human IgG3 antibody has phe (f). On the other hand, Stapleton et al (Nature Comm.599(2011) reported that human IgG3 allotypes containing an amino acid substitution according to EU numbering R435H have plasma half-lives in humans comparable to IgG1 therefore, the inventors also believe that plasma retention can be improved by increasing FcRn binding under acidic conditions by introducing an R435H amino acid substitution in combination with the amino acid substitution at position 436.

WO2013/046704 also specifically reports that the double amino acid residue substitutions Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D (according to EU numbering) when combined with amino acid substitutions that can increase FcRn binding under acidic conditions results in a significant reduction in rheumatoid factor binding.

Thus, in a preferred embodiment, the FcRn-binding domain of the Fc region variant of disclosure B may contain a combination of substituted amino acid positions selected from the group consisting of: (a) N434A/Q438R/S440E; (b) N434A/Q438R/S440D; (c) N434A/Q438K/S440E; (d) N434A/Q438K/S440D; (e) N434A/Y436T/Q438K/S440E; (f) N434A/Y436T/Q438R/S440D; (g) N434A/Y436T/Q438K/S440E; (h) N434A/Y436T/Q438K/S440D; (i) N434A/Y436V/Q438R/S440E; (j) N434A/Y436V/Q438R/S440D; (k) N434A/Y436V/Q438K/S440E; (l) N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/S440E; (N) N434A/R435H/F436T/Q438R/S440D; (o) N434A/R435H/F436T/Q438K/S440E; (p) N434A/R435H/F436T/Q438K/S440D; (Q) N434A/R435H/F436V/Q438R/S440E; (R) N434A/R435H/F436V/Q438R/S440D; (S) N434A/R435H/F436V/Q438K/S440E; (t) N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v) M428L/N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x) M428L/N434A/Q438K/S440D; (Y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/Y436T/Q438R/S440D; (aa) M428L/N434A/Y436T/Q438K/S440E; (ab) M428L/N434A/Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad) M428L/N434A/Y436V/Q438R/S440D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af) M428L/N434A/Y436V/Q438K/S440D; (ag) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (ah) L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E (by EU numbering).

In further preferred embodiments, the FcRn-binding domain of the Fc region variant of disclosure B may contain a combination of substituted amino acids selected from the group consisting of: (a) N434A/Q438R/S440E; (b) N434A/Y436T/Q438R/S440E; (c) N434A/Y436V/Q438R/S440E; (d) M428L/N434A/Q438R/S440E; (e) M428L/N434A/Y436T/Q438R/S440E; (f) M428L/N434A/Y436V/Q438R/S440E; (g) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (h) L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E (by EU numbering).

In one embodiment, it is preferred that the FcRn-binding activity of the Fc region variant of disclosure B is increased under acidic pH conditions as compared to the Fc region of a native IgG.

An increase in FcRn-binding activity (binding affinity) of the FcRn-binding domain over the pH range may correspond to an increase in the measured FcRn-binding activity (binding affinity) when compared to the measured FcRn-binding activity (binding affinity) of the native FcRn-binding domain. In this case, KD (native Fc region)/KD (Fc region variant of disclosure B), which represents a difference in binding activity (binding affinity), may be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 70-fold, 80-fold, 100-fold, 500-fold, or 1000-fold. Such an increase may occur in an acidic pH range and/or in a neutral pH range; however, from the standpoint of the mechanism of action of disclosure B, an increase in the acidic pH range may be preferred.

In some embodiments, the Fc region variant of disclosure B, which has increased FcRn-binding activity over an acidic pH range, has higher FcRn-binding activity (e.g., at pH 6.0 and 25 ℃) than the Fc region of native IgG, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more. In some embodiments, the Fc region variant may have increased FcRn-binding activity over an acidic pH range at least 5-fold or at least 10-fold greater than the FcRn-binding activity of the Fc region of a native IgG.

Manipulation of the FcRn-binding domain by introducing amino acid substitutions may occasionally reduce antibody stability (WO 2007/092772). Proteins with poor stability tend to aggregate easily during storage, and the stability of pharmaceutical proteins is highly important in the production of pharmaceutical agents. Thus, a decrease in stability caused by a substitution in the Fc region may lead to difficulty in developing a stable antibody formulation (WO 2007/092772).

The purity of pharmaceutical proteins is also important in the development of pharmaceutical agents, both in terms of monomers and high molecular weight substances. Wild-type IgG1 did not contain significant amounts of high molecular weight material after purification with protein a, whereas FcRn-binding domains could be manipulated by introduction of substitutions to produce large amounts of high molecular weight material. In this case, the high molecular weight substances may have to be removed from the drug by a purification step.

Amino acid substitutions in antibodies can lead to negative consequences, such as increased immunogenicity of the therapeutic antibody, which in turn can lead to cytokine storms and/or the production of anti-drug antibodies (ADAs). The clinical utility and effectiveness of therapeutic antibodies can be limited by ADAs, as they affect the efficacy and pharmacokinetics of therapeutic antibodies and sometimes result in serious side effects. Many factors influence the immunogenicity of therapeutic antibodies, and the presence of effector T-cell epitopes is one of the factors. Likewise, the presence of pre-existing antibodies to therapeutic antibodies can also be problematic. An example of such a pre-existing antibody is Rheumatoid Factor (RF), an auto-antibody (antibody against self-protein) directed against the Fc portion of an antibody (i.e., IgG). Rheumatoid factor is especially found in patients with Systemic Lupus Erythematosus (SLE) or rheumatoid arthritis. In arthritic patients, RF and IgG associate to form an immune complex, which promotes disease progression. Recently, it has been reported that humanized anti-CD 4 IgG1 antibody having an Asn434His mutation induces significant rheumatoid factor binding (Zheng et al, Clin. Pharmacol. Ther.89 (2): 283-. Detailed studies confirmed that Asn434His mutation of human IgG1 increases binding of rheumatoid factor to the Fc region of the antibody compared to the parent human IgG 1.

RF is a polyclonal auto-antibody against human IgG. The RF epitope of the human IgG sequence varies between clones; however, the RF epitope appears to be located at the CH2/CH3 interface region as well as in the CH3 domain, which may overlap with the FcRn-binding epitope. Thus, mutations to increase FcRn-binding activity at neutral pH may also increase binding activity to a particular RF clone.

In the context of disclosure B, the term "anti-drug antibody" or "ADA" may refer to an endogenous antibody that has binding activity against an epitope located on the therapeutic antibody and is thus capable of binding the therapeutic antibody. The term "pre-existing anti-drug antibody" or "pre-existing ADA" may refer to an anti-drug antibody that is present and detectable in the blood of a patient prior to administration of a therapeutic antibody to the patient. In some embodiments, the pre-existing ADA is a human antibody. In another embodiment, the pre-existing ADA is a rheumatoid factor.

The binding activity of an antibody Fc region (variant) against pre-existing ADA can, for example, be represented by an Electrochemiluminescence (ECL) reaction at acidic pH and/or at neutral pH. ECL assays are described, for example, in Moxness et al (Clin chem. 51: 1983-1985(2005)) and example 6. The assay can be carried out, for example, in MES buffer at 37 ℃. The antigen binding activity of the antibody can be determined, for example, by BIACORE (registered trademark) analysis.

The binding activity to pre-existing ADA can be assessed at any temperature from 10 ℃ to 50 ℃. In some embodiments, the binding activity (binding affinity) of the human Fc region to human pre-existing ADA is determined at a temperature of 15 ℃ to 40 ℃, e.g., such as 20 ℃ to 25 ℃, or 25 ℃. In another embodiment, the interaction between human pre-existing ADA and human Fc region is measured at pH 7.4 (or pH 7.0) and 25 ℃.

Within the scope of the disclosure B described herein, a significant increase in binding activity to (pre-existing) ADA or an equivalent expression may mean, (pre-existing) ADA-binding activity (binding affinity) (i.e., KD) of the Fc region variant of publication B or an antibody comprising the same measured is increased as compared to the (pre-existing) ADA-binding activity (binding affinity) of the reference Fc region variant or a reference antibody comprising the reference Fc region variant measured, for example, 0.55-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, or 2.3-fold or more. This increase in binding activity to pre-existing ADA can be observed in individual patients or groups of patients.

In one embodiment, the term "plurality of patients" or "one patient" as used in the context of disclosure B is not limited and may include all people with a disease that are being treated with a therapeutic antibody. The patient may be a human suffering from an autoimmune disease, such as arthritis or Systemic Lupus Erythematosus (SLE). The arthritis may include rheumatoid arthritis.

In one embodiment of disclosure B, a significant increase in binding activity to a preexisting ADA in an individual patient can mean that the binding activity of an antibody comprising a variant Fc region (e.g., a therapeutic antibody) to a preexisting ADA measured in the patient is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or more when compared to the binding activity of a reference antibody to the preexisting ADA. Alternatively, this may mean that the ECL response of the antibody is preferably higher than 250, or at least 500, or at least 1000, or even at least 2000. Preferably, the increase may be an increase relative to a reference antibody whose ECL response is less than 500 or 250. Specifically, the ECL reaction preferably ranges from less than 250 to at least 250, less than 250 to at least 500, less than 500 to 500 or more, less than 500 to 1000 or more, or less than 500 to at least 2000, without being limited thereto, between the binding activity of a reference antibody to a pre-existing ADA and that of an antibody having a variant Fc region.

In one embodiment, an increased binding activity to a pre-existing ADA may mean that in the patient group the proportion of patients having an ECL response of at least 500 (preferably, at least 250 or more) to an antibody comprising an Fc region variant (a) having an increased binding activity to FcRn at acidic pH and (b) having an increased binding activity to a pre-existing ADA at neutral pH is increased by, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50% (when compared to the proportion of patients having an ECL response to the reference antibody), measured compared to the proportion of patients having an ECL response of at least 500 (preferably, at least 250 or more) to the reference antibody.

In one embodiment of disclosure B, a decrease in binding activity to a pre-existing ADA can mean a decrease in the binding activity (i.e., KD or ECL response) of an antibody comprising a variant Fc region measured as compared to a reference antibody. The reduction may be observed in an individual patient or group of patients. A significant decrease in affinity of an antibody comprising a variant Fc region for pre-existing ADA at neutral pH in each patient may mean that the binding activity measured at neutral pH for pre-existing ADA measured in the patient is decreased by, e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% as compared to the binding activity measured at neutral pH of a reference antibody for pre-existing ADA.

Alternatively, for example, a significant decrease in the binding activity of an antibody comprising a variant Fc region to pre-existing ADA in an individual patient may mean that the ECL response to the antibody, once 500 or more (preferably, 1000 or more, or 2000 or more) compared to the ECL response to a reference antibody, is measured to be less than 500, preferably less than 250.

In preferred embodiments, the Fc region variants of disclosure B and antibodies comprising them have low binding activity to pre-existing ADA at neutral pH. In particular, it is preferred that an antibody comprising an Fc region variant of disclosure B has less or no significant increase in binding activity to pre-existing ADA at neutral pH (e.g., pH 7.4) compared to the binding activity of a reference antibody comprising an Fc region of a native IgG to pre-existing ADA at neutral pH. Low binding activity (binding affinity) or affinity at baseline levels to pre-existing ADA may mean, but is not limited to, an ECL response of less than 500, or less than 250 in an individual patient. For example, a low binding activity to pre-existing ADA in a patient group may mean that ECL response is less than 500 in 90%, preferably 95%, and more preferably 98% of the patients in the group.

The Fc region variants of publication B or antibodies containing them may preferably be selected for which the binding activity to (pre-existing) ADA in plasma is not significantly increased at neutral pH and for which the FcRn-binding activity is increased at neutral pH and/or at acidic pH. Preferably, the FcRn-binding activity is increased at acidic pH (e.g., pH 5.8). In one embodiment, the Fc region variant preferably does not have significantly increased binding activity to ADA under neutral pH conditions (e.g., pH 7.4) compared to the Fc region of a native IgG, and the ADA may be a pre-existing ADA, preferably Rheumatoid Factor (RF).

In one embodiment, it may be preferred that the Fc region variants of publication B have increased FcRn-binding activity under acidic pH conditions compared to the Fc region of native IgG, and thus, they exhibit reduced clearance in plasma (CL), extended retention time in plasma, or extended half-life in plasma (t 1/2). Their association is known in the art.

In one embodiment, it may be preferred that the Fc region variants of publication B have increased FcRn-binding activity under acidic pH conditions, but not significantly increased ADA-binding activity under neutral pH conditions, and that they exhibit reduced clearance in plasma (CL), extended retention time in plasma, or extended half-life in plasma (t1/2) compared to the Fc region of native IgG. The ADA may be a pre-existing ADA, preferably Rheumatoid Factor (RF).

In one embodiment, the Fc region variants of disclosure B are advantageous in that their plasma retention is improved compared to a reference Fc region variant comprising the combination of amino acid substitutions N434Y/Y436V/Q438R/S440E (according to EU numbering).

Examples 5 to 7 compare the plasma retention of two Fc region variants: the Fc region variant F1718 (Fc region with mutations introduced at four positions: N434Y/Y436V/Q438R/S440E) and the novel Fc region variant F1848m (mutations introduced at four positions: N434A/Y436V/Q438R/S440E) described in WO 2013/046704. The difference in amino acid mutations between the two Fc region variants was only at position 434 according to EU numbering, where the amino acid mutation introduced was Y (tyrosine) for F1718 and a (alanine) for F1848 m. Nevertheless, F1848m exhibited improved plasma retention when compared to native IgG1, whereas F1718 did not show such improvement in plasma retention (see example (7-2)). Thus, the Fc region variant of disclosure B may preferably have improved plasma retention compared to a reference Fc region variant comprising the amino acid substitution combination N434Y/Y436V/Q438R/S440E. The experimental results described in examples (5-2) and (7-3) herein show that, among the various Fc region variants, F1847m, F1886m, F1889m, and F1927m are further improved in plasma retention time than F1848 m. Thus, one skilled in the art will appreciate that the Fc region variants of disclosure B (including F1847m, F1886m, F1889m, or F1927m, and F1848m) have improved plasma retention compared to the reference Fc region variant containing the substitutions N434Y/Y436V/Q438R/S440E.

Binding to Fc γ R or complement proteins may also have adverse effects (e.g., inappropriate platelet activation). Do not bind effector receptors such as Fc gamma RIIaVariants of the Fc region of the body may be safer and/or more advantageous. In some embodiments, the Fc region variants of disclosure B have only weak effector receptor-binding activity or no effector receptor binding. Examples of effector receptors include activating Fc γ rs, particularly Fc γ RI, Fc γ RII, and Fc γ RIII. Fc γ RI includes Fc γ RIa, Fc γ RIb, and Fc γ RIc, and subtypes thereof. Fc γ RII includes Fc γ RIIa (with two allotypes: R131 and H131) and Fc γ RIIb. Fc γ RIII includes Fc γ RIIIa (which has two allotypes: V158 and F158) and Fc γ RIIIb (which has two allotypes: Fc γ RIIIb-NA1 and Fc γ RIIIb-NA 2). Antibodies that have only weak effector receptor-binding activity or do not bind the receptor include, for example, antibodies containing a silent Fc region and antibodies that do not have an Fc region (e.g., Fab, F (ab) ')'₂，scFv，sc(Fv)₂And diabodies).

Examples of Fc regions having only weak or no effector receptor-binding activity are described, for example, in Strohl et al (Curr. Op. Biotech.20 (6): 685-. WO2008/092117 describes antibodies comprising a silenced Fc region comprising substitutions G236R/L328R, L235G/G236R, N325A/L328R, or N325L/L328R (numbering according to EU). WO2000/042072 describes antibodies comprising a silenced Fc region comprising a substitution at one or more of positions EU233 (according to EU numbering position 233), EU234, EU235, and EU 237. WO2009/011941 describes antibodies comprising a silent Fc region lacking residues from EU231 to EU 238. Davis et al (J.Rheum.34 (11): 2204-2210(2007)) describe antibodies having a silenced Fc region with the substitutions C220S/C226S/C229S/P238S. Shields et al (j.biol. chem.276 (9): 6591-6604(2001)) describe antibodies comprising a silent Fc region containing the substitution D265A. Modifications of these amino acid residues may also be suitably introduced into the Fc region variants of disclosure B.

The expression "binding weak to an effector receptor" may mean that the effector receptor-binding activity is, for example, 95% or less, preferably 90% or less, 85% or less, 80% or less, 75% or less, more preferably 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of a native IgG or an antibody containing the Fc region of a native IgG.

A "silent Fc region" is an Fc region variant containing one or more amino acid substitutions, insertions, additions, deletions, and the like, which reduces binding to an effector receptor as compared to the native Fc region. Because effector receptor-binding activity can be significantly reduced, the silenced Fc region can no longer bind to an effector receptor. The silent Fc region may include, for example, an Fc region containing amino acid substitutions at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU 332. Modifications at these amino acid positions may also be appropriately introduced into the Fc region variants of disclosure B.

In another embodiment, the silenced Fc region has a substitution at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332, and said position is preferably selected from the group consisting of: EU235, EU237, EU238, EU239, EU270, EU298, EU325, and EU329, wherein the substitutions utilize amino acid residues selected from the following table:

the amino acid at position EU234 is preferably substituted with an amino acid selected from the group consisting of: ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, and Thr.

The amino acid at position EU235 is preferably substituted with an amino acid selected from the group consisting of: ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, and Arg.

The amino acid at position EU236 is preferably substituted with an amino acid selected from the group consisting of: arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, and Tyr.

The amino acid at position EU237 is preferably substituted with an amino acid selected from the group consisting of: ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, and Arg.

The amino acid at position EU238 is preferably substituted with an amino acid selected from the group consisting of: ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, and Arg.

The amino acid at position EU239 is preferably substituted with an amino acid selected from the group consisting of: gln, His, Lys, Phe, Pro, Trp, Tyr, and Arg.

The amino acid at position EU265 is preferably substituted with an amino acid selected from the group consisting of: ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU266 is preferably substituted with an amino acid selected from the group consisting of: ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, and Tyr.

The amino acid at position EU267 is preferably substituted with an amino acid selected from the group consisting of: arg, His, Lys, Phe, Pro, Trp, and Tyr.

The amino acid at position EU269 is preferably substituted with an amino acid selected from the group consisting of: ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU270 is preferably substituted with an amino acid selected from the group consisting of: ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU271 is preferably substituted with an amino acid selected from the group consisting of: arg, His, Phe, Ser, Thr, Trp, and Tyr.

The amino acid at position EU295 is preferably substituted with an amino acid selected from the group consisting of: arg, Asn, Asp, Gly, His, Phe, Ser, Trp, and Tyr.

The amino acid at position EU296 is preferably substituted with an amino acid selected from the group consisting of: arg, Gly, Lys, and Pro.

The amino acid in position EU297 is preferably replaced with Ala.

The amino acid at position EU298 is preferably substituted with an amino acid selected from the group consisting of: arg, Gly, Lys, Pro, Trp, and Tyr.

The amino acid at position EU300 is preferably substituted with an amino acid selected from the group consisting of: arg, Lys, and Pro.

The amino acid at position EU324 is preferably substituted with Lys or Pro.

The amino acid at position EU325 is preferably substituted with an amino acid selected from the group consisting of: ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, and Val.

The amino acid at position EU327 is preferably substituted with an amino acid selected from the group consisting of: arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU328 is preferably substituted with an amino acid selected from the group consisting of: arg, Asn, Gly, His, Lys, and Pro.

The amino acid at position EU329 is preferably substituted with an amino acid selected from the group consisting of: asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, and Arg.

The amino acid at position EU330 is preferably replaced with Pro or Ser.

The amino acid at position EU331 is preferably substituted with an amino acid selected from the group consisting of: arg, Gly, and Lys.

The amino acid at position EU332 is preferably substituted with an amino acid selected from the group consisting of: arg, Lys, and Pro.

The silent Fc region may preferably comprise a substitution by Lys or Arg in EU235, a substitution by Lys or Arg in EU237, a substitution by Lys or Arg in EU238, a substitution by Lys or Arg in EU239, a substitution by Phe in EU270, a substitution by Gly in EU298, a substitution by Gly in EU325, or a substitution by Lys or Arg in EU 329. More preferably, the silent Fc region may contain a substitution with arginine in EU235 or a substitution with lysine in EU 239. Even more preferably, the silenced Fc region can comprise the L235R/S239K substitutions. Modifications of these amino acid residues may also be suitably introduced into the Fc region variants of disclosure B.

In one embodiment, an antibody comprising a variant Fc region of disclosure B has only weak complement protein-binding activity or does not bind complement proteins. In some embodiments, the complement protein is C1 q. In some embodiments, a weak complement protein-binding activity refers to a 10-fold or greater, 50-fold or greater, or 100-fold or greater complement protein-binding activity that is reduced compared to the complement protein-binding activity of a native IgG or an antibody containing the Fc region of a native IgG. The complement protein-binding activity of the Fc region may be reduced by amino acid modifications such as amino acid substitutions, insertions, additions, or deletions.

In one embodiment, the Fc region variant of disclosure B or an antibody comprising the Fc region variant may be assessed for (human) FcRn-binding activity in the neutral pH range and/or in the acidic pH range in the same manner as described above.

In one embodiment, the method of modifying antibody constant regions to produce Fc region variants of disclosure B may be based on, for example, evaluating several constant region isotypes (IgG1, IgG2, IgG3, and IgG4) to select isotypes with reduced antigen binding activity in the acidic pH range and/or increased off-rates in the acidic pH range. Alternative methods may be based on introducing amino acid substitutions into the amino acid sequence of the native IgG isotype to reduce antigen binding activity and/or increase off-rate in the acidic pH range (e.g., pH 5.8). The hinge region sequence of the antibody constant region varies widely between isotypes (IgG1, IgG2, IgG3, and IgG4), and differences in the hinge-region amino acid sequence can have a significant effect on antigen-binding activity. Thus, isoforms having reduced antigen binding activity in the acidic pH range and/or increased off-rates in the acidic pH range may be selected by selecting appropriate isoforms depending on the type of antigen or epitope. Furthermore, because differences in the hinge region amino acid sequence can have a significant effect on antigen binding activity, amino acid substitutions in the amino acid sequence of the native isoform can be located in the hinge region.

In an alternative embodiment, disclosure B provides for the use of an antibody comprising a variant Fc region of disclosure B described above to accelerate release of an antibody internalized into a cell in an antigen-bound form, out of the cell in a form free from the antigen. Herein, "an antibody internalized into a cell in an antigen-bound form is released extracellularly in an antigen-free form" does not necessarily mean that the antibody internalized into a cell in an antigen-bound form is completely released extracellularly in an antigen-free form. It is acceptable that the proportion of antibody released extracellularly in the form free from antigen is increased compared to before modification of its FcRn-binding domain (e.g. before increasing FcRn-binding activity of the antibody in the acidic pH range). Preferably, the antibody released outside the cell retains its antigen binding activity.

"ability to remove antigen from plasma" or equivalent terms may refer to the ability to remove antigen from plasma when the antibody is administered or secreted in vivo. Thus, "an antibody has an increased ability to remove antigen from plasma" may mean that, when the antibody is administered, the rate of removal of antigen from plasma is increased, e.g., compared to before modification of its FcRn-binding domain. The increase in the antigen-removing activity of an antibody from plasma can be evaluated, for example, by administering a soluble antigen and an antibody in vivo, and measuring the concentration of the soluble antigen in plasma after administration. The soluble antigen may be antibody-bound or antibody-free antigen, and its concentration may be determined as "antibody-bound antigen concentration in plasma" and "antibody-free antigen concentration in plasma", respectively. The latter is synonymous with "concentration of free antigen in plasma". "Total antigen concentration in plasma" may refer to the sum of the concentration of antibody-bound antigen and the concentration of antibody-free antigen.

In an alternative embodiment, publication B provides a method of increasing the plasma retention time of an antibody comprising an Fc region variant of publication B. Native human IgG is capable of binding to FcRn derived from non-human animals. For example, antibodies can be administered to mice to assess the properties of the antibodies because native human IgG binds more strongly to mouse FcRn than to human FcRn (Ober et al, Intl. Immunol.13 (12): 1551-1559 (2001)). Alternatively, for example, mice that have and express as transgenes instead of, and whose own FcRn gene is disrupted (ropenian et al, meth.mol.biol.602: 93-104(2010)) are also suitable for evaluating antibodies.

Within the disclosure A and B described herein, the plasma concentration of free antigen not bound to antibody or the ratio of free antigen concentration to total antigen concentration can be determined (e.g., Ng et al, pharm. Res.23 (1): 95-103 (2006)). Alternatively, where an antigen exhibits a particular in vivo function, whether the antigen binds to an antibody (antagonist molecule) that neutralizes the antigen function can be assessed by testing whether the antigen function is neutralized. Whether antigen function is neutralized can be assessed by measuring specific in vivo markers that reflect antigen function. Whether an antigen binds to an antibody (agonist molecule) that activates the function of the antigen can be assessed by measuring specific in vivo markers that reflect the function of the antigen.

There are no particular limitations on the measurement (e.g., determination of the concentration of free antigen in plasma, determination of the ratio of the amount of free antigen to the amount of total antigen in plasma, and in vivo marker measurement); however, the measurement may preferably be performed after a certain time period after the antibody administration. Within the scope of the disclosure B, "after a certain period of time after antibody administration" is not particularly limited, and the period may be appropriately determined by those skilled in the art depending on the nature of the antibody administered and the like, and includes, for example, one day, three days, seven days, 14 days, or 28 days after antibody administration. As used herein, the term "plasma antigen concentration" can refer to either "total antigen concentration in plasma" (which is the sum of antibody-bound antigen concentration and antibody-free antigen concentration), or "free antigen concentration in plasma" (which is antibody-free antigen concentration).

The molar ratio of antigen to antibody may be determined using the following formula: c ═ a/B calculation, where the value a is the molar concentration of antigen at each time point, the value B is the molar concentration of antibody at each time point, and the value C is the molar concentration of antigen/molar concentration of antibody (molar ratio of antigen/antibody) at each time point.

A smaller C value indicates a higher efficiency of each antibody in removing antigen, and a larger C value indicates a lower efficiency of each antibody in removing antigen.

In some aspects, the antigen/antibody molar ratio is reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold or more when the antibody of disclosure B is administered compared to when a reference antibody containing a native human IgG Fc region is administered as a human FcRn-binding domain.

The reduction in total antigen concentration or antigen/antibody molar ratio in plasma can be assessed using methods known in the art, as described in examples 6, 8 and 13 of WO 2011/122011. More specifically, when the antibodies of interest in disclosure B do not cross-react with the mouse corresponding antigen, they can be evaluated using human FcRn transgenic mouse strain 32 or 276(Jackson Laboratories, Methods mol. biol. 602: 93-104(2010)) based on an antigen-antibody co-injection model or a steady-state antigen infusion model. When the antibody cross-reacts with the mouse counterpart, it can be assessed by simply injecting the antibody into human FcRn transgenic mouse strain 32 or 276(Jackson Laboratories). In the co-injection model, a mixture of antibody and antigen is administered to mice. In the steady-state antigen infusion model, an infusion pump filled with an antigen solution is implanted into mice to achieve a constant antigen concentration in plasma, and then antibodies are injected into the mice. All test antibodies were administered at the same dose. The total antigen concentration in plasma, the free antigen concentration in plasma, and the antibody concentration in plasma can be measured at appropriate time points.

To assess the long-term effect of the antibodies of disclosure B, the total or free antigen concentration, or antigen/antibody molar ratio, in plasma can be measured two days, four days, seven days, 14 days, 28 days, 56 days, or 84 days after administration. In other words, for assessing the properties of an antibody, the antigen concentration in plasma over a long period of time can be determined by measuring the total or free antigen concentration, or the antigen/antibody molar ratio, in plasma two days, four days, seven days, 14 days, 28 days, 56 days, or 84 days after administration of the antibody. Whether the concentration of antigen or the molar ratio of antigen/antibody in plasma decreases with antibody can be determined by assessing the decrease at one or more time points as described above.

To assess the short-term effects of the antibodies of disclosure B, the total or free antigen concentration, or antigen/antibody molar ratio, in the plasma can be measured 15 minutes, one hour, two hours, four hours, eight hours, 12 hours, or 24 hours after administration. In other words, for assessing the properties of an antibody, the concentration of antigen in plasma over a short period of time can be determined by measuring the total or free antigen concentration in plasma, or the molar ratio of antigen/antibody 15 minutes, one hour, two hours, four hours, eight hours, 12 hours, or 24 hours after administration. When plasma retention in humans is difficult to determine, it can be predicted based on plasma retention in mice (e.g., normal mice, transgenic mice expressing human antigens, or transgenic mice expressing human FcRn) or in monkeys (e.g., cynomolgus monkeys).

In an alternative embodiment, disclosure B relates to an antibody comprising an Fc region variant of disclosure B above. Various embodiments of the antibodies described within the scope of the disclosures a and B described herein may be employed without departing from the general technical knowledge of the field, unless the context is inconsistent.

In one embodiment, antibodies comprising Fc region variants of disclosure B are used as therapeutic antibodies for treating human patients with autoimmune diseases, transplant rejection (graft versus host disease), other inflammatory diseases, or allergic diseases, as described in WO 2013/046704.

In one embodiment, an antibody comprising an Fc region variant of disclosure B may have a modified sugar chain. The antibody having a modified sugar chain may include, for example, an antibody having modified glycosylation (WO99/54342), an antibody lacking fucose (WO00/61739, WO02/31140, WO2006/067847, WO2006/067913), and an antibody having a sugar chain in which GlcNAc is divided into two parts (WO 02/79255). In one embodiment, the antibody may be deglycosylated. In some embodiments, the antibody comprises, for example, a mutation at the heavy chain glycosylation site to inhibit glycosylation at that position, as described in WO 2005/03175. The aglycosylated antibody may be prepared by modifying the heavy chain glycosylation site, i.e. by introducing an N297Q or N297A substitution (according to EU numbering), and expressing the protein in a suitable host cell.

In an alternative embodiment, disclosure B relates to a composition or pharmaceutical composition comprising an antibody comprising a variant of said Fc region. Various embodiments of the compositions or pharmaceutical compositions described within the scope of the disclosure a and B herein may be employed without departing from the ordinary skill in the art, except where inconsistent from the context. The compositions can be used to enhance plasma retention (in a subject, when the antibody of disclosure B is administered (applied) to the subject).

In an alternative embodiment, disclosure B relates to a nucleic acid encoding an Fc region variant or an antibody comprising an Fc region variant. The various embodiments of nucleic acids described within a and B of the disclosure described herein may be applied without departing from the general technical knowledge in the field, unless the context is inconsistent. Alternatively, disclosure B relates to a vector comprising the nucleic acid. Various embodiments within the scope of the disclosures a and B described herein may be applied without departing from the general technical knowledge in the field, unless the context is inconsistent. Alternatively, disclosure B relates to a host or host cell comprising the vector. Various embodiments within the scope of the disclosure a and B described herein may be applied without departing from the general technical knowledge in the field, unless the context is inconsistent.

In an alternative embodiment, disclosure B relates to a method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising said Fc region variant, comprising culturing the above-described host cell, or culturing the above-described host and collecting the Fc region variant or the antibody comprising said Fc region variant from material secreted by the host from the cell culture. In this case, disclosure B may include a production method optionally further including any one or more of: (a) selecting a variant Fc region having enhanced FcRn-binding activity under acidic pH conditions as compared to the Fc region of native IgG; (b) selecting a variant Fc region whose binding activity to (pre-existing) ADA is not significantly enhanced under neutral pH conditions compared to the Fc region of native IgG; (c) selecting a variant Fc region having increased plasma retention compared to the Fc region of a native IgG; and (d) selecting an antibody comprising a variant Fc region capable of promoting removal of antigen from plasma compared to a reference antibody comprising the Fc region of a native IgG.

From the viewpoint of evaluating plasma retention of the Fc region variant of disclosure B, without limitation, it may be preferable that the antibody comprising the Fc region variant and the "reference antibody comprising an Fc region of natural IgG" produced in disclosure B are identical to each other (except for the Fc region to be compared). FcRn may be human FcRn.

For example, after an antibody comprising an Fc region variant of disclosure B is produced, the antibody can be compared to a reference antibody comprising a native IgG Fc region for FcRn-binding activity under acidic pH conditions (e.g., pH 5.8) using BIACORE (registered trademark) or other known techniques to select an Fc region variant or an antibody comprising the Fc region variant whose FcRn-binding activity increases under acidic pH conditions.

Alternatively, for example, after an antibody comprising an Fc region variant of disclosure B is produced, the antibody can be compared to a reference antibody comprising a native IgG Fc region for ADA-binding activity at neutral pH conditions by Electrochemiluminescence (ECL) or known techniques to select an Fc region variant or an antibody comprising the Fc region variant whose ADA-binding activity is not significantly increased at neutral pH conditions.

Alternatively, for example, after an antibody comprising an Fc region variant of disclosure B is produced, the antibody can be compared to a reference antibody comprising a native IgG Fc region by antibody pharmacokinetic testing using plasma (e.g., from mouse, rat, rabbit, dog, monkey, or human) to select an Fc region variant or an antibody comprising the Fc region variant that demonstrates improved plasma retention in a subject.

Alternatively, for example, after an antibody comprising an Fc region variant of disclosure B is produced, the antibody can be compared to a reference antibody comprising a native IgG Fc region by antibody pharmacokinetic testing using plasma (e.g., from mouse, rat, rabbit, dog, monkey, or human) to select an Fc region variant or an antibody comprising the Fc region variant with enhanced antigen removal from plasma.

Alternatively, for example, the above selection methods may be appropriately combined, if necessary.

In one embodiment, disclosure B relates to a method of producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising said variant, wherein said method comprises making amino acid substitutions in such a way that the resulting Fc region variant or antibody comprising said variant comprises Ala at position 434; glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440 (according to EU numbering). In a further embodiment, the method comprises substituting amino acids in such a way that the resulting Fc-region variant or antibody comprising said variant further comprises Ile or Leu at position 428 and/or Ile, Leu, Val, Thr, or Phe (numbering according to EU) at position 436. In another embodiment, the amino acids are substituted in such a way that the resulting variant of the Fc-region produced according to said method or an antibody comprising said variant further comprises Leu at position 428 and/or Val or Thr at position 436 (numbering according to EU).

In one embodiment, disclosure B relates to a method for producing a variant Fc region comprising an FcRn-binding domain or an antibody comprising said variant, wherein said method comprises performing amino acid substitutions in such a way that the resulting variant Fc region or antibody comprising said variant comprises an Ala at position 434; arg or Lys at position 438; and Glu or Asp at position 440 (according to EU numbering). In a further embodiment, the method comprises substituting amino acids in such a way that the resulting Fc-region variant or antibody comprising said variant further comprises Ile or Leu at position 428 and/or Ile, Leu, Val, Thr, or Phe (numbering according to EU) at position 436. In another embodiment, the amino acids are substituted in such a way that the resulting variant of the Fc-region produced according to the method or an antibody comprising said variant further comprises Leu at position 428 and/or Val or Thr at position 436 (numbering according to EU).

In one embodiment, the method comprises contacting the cell with Ala at positions 434, 438, and 440, respectively; glu, Arg, Ser, or Lys; and Glu, Asp, or Gln for all amino acids. In a further embodiment, the method comprises substituting amino acids in such a way that the resulting Fc-region variant or antibody comprising said variant further comprises Ile or Leu at position 428 and/or Ile, Leu, Val, Thr, or Phe (numbering according to EU) at position 436. In another embodiment, the amino acids are substituted in such a way that the resulting variant of the Fc-region produced according to the method or an antibody comprising said variant further comprises Leu at position 428 and/or Val or Thr at position 436 (numbering according to EU).

In an alternative embodiment, the disclosure B relates to an Fc region variant or an antibody comprising said Fc region variant obtained by any one of the above methods of production of disclosure B.

In an alternative embodiment, disclosure B provides a method for reducing the (pre-existing) ADA-binding activity of an antibody comprising a variant Fc region having increased FcRn-binding activity at acidic pH; and methods for producing a variant Fc region having increased FcRn-binding activity and decreased pre-existing ADA-binding activity at acidic pH (e.g., pH 5.8), the method comprising: (a) providing an antibody comprising an Fc region (variant) with increased FcRn-binding activity at acidic pH as compared to a reference antibody; and (b) introducing into the Fc region according to EU numbering, (i) an amino acid substitution at position 434 with Ala; (ii) an amino acid substitution at position 438 with any one of Glu, Arg, Ser, and Lys; and (iii) an amino acid substitution at position 440 with any one of Glu, Asp, and Gln, (iv) optionally, an amino acid substitution at position 428 with Ile or Leu; and/or (v) optionally, an amino acid substitution at position 436 with any one of Ile, Leu, Val, Thr, and Phe.

In some embodiments, the Fc domain (variant) in step (a) is preferably a human IgG Fc domain (variant). Furthermore, in order to increase FcRn-binding activity at acidic pH and decrease (pre-existing) ADA-binding activity within a neutral pH range (e.g., pH 7.4), the Fc region (variant) contains a combination of amino acid substitutions selected from the group consisting of: (a) N434A/Q438R/S440E; (b) N434A/Q438R/S440D; (c) N434A/Q438K/S440E; (d) N434A/Q438K/S440D; (e) N434A/Y436T/Q438R/S440E; (f) N434A/Y436T/Q438R/S440D; (g) N434A/Y436T/Q438K/S440E; (h) N434A/Y436T/Q438K/S440D; (i) N434A/Y436V/Q438R/S440E; (j) N434A/Y436V/Q438R/S440D; (k) N434A/Y436V/Q438K/S440E; (l) N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/S440E; (N) N434A/R435H/F436T/Q438R/S440D; (o) N434A/R435H/F436T/Q438K/S440E; (p) N434A/R435H/F436T/Q438K/S440D; (Q) N434A/R435H/F436V/Q438R/S440E; (R) N434A/R435H/F436V/Q438R/S440D; (S) N434A/R435H/F436V/Q438K/S440E; (t) N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v) M428L/N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x) M428L/N434A/Q438K/S440D; (Y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/Y436T/Q438R/S440D; (aa) M428L/N434A/Y436T/Q438K/S440E; (ab) M428L/N434A/Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad) M428L/N434A/Y436V/Q438R/S440D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af) M428L/N434A/Y436V/Q438K/S440D; (ag) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (ah) L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

The method may optionally further comprise: (c) assessing whether the (pre-existing) ADA-binding activity of an antibody comprising the produced Fc region variant is reduced compared to the binding activity of a reference antibody.

Alternatively, the method may be used as a method of enhancing the release of an antibody internalized into a cell in an antigen-bound form, out of the cell in an antigen-free form, without significantly increasing the (pre-existing) ADA-binding activity of the antibody at neutral pH.

Disclosure C

Disclosure C also relates to anti-IL-8 antibodies, nucleic acids encoding the antibodies, pharmaceutical compositions comprising the antibodies, methods of producing the antibodies, and uses of the antibodies in the treatment of IL-8 related diseases, as described below. The meanings of the terms given below apply in the description of the disclosure C herein, without departing from the general technical knowledge of the person skilled in the art and the embodiments known to the person skilled in the art.

I. Definitions within the scope of disclosure C

Within the scope of disclosure C described herein, "acidic pH" refers to a pH that may be selected from, for example, pH 4.0 to pH 6.5. In one embodiment, acidic pH refers to, but is not limited to, pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, or pH 6.5. In a particular embodiment, the term acidic pH refers to pH 5.8.

Within the scope of disclosure C described herein, "neutral pH" refers to a pH that may be selected from, for example, pH 6.7 to pH 10.0. In one embodiment, neutral pH refers to, but is not limited to, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.5, or pH 10.0. In a particular embodiment, the term neutral pH refers to pH 7.4.

The term "IL-8", as used in disclosure C, refers to any native IL-8 derived from any vertebrate, primate (e.g., human, cynomolgus monkey, macaque monkey) and other mammals (e.g., dogs and rabbits), unless otherwise specified. The term "IL-8" includes full-length IL-8, unprocessed IL-8, as well as any form of IL-8 that is processed in cells. The term "IL-8" also includes derivatives of native IL-8, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human IL-8 is set forth in SEQ ID NO: 66, respectively.

The terms "anti-IL-8 antibody" and "antibody that binds IL-8" refer to an antibody that is capable of binding IL-8 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting IL-8.

In one embodiment, the anti-IL-8 antibody to irrelevant, non-IL-8 protein binding degree, for example, less than the antibody to IL-8 binding about 10%.

"affinity" within the context of the description of disclosure C herein generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used within the context of the description of disclosure C herein refers to intrinsic binding affinity, which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can be generally represented by the dissociation constant (KD). Binding affinity can be measured using methods known in the art, including those described within the scope of the description of disclosure C herein.

In certain embodiments, an antibody that binds IL-8 can have a dissociation constant (KD), e.g.,

less than or equal to 1000nM, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM or less than or equal to 0.001nM

(e.g., 10)^-8M or less, 10^-8M to 10^-13M，10^-9M to 10^-13M)。

The term "antibody" is used in the broadest sense within the description of disclosure C herein and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen binding activity.

An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by, for example, 50%, 60%, 70%, or 80% or more; and, conversely, the reference antibody blocks binding of the antibody to its antigen by, for example, 50%, 60%, 70%, or 80% or more. Herein, exemplary competition assays may be used, but are not limited thereto.

By "chimeric antibody" is meant an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder is derived from a different source or species.

"humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody may comprise substantially at least one, and typically two, variable regions, in which (or substantially all) HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all (or substantially all) FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

The term "monoclonal antibody" as used within the context of the description of disclosure C herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., those produced during production of naturally occurring mutant or monoclonal antibody preparations, which are typically present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with disclosure C can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals comprising all or part of a human immunoglobulin locus, and other exemplary methods of preparing monoclonal antibodies are described herein.

Within the context of the disclosure C described herein, "natural antibody" refers to an immunoglobulin molecule having a variety of naturally occurring structures. In one embodiment, a native IgG antibody, for example, a heterotetrameric glycoprotein of about 150,000 daltons, consists of two identical light chains and two identical heavy chains that are disulfide bonded. Each heavy chain has, in N-to C-terminal order, a variable region (VH), also known as the variable heavy or variable domain, followed by three constant domains (CH1, CH2, and CH 3). Likewise, each light chain has, in N-to C-terminal order, a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain. Antibody light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The constant domains used in disclosure C include those of any reported allotype (allele) or any subclass/isotype. Heavy chain constant regions include, but are not limited to, the constant regions of natural IgG antibodies (IgG1, IgG2, IgG3, and IgG 4). IgG1 alleles are known to include, for example, IGHG1 x 01, IGHG1 x 02, IGHG1 x 03, IGHG1 x 04, and IGHG1 x 05 (see imgt. org), and any of these can be used as a native human IgG1 sequence. The constant domain sequence may be derived from a single allele or subclass/isoform or from multiple alleles or subclasses/isoforms. In particular, the antibodies include, but are not limited to, antibodies whose CH1 is derived from IGHG1 x 01 and CH2 and CH3 are derived from IGHG1 x 02 and IGHG1 x 01, respectively.

"effector function" within the context of the description of disclosure C herein refers to a biological activity attributable to the Fc region of an antibody, which may vary with antibody isotype. Examples of antibody effector functions include: c1q binds to complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation, but is not limited thereto.

The term "Fc region" is used within the context of the description of disclosure C herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native Fc regions and variant Fc regions. The native Fc region represents the Fc region of a native antibody.

In one embodiment, the human IgG heavy chain Fc region extends from amino acid residues Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residue 446-447) of the Fc region may or may not be present. Unless otherwise indicated within the context of the description of disclosure C herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequence of Proteins of Immunological Interest, published Health Service, National Institutes of Health, Bethesda, MD, 1991.

"framework region" or "FR" in the context of the description of disclosure C herein refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3, FR 4. Thus, HVR and FR sequences typically occur in VH (or VL) in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

"human consensus framework region" is within the scope of the description of disclosure C herein a framework region representing the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework regions sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, a subset of sequences is a subset according to Kabat et al, Sequence of Proteins of Immunological Interest, 5 th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

In one embodiment, the subgroup of VLs is subgroup kappa I, as in Kabat et al, supra. In one embodiment, the subgroup of VH is subgroup III, as in Kabat et al, supra.

"acceptor (acceptor) human framework region" is, for the purposes of the description of disclosure C herein, a framework region comprising the amino acid sequence of a VL or VH framework region derived from a human immunoglobulin framework region or a human consensus framework region. An acceptor human framework region "derived from" a human immunoglobulin framework region or a human consensus framework region may comprise the same amino acid sequence thereof, or it may contain existing amino acid sequence substitutions. In some embodiments, the number of existing amino acid substitutions is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In one embodiment, the VL acceptor human framework region is identical to a VL human immunoglobulin framework region sequence or a human consensus framework region sequence.

The term "variable region" or "variable domain" within the context of the description of disclosure C herein refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable regions of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved Framework Regions (FRs) and three hypervariable regions (HVRs). (see, but not limited to, Kindt et al, Kuby Immunology, 6 th edition, W.H.Freeman & Co., page 91 (2007)). Furthermore, antibodies that bind a particular antigen can be isolated using VH or VL domains, respectively, from the antibody that binds the antigen to screen a library of complementary VL or VH domains. See, e.g., Portolano et al, j.immunol.150: 880- & ltwbr & gt 887 & gt (1993); clarkson et al, Nature 352: 624-628(1991).

The term "hypervariable region" or "HVR" as used within the context of the description of disclosure C herein refers to each of the antibody variable domains in the sequence that are hypervariable ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen contacts"). Typically, antibodies comprise six hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

Without being limited thereto, exemplary HVRs herein include: (a) hypervariable loops in which the amino acid residues are 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196: 901-917 (1987)); (b) CDRs in which the amino acid residues are 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2), and 95-102(H3) (Kabat et al, Sequence of Proteins of Immunological Interest, published Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts in which the amino acid residues are 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2), and 93-101(H3) (MacCallum et al, J.mol.biol.262: 732-745 (1996)); and (d) combinations of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3), and 94-102 (H3).

Unless otherwise indicated, HVRs and other residues in the variable region (e.g., FR residues) are numbered as in Kabat et al, supra.

An "individual" is a mammal within the scope of the description of disclosure C herein. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an "individual" is a human.

An "isolated" antibody is within the scope of the description of disclosure C herein an antibody that is isolated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromatogr.b 848: 79-87(2007).

An "isolated" nucleic acid, within the context of the description of disclosure C herein, refers to a nucleic acid molecule that is separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"isolated nucleic acid encoding an anti-IL-8 antibody" within the context of the description of disclosure C herein refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an anti-IL-8 antibody, including the one or more nucleic acids in a single vector or in separate vectors, one or more nucleic acids present at one or more locations in a host cell.

Within the context of the description of disclosure C herein, the terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of progeny. Progeny may not have exactly the same nucleic acid content as their parent cell, but may contain mutations. Progeny of mutants screened or selected for the same function or biological activity in the originally transformed cell are included herein.

The term "vector", as used within the context of the description of disclosure C herein, refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are introduced into a host cell and become integrated in its genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Within the context of the description of disclosure C herein, the term "package insert" is used to refer to instructions for use, typically included in commercial packaging for a therapeutic product, that contain information about the indications, use, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of the therapeutic product.

Within the context of the description of disclosure C, "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after alignment of the sequences by introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity in order to obtain the maximum percent sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the ability of those of ordinary skill in the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, megalign (dnastar) software, or GENETYX (registered trademark) (GENETYX co., Ltd.). One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, a value for% amino acid sequence identity is generated, for example, using the sequence comparison computer program ALIGN-2. ALIGN-2 is authorized by Genentech, inc, and the source code is submitted with the user profile at the us copyright office, Washington d.c., 20559, which is registered under us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX (registered trademark) operating system, including the digital UNIX (registered trademark) v4.0d. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity% (which may alternatively be expressed as a given amino acid sequence a having or comprising a certain% amino acid sequence identity with a given amino acid sequence B) of a given amino acid sequence a with a given amino acid sequence B is calculated as follows: 100 times the score X/Y, where X is the number of amino acid residues found to be an identical match in the program alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. Unless specifically stated otherwise, all% values of amino acid sequence identity are obtained using the ALIGN-2 computer program indicated within the scope of the description of disclosure C herein.

Within the context of the disclosure C described herein, "pharmaceutical composition" generally refers to an agent that treats, prevents, examines or diagnoses a disease. By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Such pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, and preservatives.

As used within the scope of disclosure C described herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural process of the individual being treated. The clinical intervention may be performed for prophylaxis or during clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or lessening the disease state and or improving prognosis. In one embodiment, the antibody of disclosure C may be used to slow the progression of a disease or disorder.

Within the scope of disclosure C described herein, an "effective amount" of an antibody or pharmaceutical composition refers to an amount that is effective when used at the dosages and for the period of time required to achieve the desired therapeutic or prophylactic result.

Compositions and methods

In one embodiment, disclosure C is based on the applicability of an anti-IL-8 antibody with pH-dependent affinity for IL-8 as a pharmaceutical composition. The antibodies of disclosure C, for example, are useful in diagnosing or treating diseases in which IL-8 is present in excess.

A. Exemplary anti-IL-8 antibodies

In one embodiment, the disclosure C provides anti-IL-8 antibodies with pH-dependent affinity for IL-8.

In one embodiment, disclosure C provides an anti-IL-8 antibody having a pH-dependent affinity for IL-8 comprising a sequence having at least one, two, three, four, five, six, seven, or eight amino acid substitutions within the following amino acid sequences: (a) comprises the amino acid sequence of SEQ ID NO: 67, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 68, HVR-H2 of the amino acid sequence of seq id no; (c) comprises the amino acid sequence of SEQ ID NO: 69, HVR-H3 of the amino acid sequence of; (d) comprises the amino acid sequence of SEQ ID NO: 70, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 71 amino acid sequence HVR-L2; and (f) comprises SEQ ID NO: 72, HVR-L3 of the amino acid sequence of.

In another embodiment, the disclosure C provides an anti-IL-8 antibody having pH-dependent affinity for IL-8 comprising at least one amino acid substitution in at least one of the following amino acid sequences: (a) comprises the amino acid sequence of SEQ ID NO: 67, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 68, HVR-H2 of the amino acid sequence of seq id no; (c) comprises the amino acid sequence of SEQ ID NO: 69, HVR-H3 of the amino acid sequence of; (d) comprises the amino acid sequence of SEQ ID NO: 70, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 71 amino acid sequence HVR-L2; and (f) comprises SEQ ID NO: 72, HVR-L3 of the amino acid sequence of.

Unless otherwise indicated, an amino acid may be substituted with any other amino acid. In one embodiment, the anti-IL-8 antibody of disclosure C comprises one or more amino acid substitutions at a position selected from the group consisting of: (a) sequence SEQ ID NO: 67, aspartic acid at position 1; (b) sequence SEQ ID NO: tyrosine at position 2 in 67; (c) sequence SEQ ID NO: tyrosine at position 3 in 67; (d) sequence SEQ ID NO: 67 leucine at position 4; (e) sequence SEQ ID NO: 67 serine at position 5; (f) sequence SEQ ID NO: 68 at position 1; (g) sequence SEQ ID NO: isoleucine at position 2 in 68; (h) sequence SEQ ID NO: arginine at position 3 in 68; (i) sequence SEQ ID NO: 68 asparagine at position 4; (j) sequence SEQ ID NO: lysine at position 5 in 68; (k) sequence SEQ ID NO: alanine at position 6 of 68; (l) Sequence SEQ ID NO: 68 asparagine at position 7; (m) sequence SEQ ID NO: glycine at position 8 in 68; (n) sequence SEQ ID NO: tyrosine at position 9 in 68; (o) sequence SEQ ID NO: threonine at position 10 in 68; (p) sequence SEQ ID NO: arginine at position 11 in 68; (q) sequence SEQ ID NO: glutamic acid at position 12 in 68; (r) sequence SEQ ID NO: tyrosine at position 13 in 68; (s) SEQ ID NO: 68 at position 14; (t) sequence SEQ ID NO: alanine at position 15 in 68; (u) sequence SEQ ID NO: 68 serine at position 16; (v) sequence SEQ ID NO: valine at position 17 of 68; (w) sequence SEQ ID NO: 68 lysine at position 18; (x) Sequence SEQ ID NO: 68 glycine at position 19; (y) sequence SEQ ID NO: 69 glutamic acid at position 1; (z) sequence SEQ ID NO: asparagine at position 2 in 69; (aa) sequence SEQ ID NO: tyrosine at position 3 in 69; (ab) sequence SEQ ID NO: 69 arginine at position 4; (ac) sequence SEQ ID NO: tyrosine at position 5 in 69; (ad) sequence SEQ ID NO: 69 aspartic acid at position 6; (ae) sequence SEQ ID NO: valine at position 7 of 69; (af) sequence SEQ ID NO: 69 glutamic acid at position 8; (ag) sequence SEQ ID NO: 69 leucine at position 9; (ah) sequence SEQ ID NO: 69 alanine at position 10; (ai) sequence SEQ ID NO: tyrosine at position 11 in 69; (aj) sequence SEQ ID NO: arginine at position 1 in 70; (ak) sequence SEQ ID NO: alanine at position 2 in 70; (al) sequence SEQ ID NO: serine at position 3 in 70; (am) sequence SEQ ID NO: glutamic acid at position 4 in 70; (an) sequence SEQ ID NO: isoleucine at position 5 in 70; (ao) sequence SEQ ID NO: isoleucine at position 6 in 70; (ap) sequence SEQ ID NO: tyrosine at position 7 in 70; (aq) sequence SEQ ID NO: serine at position 8 in 70; (ar) sequence SEQ ID NO: tyrosine at position 9 in 70; (as) sequence SEQ ID NO: 70 leucine at position 10; (at) sequence SEQ ID NO: alanine at position 11 in 70; (au) sequence SEQ ID NO: 71 asparagine at position 1; (av) sequence SEQ ID NO: alanine at position 2 in 71; (aw) sequence SEQ ID NO: 71 lysine at position 3; (ax) sequence SEQ ID NO: threonine at position 4 in 71; (ay) sequence SEQ ID NO: 71 leucine at position 5; (az) sequence SEQ ID NO: 71 alanine at position 6; (ba) sequence SEQ ID NO: 71 aspartic acid at position 7; (bb) sequence SEQ ID NO: glutamine at position 1 in 72; (bc) sequence SEQ ID NO: histidine at position 2 in 72; (bd) sequence SEQ ID NO: histidine at position 3 in 72; (be) sequence SEQ ID NO: phenylalanine at position 4 in 72; (bf) sequence SEQ ID NO: glycine at position 5 in 72; (bg) sequence SEQ ID NO: phenylalanine at position 6 in 72; (bh) sequence of SEQ ID NO: (iii) proline at position 7 in 72; (bi) sequence SEQ ID NO: arginine at position 8 in 72; and (bj) the sequence SEQ ID NO: threonine at position 9 in 72.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises one or more amino acid substitutions at positions selected from the group consisting of: (a) sequence SEQ ID NO: alanine at position 6 of 68; (b) sequence SEQ ID NO: glycine at position 8 in 68; (c) sequence SEQ ID NO: tyrosine at position 9 in 68; (d) sequence SEQ ID NO: arginine at position 11 in 68; and (e) the sequence SEQ ID NO: tyrosine at position 3 in 69.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises one or more combinations of amino acid substitutions at positions selected from the group consisting of: (a) sequence SEQ ID NO: alanine at position 6 of 68; (b) sequence SEQ ID NO: glycine at position 8 in 68; (c) sequence SEQ ID NO: tyrosine at position 9 in 68; (d) sequence SEQ ID NO: arginine at position 11 in 68; and (e) the sequence SEQ ID NO: tyrosine at position 3 in 69.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises amino acid substitutions at the following positions: (a) sequence SEQ ID NO: tyrosine at position 9 in 68; (b) sequence SEQ ID NO: arginine at position 11 in 68; and (c) the sequence SEQ ID NO: tyrosine at position 3 in 69.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises amino acid substitutions at the following positions: (a) sequence SEQ ID NO: alanine at position 6 of 68; (b) sequence SEQ ID NO: glycine at position 8 in 68; (c) sequence SEQ ID NO: tyrosine at position 9 in 68; (d) sequence SEQ ID NO: arginine at position 11 in 68; and (e) the sequence SEQ ID NO: tyrosine at position 3 in 69.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises: (a) in the sequence SEQ ID NO: 68 by replacement of alanine with aspartic acid at position 6; (b) in the sequence SEQ ID NO: substitution of arginine with proline at position 11 in 68; and (c) a sequence as set forth in SEQ ID NO: substitution of histidine for tyrosine at position 3 in 69.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises: (a) in the sequence SEQ ID NO: substitution of glycine at position 8 with tyrosine in 68; and (b) a sequence as set forth in SEQ ID NO: position 9 in 68 replaces tyrosine with histidine.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises: (a) in the sequence SEQ ID NO: 68 by replacement of alanine with aspartic acid at position 6; (b) in the sequence SEQ ID NO: substitution of glycine at position 8 with tyrosine in 68; (c) in the sequence SEQ ID NO: 68 by histidine for tyrosine at position 9; (d) in the sequence SEQ ID NO: substitution of arginine with proline at position 11 in 68; and (e) a sequence as set forth in SEQ ID NO: substitution of histidine for tyrosine at position 3 in 69.

In one embodiment, an anti-IL-8 antibody of disclosure C comprises HVR-H2 comprising SEQ ID NO: 73.

In one embodiment, an anti-IL-8 antibody of disclosure C comprises HVR-H3 comprising SEQ ID NO: 74.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises a heavy chain variable region comprising SEQ ID NO: 67, comprising the amino acid sequence of SEQ ID NO: 73, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74, HVR-H3.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises one or more amino acid substitutions at positions selected from the group consisting of: (a) sequence SEQ ID NO: serine at position 8 in 70; (b) sequence SEQ ID NO: 71 asparagine at position 1; (c) sequence SEQ ID NO: 71 leucine at position 5; and (d) the sequence SEQ ID NO: glutamine at position 1 in 72. In another embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, or all 4 of these substitutions.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises one or more combinations of amino acid substitutions at positions selected from the group consisting of: (a) sequence SEQ ID NO: serine at position 8 in 70; (b) sequence SEQ ID NO: 71 asparagine at position 1; (c) sequence SEQ ID NO: 71 leucine at position 5; and (d) the sequence SEQ ID NO: glutamine at position 1 in 72. In another embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, or all 4 of these substitutions.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises amino acid substitutions at the following positions: (a) sequence SEQ ID NO: 71 asparagine at position 1; (b) sequence SEQ ID NO: 71 leucine at position 5; and (c) the sequence SEQ ID NO: glutamine at position 1 in 72.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises amino acid substitutions at the following positions: (a) sequence SEQ ID NO: serine at position 8 in 70; (b) sequence SEQ ID NO: 71 asparagine at position 1; (c) sequence SEQ ID NO: 71 leucine at position 5; and (d) the sequence SEQ ID NO: glutamine at position 1 in 72.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises:

(a) in the sequence SEQ ID NO: substitution of lysine for asparagine at position 1 in 71; (b) in the sequence SEQ ID NO: replacement of leucine with histidine at position 5 in 71; and (c) a sequence as set forth in SEQ ID NO: substitution of lysine for glutamine at position 1 of 72.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises: (a) in the sequence SEQ ID NO: substitution of glutamic acid for serine at position 8 of 70; (b) in the sequence SEQ ID NO: substitution of lysine for asparagine at position 1 in 71; (c) in the sequence SEQ ID NO: 71 replacement of leucine with histidine at position 5; and (d) a sequence as set forth in SEQ ID NO: substitution of lysine for glutamine at position 1 of 72.

In one embodiment, an anti-IL-8 antibody of disclosure C comprises HVR-L2, comprising SEQ ID NO: 75.

In one embodiment, an anti-IL-8 antibody of disclosure C comprises HVR-L3, comprising SEQ ID NO: 76.

In one embodiment, the anti-IL-8 antibody of disclosure C has an amino acid sequence comprising SEQ ID NO: 70 comprising the amino acid sequence of SEQ ID NO: 75, and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 76, HVR-L3.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises amino acid substitutions at the following positions: (a) sequence SEQ ID NO: alanine at position 55 in 77; (b) sequence SEQ ID NO: 77 at position 57; (c) sequence SEQ ID NO: tyrosine at position 58 in 77; (d) sequence SEQ ID NO: arginine at position 60 in 77; (e) sequence SEQ ID NO: (vii) glutamine at position 84 in 77; (f) sequence SEQ ID NO: 77 serine at position 87; and (g) the sequence SEQ ID NO: tyrosine at position 103 in 77.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises: (a) in the sequence SEQ ID NO: 77 with aspartic acid for alanine at position 55; (b) in the sequence SEQ ID NO: 77 by substitution of glycine at position 57 with tyrosine; (c) in the sequence SEQ ID NO: 77 by replacement of tyrosine with histidine at position 58; (d) in the sequence SEQ ID NO: substitution of arginine with proline at position 60 in 77; (e) in the sequence SEQ ID NO: 77 by substitution of glutamine with threonine at position 84; (f) in the sequence SEQ ID NO: 77 by aspartic acid for serine at position 87; (g) and in the sequence SEQ ID NO: position 103 in 77 with histidine.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 78, or a heavy chain variable region of the amino acid sequence of seq id no.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 79.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 78 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 79. Comprises a polypeptide having the sequence of SEQ ID NO: 78 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 79 may be an anti-IL-8 antibody that binds IL-8 in a pH-dependent manner. Comprises a polypeptide having the sequence of SEQ ID NO: 78 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 79 may be an anti-IL-8 antibody that stably maintains IL-8-neutralizing activity in vivo (e.g., in plasma). Comprises a polypeptide having the sequence of SEQ ID NO: 78 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 79 may be an antibody with low immunogenicity.

In an alternative aspect, the anti-IL-8 antibodies of disclosure C also include those having pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any one of the amino acid sequences of: (a) comprises the amino acid sequence of SEQ ID NO: 102, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 103, HVR-H2 of the amino acid sequence of seq id no; (c) comprises the amino acid sequence of SEQ ID NO: 104, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 105 amino acid sequence HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 106 amino acid sequence HVR-L2; and (f) comprises SEQ ID NO: 107, HVR-L3.

In an alternative aspect, the anti-IL-8 antibodies of disclosure C also include those having pH-dependent affinity for IL-8 and containing at least one amino acid substitution in each of the following amino acid sequences: (a) comprises the amino acid sequence of SEQ ID NO: 108, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 109, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 110, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 111, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 112, HVR-L2; and (f) comprises SEQ ID NO: 113, HVR-L3.

In an alternative aspect, the anti-IL-8 antibodies of disclosure C also include those having pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any one of the amino acid sequences of: (a) comprises the amino acid sequence of SEQ ID NO: 114, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 115, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 116, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 117 of the amino acid sequence of HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 118, HVR-L2; and (f) comprises SEQ ID NO: 119 and HVR-L3 of the amino acid sequence of seq id no.

In an alternative aspect, the anti-IL-8 antibodies of disclosure C also include those having pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any one of the amino acid sequences of: (a) comprises the amino acid sequence of SEQ ID NO: 120, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 121, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 122, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 123, HVR-L1 of the amino acid sequence of seq id no; (e) comprises the amino acid sequence of SEQ ID NO: 124, HVR-L2; and (f) comprises SEQ ID NO: 125, and HVR-L3.

In an alternative aspect, the anti-IL-8 antibodies of disclosure C also include those having pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any one of the amino acid sequences of: (a) comprises the amino acid sequence of SEQ ID NO: 126, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 127, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 128, HVR-H3 of the amino acid sequence of seq id no; (d) comprises the amino acid sequence of SEQ ID NO: 129, HVR-L1 of the amino acid sequence of 129; (e) comprises the amino acid sequence of SEQ ID NO: 130, HVR-L2; and (f) comprises SEQ ID NO: 131, HVR-L3.

In an alternative aspect, the anti-IL-8 antibodies of disclosure C also include those having pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any one of the amino acid sequences of: (a) comprises the amino acid sequence of SEQ ID NO: 132 HVR-H1; (b) comprises the amino acid sequence of SEQ ID NO: 133, HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 134, HVR-H3; (d) comprises the amino acid sequence of SEQ ID NO: 135, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 136, HVR-L2; and (f) comprises SEQ ID NO: 137, or a pharmaceutically acceptable salt thereof, and HVR-L3.

In an alternative aspect, the anti-IL-8 antibodies of disclosure C also include those having pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least any one of the amino acid sequences of: (a) comprises the amino acid sequence of SEQ ID NO: 138, HVR-H1 of the amino acid sequence of seq id no; (b) comprises the amino acid sequence of SEQ ID NO: 139 of HVR-H2; (c) comprises the amino acid sequence of SEQ ID NO: 140, HVR-H3 of the amino acid sequence of; (d) comprises the amino acid sequence of SEQ ID NO: 141, HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 142, HVR-L2; and (f) comprises SEQ ID NO: 143 of the amino acid sequence of HVR-L3.

In one embodiment, the anti-IL-8 antibodies of disclosure C have IL-8-neutralizing activity. IL-8-neutralizing activity refers to activity that inhibits the biological activity of IL-8, or may refer to activity that inhibits receptor binding of IL-8.

In an alternative aspect, the anti-IL-8 antibody of disclosure C is an anti-IL-8 antibody that binds IL-8 in a pH-dependent manner. In the context of disclosure C, an anti-IL-8 antibody that binds IL-8 in a pH-dependent manner refers to an antibody that has a reduced binding affinity for IL-8 at acidic pH compared to its binding affinity for IL-8 at neutral pH. For example, pH-dependent anti-IL-8 antibodies include antibodies that have a higher affinity for IL-8 at neutral pH than at acidic pH. In one embodiment, the anti-IL-8 antibody of disclosure C has at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 200-fold, 400-fold, 1000-fold, 10000-fold or more higher affinity for IL-8 at neutral pH than at acidic pH. The binding affinity can be measured using, without particular limitation, a surface plasmon resonance method such as BIACORE (registered trademark). The association rate constant (kon) and dissociation rate constant (koff) can be calculated by matching the association and dissociation sensorgrams simultaneously, based on a simple one-to-one Langmuir association model, using BIACORE (registered trademark) T200 evaluation software (GE Healthcare). The equilibrium dissociation constant (KD) is calculated as the ratio of koff/kon. For screening of antibodies whose binding affinity changes depending on pH, there is no particular limitation, and ELISA, kinetic exclusion assay (KinExA) may be used ^TM) And the like, and surface plasmon resonance methods (such as BIACORE (registered trademark)). pH-dependent IL-8-binding capacity refers to the property of binding IL-8 in a pH-dependent manner. Meanwhile, whether an antibody is capable of binding to IL-8 multiple times can be evaluated by the method described in WO 2009/125825.

In one embodiment, it is preferred that the anti-IL-8 antibodies of disclosure C have a small dissociation constant (KD) for IL-8 at neutral pH. In one embodiment, the dissociation constant of the antibody of disclosure C at neutral pH for IL-8 is, for example, 0.3nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C at neutral pH for IL-8 is, for example, 0.1nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C at neutral pH for IL-8 is, for example, 0.03nM or less, but is not limited thereto.

In one embodiment, preferably, the disclosure C anti IL-8 antibody at pH 7.4 to IL-8 with small dissociation constant (KD). In one embodiment, the dissociation constant of the antibody of disclosure C for IL-8 at pH 7.4 is, for example, 0.3nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C for IL-8 at pH 7.4 is, for example, 0.1nM or less, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C for IL-8 at pH 7.4 is, for example, 0.03nM or less, but is not limited thereto.

In one embodiment, it is preferred that the disclosure C anti-IL-8 antibodies have a large dissociation constant (KD) for IL-8 at acidic pH. In one embodiment, the dissociation constant of the antibody of disclosure C at acidic pH for IL-8 is, for example, 3nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C at acidic pH for IL-8 is, for example, 10nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C at acidic pH for IL-8 is, for example, 30nM or more, but is not limited thereto.

In one embodiment, it is preferred that the disclosure C anti-IL-8 antibodies at pH5.8 with IL-8 with a large dissociation constant (KD). In one embodiment, the dissociation constant of the antibody of disclosure C for IL-8 at pH5.8 is, for example, 3nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C for IL-8 at pH5.8 is, for example, 10nM or more, but is not limited thereto. In one embodiment, the dissociation constant of the antibody of disclosure C for IL-8 at pH5.8 is, for example, 30nM or more, but is not limited thereto.

In one embodiment, preferably, the disclosure C anti IL-8 antibody to IL-8 binding affinity at neutral pH than at acidic pH is higher.

In one embodiment, the dissociation constant ratio between acidic pH and neutral pH, [ KD (acidic pH)/KD (neutral pH) ], of the anti-IL-8 antibody of disclosure C is, for example, 30 or more, but is not limited thereto. In one embodiment, the dissociation constant ratio between acidic pH and neutral pH, [ KD (acidic pH)/KD (neutral pH) ], of the anti-IL-8 antibody of disclosure C is, for example, 100 or more, e.g., 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, but is not limited thereto.

In one embodiment, the dissociation constant ratio between pH 5.8 and pH 7.4, of the anti-IL-8 antibody of disclosure C, [ KD (pH 5.8)/KD (pH 7.4) ], is 30 or more, but is not limited thereto. In one embodiment, the dissociation constant ratio of the antibody of disclosure C between pH 5.8 and pH 7.4, [ KD (pH 5.8)/KD (pH 7.4) ], is, for example, 100 or more, e.g., 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, but is not limited thereto.

In one embodiment, it is preferred that the anti-IL-8 antibodies of disclosure C have a large off-rate constant (koff) at acidic pH. In one embodiment, the antibody of disclosure C has an off-rate constant at acidic pH of, for example, 0.003(1/s) or more, but is not limited thereto. In one embodiment, the antibody of disclosure C has an off-rate constant at acidic pH of, for example, 0.005(1/s) or more, but is not limited thereto. In one embodiment, the antibody of disclosure C has an off-rate constant at acidic pH of, for example, 0.01(1/s) or more, but is not limited thereto.

In one embodiment, it is preferred that the anti-IL-8 antibodies of disclosure C have a large off-rate constant (koff) at pH 5.8. In one embodiment, the antibody of disclosure C has an off-rate constant at pH 5.8 of, for example, 0.003(1/s) or more, but is not limited thereto. In one embodiment, the antibody of disclosure C has an off-rate constant at pH 5.8 of, for example, 0.005(1/s) or more, but is not limited thereto. In one embodiment, the antibody of disclosure C has an off-rate constant at pH 5.8 of, for example, 0.01(1/s) or more, but is not limited thereto.

In one embodiment, preferably, the disclosure C anti IL-8 antibody in solution (for example, in PBS) stably maintain IL-8-neutralizing activity. Whether activity is stably maintained in solution can be assessed by measuring whether the IL-8-neutralizing activity of an antibody of disclosure C added to solution changes before and after storage for a period of time at a certain temperature. In one embodiment, the storage period is, for example, one, two, three or four weeks, but is not limited thereto. In one embodiment, the storage temperature is, for example, 25 ℃, 30 ℃, 35 ℃, 40 ℃, or 50 ℃, but is not limited thereto. In one embodiment, the storage temperature is, for example, 40 ℃, but is not limited thereto; and the storage period is, for example, two weeks, but is not limited thereto. In one embodiment, the storage temperature is, for example, 50 ℃, but is not limited thereto; and the storage period is, for example, one week, but is not limited thereto.

In one embodiment, preferably, the disclosure C anti IL-8 antibody in vivo (e.g., in plasma) stably maintain IL-8-neutralizing activity. Whether activity remains stable in vivo can be assessed by measuring whether the IL-8-neutralizing activity of an antibody of disclosure C added to the plasma of an animal (e.g., mouse) or human is altered before and after storage for a certain period at a certain temperature. In one embodiment, the storage period is, for example, one, two, three or four weeks, but is not limited thereto. In one embodiment, the storage temperature is, for example, 25 ℃, 30 ℃, 35 ℃, or 40 ℃, but is not limited thereto. In one embodiment, the storage temperature is, for example, 40 ℃, but is not limited thereto; and the storage period is, for example, two weeks, but is not limited thereto.

In one embodiment, the rate of cellular uptake of the anti-IL-8 antibody of disclosure C is greater when the antibody forms a complex with IL-8 compared to the antibody alone. The IL-8 antibodies of disclosure C are more readily taken into cells when they are complexed with extracellular (e.g., in plasma) IL-8 than when not complexed with IL-8.

In one embodiment, preferably, the disclosure C anti IL-8 antibody predicted immunogenicity (which in human host prediction) reduced. "Low immunogenicity" can mean, without limitation, wherein, for example, an administered anti-IL-8 antibody does not elicit an immune response in vivo in at least half or more of the individuals administered with a sufficient amount of the antibody for a sufficient period of time to achieve therapeutic efficacy. Induction of an immune response may include the production of anti-drug antibodies. "Low anti-drug antibody production" may be interchangeable with "low immunogenicity". Immunogenicity in humans can be assessed using T cell epitope prediction programs. The T cell epitope prediction programs include Epibase (Lonza), iTope/TCED (Anticope), EpiMatrix (EpiVax), and the like. EpiMatrix is a system for predicting the immunogenicity of proteins studied, in which the sequence of peptide fragments is automatically designed by dividing the amino acid sequence of the protein for analysis of its immunogenicity into nine amino acids each, in order to predict its ability to bind eight major MHC class II alleles (DRB1 × 0101, DRB1 × 0301, DRB1 × 0401, DRB1 × 0701, DRB1 × 0801, DRB1 × 1101, DRB1 × 1301, and DRB1 × 1501) (De Groot et al, clin. The sequences in which the amino acids of the amino acid sequence of the anti-IL-8 antibody are modified can be analyzed using the T cell epitope prediction program described above to design sequences with reduced immunogenicity. Preferred amino acid modification sites that reduce the immunogenicity of anti-IL-8 antibodies of disclosure C include, but are not limited to, SEQ ID NO: 78 of the heavy chain sequence of the anti-IL-8 antibody according to Kabat numbering at position 81 and/or position 82 b.

In one embodiment, disclosure C provides a method of enhancing removal of IL-8 from an individual compared to when using a reference antibody, comprising administering to the individual an anti-IL-8 antibody of disclosure C. In one embodiment, the disclosure C relates to the use of an anti-IL-8 antibody of disclosure C to enhance the removal of IL-8 from an individual compared to when using a reference antibody. In one embodiment, disclosure C is directed to an anti-IL-8 antibody of disclosure C for enhancing the removal of IL-8 from an individual compared to when using a reference antibody. In one embodiment, the disclosure C relates to the use of an anti-IL-8 antibody of disclosure C in the manufacture of a pharmaceutical composition that enhances the in vivo removal of IL-8 compared to when using a reference antibody. In one embodiment, disclosure C relates to a pharmaceutical composition comprising an anti-IL-8 antibody of disclosure C that enhances removal of IL-8 compared to when using a reference antibody. In one embodiment, disclosure C is directed to a method of enhancing removal of IL-8 compared to when using a reference antibody, comprising administering to a subject an anti-IL-8 antibody of disclosure C. In embodiments of disclosure C, a reference antibody refers to an anti-IL-8 antibody prior to modification to obtain an antibody of disclosure C, or an antibody whose IL-8 binding affinity is strong at both acidic and neutral pHs. The reference antibody may be a polypeptide comprising SEQ ID NOs: 83 and 84, or SEQ ID NOs: 89 and 87.

In one embodiment, disclosure C provides a pharmaceutical composition comprising an anti-IL-8 antibody of disclosure C, characterized in that the anti-IL-8 antibody of disclosure C binds IL-8 and subsequently binds extracellular matrix. In one embodiment, disclosure C relates to the use of an anti-IL-8 antibody of disclosure C in the manufacture of a pharmaceutical composition, characterized in that the anti-IL-8 antibody of disclosure C binds IL-8 and subsequently to the extracellular matrix.

In any of the above embodiments, the anti-IL-8 antibody can be a humanized antibody.

In one aspect, the antibody of disclosure C comprises a heavy chain variable region of any one of the above embodiments and a light chain variable region of any one of the above embodiments. In one embodiment, the antibody of disclosure C comprises SEQ ID NO: 78 and SEQ ID NO: 79 and may further comprise post-translational modifications in the sequence thereof.

In a further aspect, an anti-IL-8 antibody according to any of the above embodiments can bind, singly or in combination, any of the features described in sections 1 to 7 below.

1. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided in disclosure C can be chimeric antibodies. Certain chimeric antibodies, for example, are described in U.S. Pat. nos. 4,816,567; and Morrison et al, proc.natl.acad.sci.usa 81: 6851 (1984). In one example, a chimeric antibody can comprise a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable regions in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody can be replaced with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), e.g., to retain or improve antibody specificity or affinity.

Humanized antibodies and methods for making them, e.g., as described in Almagro et al, front.biosci.13: 1619-: 323-329 (1988); queen et al, proc.natl acad.sci.usa 86: 10029-10033 (1989); U.S. Pat. nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; kashmiri et al, Methods 36: 25-34(2005) (describing Specificity Determining Region (SDR) grafting); padlan, mol.immunol.28: 489-498(1991) (description "resurfacing"); dall' Acqua et al, Methods 36: 43-60(2005) (describing "FR shuffling"); and Osbaum et al, Methods 36: 61-68(2005) and Klimka et al, Br.J. cancer 83: 252-260(2000) (describing the "guided selection" method for FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al, J.Immunol.151: 2296 (1993); the framework regions of human antibody consensus sequences derived from a specific subset of light or heavy chain variable regions (see, e.g., Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al, J.Immunol., 151: 2623 (1993)); and the framework regions derived from screening FR libraries (see, e.g., Baca et al, J.biol.chem.272: 10678-10684(1997) and Rosok et al, J.biol.chem.271: 22611-22618 (1996)).

2. Antibody fragments

In certain embodiments, the antibodies provided in disclosure C can be antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab ', Fab ' -SH, F (ab ')₂Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al, nat. med.9: 129-134(2003). For reviews on scFv fragments see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458.

Diabodies (Diabodies) are antibody fragments with two antigen binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proc.natl.acad.sci.usa 90: 6444-6448(1993). Triabodies and tetrabodies are also described in Hudson et al, nat. med.9: 129-134 (2003).

A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody can be a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1). Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., e.coli or phage), as described within the scope of the description of disclosure C herein.

3. Human antibodies

In certain embodiments, the antibodies provided in disclosure C can be human antibodies. Human antibodies can be prepared by various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, C urr, opin, pharmacol.5: 368-74(2001) and Lonberg, curr. opin. immunol.20: 450-. Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to antigen challenge. The animal typically contains all or part of a human immunoglobulin locus, which replaces the animal (non-human) immunoglobulin locus, or is present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, the immunoglobulin loci of the animal (non-human) are typically inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. biotech.23: 1117-1125(2005). For XENOMOUSE^TMSee also U.S. Pat. nos. 6,075,181 and 6,150,584; for HUMAB^TMSee U.S. patent No. 5,770,429; for K-M MOUSE^TMSee U.S. Pat. No. 7,041,870 for technical reference, and for VELOCIMOUSE^TMSee U.S. patent application publication No. US 2007/0061900 for technical reference.

The human variable regions from the whole antibody produced by the animal may be further modified, for example, by combination with different human constant regions. Human antibodies can also be made based on hybridoma methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies, e.g., as described in Kozbor, j.immunol.133: 3001 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al, j.immunol.147: 86 (1991). Human antibodies produced via human B-cell hybridomas are described in Li et al, proc.natl.acad.sci.usa 103: 3557 and 3562 (2006). Other methods include, for example, the method described in U.S. Pat. No. 7,189,826 for the production of monoclonal human IgM antibodies from hybridoma cell lines, and, for example, the methods described in Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006). Human hybridoma technology (trioma) technology) also in Vollmers et al, Histol. & histopath.20 (3): 927-937(2005) and Vollmers et al, Methods and dressings in Experimental and Clinical Pharmacology 27 (3): 185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. The variable domain sequence may be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

4. Library-derived antibodies

The antibodies of disclosure C can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods known in the art are used to generate phage display libraries and to screen the libraries for antibodies with desired binding properties. The method is described in Hoogenboom et al, meth.mol.biol.178: 1-37 (O' Brien et al, ed., human Press, Totowa, NJ, 2001), and, for example, in McCafferty et al, Nature 348: 552 (1990); clackson et al, Nature 352: 624-628 (1991); marks et al, j.mol.biol.222: 581-597 (1992); marks and Bradbury, in meth.mol.biol.248: 161-175(Lo, ed., Human Press, Totowa, NJ, 2003); sidhu et al, j.mol.biol.338 (2): 299-310 (2004); lee et al, j.mol.biol.340 (5): 1073-1093 (2004); fellouse, proc.natl.acad.sci.usa 101 (34): 12467-12472 (2004); and Lee et al, j.immunol.meth.284 (1-2): 119, 132 (2004).

In certain phage display methods, libraries of VH and VL coding sequences (reporters) can be separately cloned by Polymerase Chain Reaction (PCR) and randomly recombined in phage libraries. The resulting phage library is screened for antigen-binding phage, such as Winter et al, ann.rev.immunol.12: 433 and 455 (1994). Phage typically display antibody fragments, either single chain fv (scfv) fragments or Fab fragments.

Alternatively, a non-immune (nave) repertoire can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self as well as self antigens without any immunization, such as Griffiths et al, EMBO j.12: 725, 734 (1993).

Finally, the non-immune library can also be constructed synthetically by cloning unrearranged V gene fragments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and accomplish in vitro rearrangement (see Hoogenboom and Winter, J.mol.biol., 227: 381-388(1992), infra; patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373; and U.S. application publications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. herein, an antibody or antibody fragment isolated from a human antibody library is considered to be a human antibody or human antibody fragment.

5. Multispecific antibodies

In certain embodiments, the antibody provided according to disclosure C can be, for example, a multispecific antibody such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.

In certain embodiments, one of the binding specificities is for IL-8 and the other is for any other antigen.

In certain embodiments, bispecific antibodies can bind to two different epitopes on IL-8. Bispecific antibodies can also be used to target cytotoxic agents to IL-8 expressing cells. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)); WO 93/08829; and Traunecker et al, EMBO j.10: 3655(1991)) and "knob-in-hole" methods (see U.S. Pat. No. 5,731,168). Multispecific antibodies may be prepared by using electrostatic control effects to prepare Fc-heterodimeric molecules (WO2009/089004A1), by cross-linking two or more antibodies or fragments (U.S. Pat. No. 4,676,980; and Brennan et al, Science 229: 81(1985)), by using leucine zippers to generate bispecific antibodies (Kostelny et al, J.Immunol.148 (5): 1547. 1553(1992)), by using the "diabody" technique to prepare bispecific antibody fragments (Hollinger et al, Proc.Natl.Acad.Sci.USA 90: 6444. 6448(1993)), by using single chain fv (scFv) dimers (Gruber et al, J.Immunol.152: 5368(1994)), or other methods. Preparation of trispecific antibodies, for example, in Tutt et al, j.immunol.147: 60 (1991).

Engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, e.g., US 2006/0025576).

Within the context of the description of disclosure C herein, an antibody or antibody fragment also includes a "dual action FAb" or "DAF" comprising an antigen binding site that binds IL-8 as well as another, different antigen (see US 2008/0069820, for example).

6. Antibody variants

Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues in the amino acid sequence of the antibody. The final construct may be achieved using any combination of deletions, insertions and substitutions, as long as the final construct is an antibody having the desired properties described in the context of disclosure C.

In one embodiment, disclosure C provides antibody variants having one or more amino acid substitutions. The substitution site may be at any position in the antibody. Amino acids used for conservative substitutions are shown under the heading "conservative substitutions" in table 10. Typical substitutions of amino acids that result in more substantial changes are shown in table 10 under the heading "typical substitutions" and are further described with reference to amino acid side chain types.

[ Table 10]

Initial residue	Exemplary permutation	Conservative substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp，Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Ash；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

Amino acids can be classified according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: cys, Ser, Thr, Asn, Gln; (3) acidic: asp, Glu; (4) basic: his, Lys, Arg; (5) residues that influence chain orientation: gly, Pro; and (6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will require the exchange of members of one of these classes for another.

Amino acid insertions include fusion to polypeptides comprising one, two, or three to one hundred or more residues at the N-terminus and/or C-terminus, and insertion of one or more amino acid residues into the sequence. Antibodies having such terminal insertions include, for example, antibodies having an N-terminal methionyl residue. Other insertional variants of antibody molecules include those in which the antibody is fused at the N-or C-terminus to an enzyme (e.g., ADEPT) or polypeptide that increases the plasma half-life of the antibody.

7. Variants of glycosylation

In one embodiment, the antibody provided according to disclosure C may be a glycosylated antibody. Glycosylation sites can be added to or deleted from an antibody by altering the amino acid sequence in such a way that glycosylation sites are created or removed.

When the antibody contains an Fc region, the sugar chain attached thereto may be changed. Natural antibodies produced by animal cells typically contain a branched, bifurcated oligosaccharide linked through an N-linkage to Asn297 of the CH2 domain of the Fc region (see Wright et al TIBTECH 15: 26-32 (1997)). Oligosaccharides include, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose, which is attached to GlcNAc in the "stem" of a bifurcated oligosaccharide structure. In one embodiment, the oligosaccharides in the antibody of disclosure C are modified to produce antibody variants with certain improved properties.

Fc region variants

In one embodiment, one or more amino acid modifications are introduced into the Fc region of an antibody provided according to disclosure C, thereby generating an Fc region variant. Fc region variants include those having modifications (e.g., substitutions) of one, two, three, or more amino acids in the native human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG 4).

The anti-IL-8 antibodies of disclosure C may contain an Fc region having at least one of the following five properties, not limited thereto: (a) increased binding affinity of the Fc region for FcRn relative to the binding affinity of the native Fc region for FcRn at acidic pH; (b) a reduced binding affinity of the Fc region for the pre-existing ADA relative to the binding affinity of the native Fc region for the pre-existing ADA; (c) increased plasma half-life of the Fc region relative to the plasma half-life of the native Fc region; (d) reduced plasma clearance of the Fc region relative to plasma clearance of the native Fc region; and (e) reduced binding affinity of the Fc region for the effector receptor relative to the binding affinity of the native Fc region for the effector receptor. In some embodiments, the Fc region has 2, 3, or 4 of the above properties. In one embodiment, Fc region variants include those having increased FcRn-binding affinity at acidic pH. Fc region variants with increased FcRn-binding affinity include, but are not limited to, Fc region variants with an FcRn-binding affinity that is increased up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, or 100-fold as compared to the FcRn-binding affinity of an antibody comprising a native IgG Fc region.

In one embodiment, Fc region variants include safe and advantageous Fc region variants that do not bind pre-existing ADA and at the same time have improved plasma retention. As used in the context of disclosure C, the term "ADA" refers to an endogenous antibody having binding affinity for an epitope on a therapeutic antibody. As used within the scope of disclosure C, the term "pre-existing ADA" refers to a detectable anti-drug antibody that is present in the blood of a patient prior to administration of a therapeutic antibody to the patient. The pre-existing ADA includes rheumatoid factors. Fc region variants with low binding affinity for pre-existing ADA include, but are not limited to, Fc region variants with ADA-binding affinity reduced to 1/10 or less, 1/50 or less, or 1/100 or less, as compared to the ADA-binding affinity of an antibody comprising a native IgG Fc region.

In one embodiment, the Fc region variant comprises an Fc region variant that has low binding affinity for a complement protein or does not bind a complement protein. Complement proteins include C1 q. Fc region variants with low binding affinity for complement proteins include, but are not limited to, Fc region variants with reduced binding affinity for complement proteins to 1/10 or less, 1/50 or less, or 1/100 or less, as compared to the complement protein-binding affinity of an antibody comprising a native IgG Fc region.

In one embodiment, the Fc region variant comprises an Fc region variant that has low binding affinity for an effector receptor or no binding affinity for an effector receptor. Effector receptors include, but are not limited to, Fc γ RI, Fc γ RII, and Fc γ RIII. Fc γ RI includes, but is not limited to, Fc γ RIa, Fc γ RIb, and Fc γ RIc, and subtypes thereof. Fc γ RII includes, but is not limited to, Fc γ RIIa (which has two allotypes: R131 and H131) and Fc γ RIIb. Fc γ RIII includes, but is not limited to, Fc γ RIIIa (which has two allotypes: V158 and F158) and Fc γ RIIIb (which has two allotypes: Fc γ RIIIb-NA1 and Fc γ RIIIb-NA 2). Fc region variants having low binding affinity for effector receptors include, but are not limited to, Fc region variants having a reduced binding affinity for effector receptors compared to the binding affinity of an antibody comprising a native IgG Fc region to at least 1/10 or less, 1/50 or less, or 1/100 or less.

In one embodiment, the Fc region variant comprises an Fc region comprising one or more amino acid substitutions at any one of the positions of the group consisting of: positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438, and 440 (according to EU numbering).

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions at positions 235, 236, 239, 428, 434, 436, 438, and 440, according to EU numbering, as compared to a native Fc region.

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions at positions 235, 236, 327, 330, 331, 428, 434, 436, 438, and 440 according to EU numbering as compared to a native Fc region.

In one embodiment, the Fc region variant comprises an Fc region comprising one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, a327G, a330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions of L235R, G236R, a327G, a330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82. Comprises the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 can be an anti-IL-8 antibody that binds IL-8 in a pH-dependent manner. Comprises the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 can stably maintain IL-8-neutralizing activity in vivo (e.g., in plasma). Comprises the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 can be an antibody with low immunogenicity. Comprises the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 may comprise an Fc region having increased FcRn-binding affinity at acidic pH (e.g., pH 5.8) as compared to the FcRn-binding affinity of a native Fc region at acidic pH. Comprises the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 may comprise an Fc region having reduced binding affinity for pre-existing ADA compared to the binding affinity of the native Fc region for pre-existing ADA. Comprises the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 can contain an Fc region having an increased half-life in plasma compared to the native Fc region. Comprises the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region having reduced binding affinity for an effector receptor as compared to the native Fc region. In another embodiment, the anti-IL-8 antibody comprises any of the above properties of 2, 3, 4, 5, 6, or all 7 combinations.

In one embodiment, the anti-IL-8 antibody of disclosure C comprises SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82. Comprises the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 can be an anti-IL-8 antibody that binds IL-8 in a pH-dependent manner. Comprises the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 can stably maintain IL-8-neutralizing activity in vivo (e.g., in plasma). Comprises the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 can be an antibody with low immunogenicity. Comprises the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region having increased FcRn-binding affinity at acidic pH compared to the FcRn-binding affinity of a native Fc region. Comprises the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region having a reduced binding affinity for pre-existing ADA compared to the binding affinity of the native Fc region for pre-existing ADA. Comprises the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 can contain an Fc region whose half-life in plasma is extended compared to the native Fc region. Comprises the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 may contain an Fc region having reduced binding affinity for an effector receptor as compared to the native Fc region. In another embodiment, the anti-IL-8 antibody comprises any of the above properties of 2, 3, 4, 5, 6, or all 7 combinations.

In certain embodiments, disclosure C includes antibody variants that have some, but not all, effector functions. Antibody variants may be desirable candidates for situations in which certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays known in the art can be routinely performed to confirm the reduction/complete loss of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to identify antibodies that lack fcyr binding (and thus ADCC activity), but retain FcRn binding ability.

Cells of primary culture that mediate ADCC and NK cells express only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is described in ravatch et al, annu. 457, 492(1991) are summarized in Table 3 on page 464. Non-limiting examples of in vitro assays to assess ADCC activity of the molecules studied are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al, Proc. Natl Acad. Sci. USA 83: 7059-. Alternatively, non-radioactive isotope assays can be used to assess effector cell function (see, e.g., ACTI (TM) for flow cytometry) non-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox 96 ^TMNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells used in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells.

Alternatively or additionally, the ADCC activity of the antibody variant of interest may be determined, for example, in the methods described by Clynes et al, proc.natl.acad.sci.usa 95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity. See, for example, WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. meth.202: 163 (1996); Cragg et al, Blood 101: 1045-. Determination of FcRn binding and in vivo clearance/half-life may be performed using methods known in the art (see, e.g., Petkova et al, int. immunol.18 (12): 1759-.

Antibodies with reduced effector function include those having one or more Fc region residues substituted at positions 238, 265, 269, 270, 297, 327 or 329 (U.S. Pat. No. 6,737,056). Such Fc region variants include those having substitutions of two or more residues at positions 265, 269, 270, 297 or 327, including so-called "DANA" Fc region variants with substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Antibody variants with improved or reduced binding to the FcR group are described below. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al, J.biol.chem.9 (2): 6591-6604 (2001))

Antibodies with increased blood half-life and improved FcRn binding at acidic pH are described in US 2005/0014934. Antibodies are described that comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc region variants include those having a substitution at one or more positions selected from 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 in the Fc region, for example, a substitution at position 434 in the Fc region (U.S. patent No. 7,371,826).

For further examples of Fc region variants, see also Duncan et al, Nature 322: 738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351.

9. Antibody derivatives

In certain embodiments, the antibodies provided in disclosure C can be further modified to contain additional non-protein moieties known and readily available in the art. Binding moieties suitable for derivatizing antibodies include, but are not limited to, water soluble polymers. Examples of soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol or propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic acid glycoside copolymers, polyaminoacids (homopolymer or random copolymers), poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, prolyl propylene oxide/ethylene oxide copolymers, polyoxyethylated alcohols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

Polyethylene glycol propionaldehyde is industrially advantageous because of its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the amount and/or type of polymer used for derivatization may be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved when the antibody derivative is used in a defined therapy, and the like.

In another embodiment, can provide the disclosure C anti IL-8 antibody and can be exposed to irradiation selective heating of the non protein moiety conjugates. In one embodiment, the non-protein moiety is, for example, a carbon nanotube (see, e.g., Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-. The irradiation can be of any wavelength and includes, without limitation, wavelengths that are not harmful to humans but that can heat the non-protein moiety to a temperature that kills cells proximate to the antibody-non-protein moiety.

B. Recombinant methods and compositions

The anti-IL-8 antibodies of disclosure C can be produced using recombinant methods and compositions, for example, as described in U.S. patent No. 4,816,567. One embodiment provides one or more isolated nucleic acids encoding the anti-IL-8 antibodies provided as disclosure C. The one or more nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acids are provided. In one embodiment, a host cell comprising the one or more nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising nucleic acids encoding a VL of an antibody and a VH of an antibody, or (2) a first vector comprising nucleic acids encoding a VL of an antibody and a second vector comprising nucleic acids encoding a VH of an antibody.

In one embodiment, the host is a eukaryote (e.g., Chinese Hamster Ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, SP20 cells)).

In one embodiment, a method of producing an anti-IL-8 antibody of disclosure C is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-IL-8 antibody provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody (e.g., from the host cell or host cell culture medium).

For recombinant production of anti-IL-8 antibodies, one or more nucleic acids encoding the antibodies are isolated, e.g., as described above, and inserted into one or more vectors for further cloning and/or expression in a host cell. The one or more nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to nucleic acids encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described within the scope of description of disclosure C herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol.248(B.K.C.Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254 for expression of antibody fragments in E.coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for cloning or expressing antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", which are capable of producing antibodies with partially or fully human glycosylation patterns. See gemdross, nat. biotech.22: 1409-: 210-215(2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Without particular limitation, baculovirus has been used in conjunction with insect cells to transfect Spodoptera frugiperda (Spodoptera frugiperda) cells and various baculovirus strains have been identified.

Plant cell cultures may also be used as hosts. See U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the plantibodies technique for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension are useful. Other examples of mammalian host cells that may be used are the SV40 transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell lines (293 cells described in Graham et al, J.Gen Virol.36: 59 (1977)); baby hamster kidney cells (BHK); mouse testicular support cells (TM 4 cells as described in Mather, biol. reprod.23: 243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, e.g., Mather et al, Annals n.y acad.sci.383: 44-68 (1982); MRC5 cells; and FS4 cells.

Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp20, but are not limited thereto. For a review of other mammalian host cells suitable for antibody production, see Yazaki and Wu, Methods in Molecular Biology, Vol.248(B.K.C.Lo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).

The antibody of disclosure C produced by culturing a host cell carrying a nucleic acid encoding said antibody as described above under conditions suitable for expression of the antibody can be isolated from the interior or exterior of the host cell (medium, milk, etc.) and purified as a substantially pure homogeneous antibody. Separation/purification methods generally used for purifying polypeptides may be appropriately used for separating and purifying the antibody; however, the method is not limited to the above example. The antibody can be suitably separated and purified, for example, by appropriately selecting and combining column chromatography, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis and recrystallization (without being limited thereto). Chromatography includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. The chromatography may be performed using liquid chromatography, e.g., HPLC and FPLC. Columns for affinity chromatography include, but are not limited to, protein a columns and protein G columns. Protein A columns include, but are not limited to, Hyper D, POROS, Sepharose F.F (Pharmacia), and the like.

Focusing on the properties of anti-IL-8 antibodies (such as increased extracellular matrix binding activity and enhanced cellular uptake of the above-described complexes), disclosure C provides methods for selecting antibodies with increased extracellular matrix binding and antibodies with enhanced cellular uptake. In one embodiment, disclosure C provides a method for producing an anti-IL-8 antibody comprising a variable region whose binding activity to IL-8 is in a pH-dependent manner, comprising the steps of: (a) assessing binding between the anti-IL-8 antibody and the extracellular matrix; (b) selecting an anti-IL-8 antibody that strongly binds extracellular matrix; (c) culturing a host comprising a vector carrying a nucleic acid encoding an antibody; and (d) isolating the antibody from the culture medium.

Binding to the extracellular matrix can be evaluated by any method without particular limitation as long as they are known to those skilled in the art. For example, the assay can be performed using an ELISA system for detecting binding between an antibody and extracellular matrix, in which the antibody is added to a plate on which extracellular matrix is immobilized, and a labeled antibody against the antibody is added thereto. Alternatively, the assay can be performed, for example, using an Electrochemiluminescence (ECL) method, in which a mixture of an antibody and a ruthenium antibody is added to a plate immobilized with an extracellular matrix and binding between the antibody and the extracellular matrix is evaluated based on electrochemiluminescence of ruthenium.

The anti-IL-8 antibody that is evaluated for binding to the extracellular matrix in step (a) above may be the antibody itself or in contact with IL-8. "selecting an anti-IL-8 antibody that strongly binds extracellular matrix" in step (b) means that, in the assessment of extracellular matrix binding, an anti-IL-8 antibody is selected based on the criterion that the value representing the binding between extracellular matrix and anti-IL-8 antibody is higher than the value representing the binding between extracellular matrix and a control antibody, and may be, for example, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more; however, the ratio is not particularly limited to the above example. Except for the presence of IL-8, the conditions are preferably the same as in the step of assessing the binding between the anti-IL-8 antibody and the extracellular matrix. The control anti-IL-8 antibody used to compare several modified anti-IL-8 antibodies may be an unmodified anti-IL-8 antibody. In this case, the conditions are preferably the same except for the presence of IL-8. Specifically, in one embodiment, disclosure C includes selecting an antibody having a higher value representative of extracellular matrix binding from a plurality of anti-IL-8 antibodies that are not contacted with IL-8. In another embodiment, disclosure C includes selecting an antibody from a plurality of anti-IL-8 antibodies that are contacted with IL-8 that represents a higher value of extracellular matrix binding. In an alternative embodiment, "selecting an anti-IL-8 antibody that strongly binds extracellular matrix" in step (b) means that, when assessing extracellular matrix binding, the antibody can be selected based on the criterion that the binding between the antibody and extracellular matrix is altered by the presence of IL-8. The ratio of the value representing extracellular matrix binding of an anti-IL-8 antibody contacted with IL-8 to the value representing extracellular matrix binding of an anti-IL-8 antibody not contacted with IL-8 can be, for example, 2 to 1000. Further, the ratio between values may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.

C. Measurement of

The anti-IL-8 antibodies provided within the scope of the disclosure C described herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by various methods known in the art.

1. Binding assays and other assays

In one aspect, antibodies of disclosure C can be evaluated for antigen binding activity by known methods, e.g., ELISA, western blot, kinetic exclusion assay (KinExA)^TM) And surface plasmon resonance using devices such as biacore (ge healthcare).

In one embodiment, binding affinity can be measured using BIACORE T200(GE Healthcare) in the following manner. An appropriate amount of capture protein (e.g., protein a/g (pierce)) is immobilized on the sensor chip CM4(GE Healthcare) by an amine coupling method, and the target antibody is allowed to be captured. Then, the diluted antigen solution and a running buffer (as a reference solution: e.g., 0.05% Tween 20, 20mM ACES, 150mM NaCl, pH 7.4) were injected to interact the antigen molecules with the antibody captured on the sensor chip. The sensor chip was regenerated using 10mM glycine HCl solution (pH 1.5). The measurement is performed at a predetermined temperature (e.g., 37 ℃, 25 ℃, or 20 ℃). The association rate constant kon (1/Ms) and dissociation rate constant koff (1/s) (both kinetic parameters) were calculated from sensorgrams obtained by measurement. Kd (m) for each antibody to the antigen was calculated based on these constants. Each parameter was calculated using BIACORE T200 evaluation software (GE Healthcare).

In one embodiment, IL-8 can be as follows quantitative. An anti-human IL-8 antibody comprising a mouse IgG constant region was immobilized on the plate. Containing humanized anti IL-8 antibody IL-8 solution (which does not with the anti IL-8 antibody competition), aliquoting to the immobilized plate. After stirring, biotinylated anti-human Ig kappa light chain antibody was added and allowed to react for a period of time. Then, SULFO-Tag-labeled streptavidin was further added and allowed to react for a certain period of time. Assay buffer was then added and immediately measured using SECTOR Imager 2400(Meso Scale Discovery).

2. Activity assay

In one aspect, assays are provided to identify anti-IL-8 antibodies having biological activity. Biological activities include, for example, IL-8-neutralizing activity and activity that blocks IL-8 signaling. Disclosure C also provides antibodies having such biological activity in vivo and/or in vitro.

In one embodiment, the method of determining the level of neutralization of IL-8 is not particularly limited and it can also be determined by the methods described below. PathHunter^TMThe CHO-K1 CXCR2 β -Arrestin cell line (DiscovexX, Cat. #93-0202C2) is an artificial cell line that is generated to express human CXCR2, known as the human IL-8 receptor, and that emits chemiluminescence upon receiving a signal caused by human IL-8. When human IL-8 is added to the culture medium of cells, chemiluminescence is emitted from the cells in a manner that depends on the concentration of human IL-8 added. When human IL-8 is added to the medium in combination with an anti-human IL-8 antibody, the chemiluminescence of the cells is reduced or undetectable compared to when no antibody is added, as anti-human IL-8 antibodies can block IL-8 signaling. Specifically, the stronger the human IL-8-neutralizing activity of the antibody, the weaker the level of chemiluminescence; and the weaker the human IL-8-neutralizing activity of the antibody, the higher the level of chemiluminescence. Therefore, anti human IL-8 antibody human IL-8-neutralizing activity can be evaluated by examining the difference.

D. Pharmaceutical preparation

Pharmaceutical formulations comprising anti-IL-8 antibodies described within the scope of disclosure C herein can be prepared as lyophilized formulations or aqueous solution formulations by mixing an anti-IL-8 antibody of the desired purity with one or more optional pharmaceutical carriers (see, e.g., Remington's pharmaceutical Sciences 16 th edition, Osol, a.ed. (1980)).

Pharmaceutically acceptable carriers are generally nontoxic at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, histidine, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^TM，PLURONICS^TMOr polyethylene glycol (PEG).

Exemplary pharmaceutical carriers herein also include interstitial drug dispersing agents such as soluble hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (HYLENEX)^TMBaxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. application publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more glycosaminoglycanases, such as chondroitinases.

An exemplary lyophilized antibody formulation is described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908; and WO2006/044908 formulations include histidine-acetate buffer.

Formulations within the scope of disclosure C herein may also contain more than one active ingredient necessary for a particular indication of treatment, preferably those with complementary activities that do not adversely affect each other. The active ingredients are suitably present in combination in an amount effective for the intended purpose.

The active ingredients can be loaded separately in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules. The technique is disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, A.Ed. (1980).

Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the anti-IL 8 antibody of disclosure C, wherein the matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

E. Therapeutic methods and compositions

In some embodiments, the anti-IL-8 antibodies provided according to disclosure C are used in methods of treatment.

In one aspect, provided is an anti-IL-8 antibody for use as a pharmaceutical composition. In an alternative aspect, anti-IL-8 antibodies are provided for the treatment of diseases in which IL-8 is present in excess. In one embodiment, provided is an anti-IL-8 antibody for use in a method of treating a disease in which IL-8 is present in excess. In one embodiment, disclosure C provides a method for treating a subject having a disease in which IL-8 is present in excess (e.g., a disease resulting from the presence of excess IL-8), comprising administering to the subject an effective amount of an anti-IL-8 antibody. In another embodiment, disclosure C provides anti-IL-8 antibodies for use in the methods. In one embodiment, the disclosure C relates to pharmaceutical compositions comprising an effective amount of an anti-IL-8 antibody for treating diseases in which IL-8 is present in excess. In one embodiment, the disclosure C relates to the use of anti-IL-8 antibodies in the preparation of pharmaceutical compositions for diseases in which IL-8 is present in excess. In one embodiment, disclosure C relates to the use of an effective amount of an anti-IL-8 antibody in the treatment of a disease in which IL-8 is present in excess. Diseases in which IL-8 is present in excess include, but are not limited to, inflammatory skin diseases such as inflammatory keratosis (psoriasis, etc.), atopic dermatitis (atopic dermatitis), and contact dermatitis; autoimmune diseases such as chronic inflammatory diseases including chronic rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), and Behcet's disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis (ulcerative colitis); inflammatory liver diseases such as hepatitis B (hepatitis B), hepatitis C (hepatitis C), alcoholic hepatitis (alcoholic hepatitis), and drug-induced allergic hepatitis; inflammatory renal diseases such as glomerulonephritis (glomerular nephritis); inflammatory respiratory diseases such as bronchitis and asthma; chronic inflammatory vascular diseases such as atherosclerosis (atherosclerosis); multiple sclerosis; aphtha; vocal cord inflammation (chorditis); inflammation caused by artificial organs/artificial blood vessels; malignant tumors such as ovarian cancer, lung cancer, prostate cancer, gastric cancer, breast cancer, melanoma, head and neck cancer, and renal cancer; sepsis due to infection; cystic fibrosis (cystic fibrosis); pulmonary fibrosis; and acute lung injury.

In an alternative embodiment, the disclosure C provides an anti-IL-8 antibody for inhibiting the accumulation of biologically active IL-8. "inhibiting the accumulation of IL-8" can be achieved by preventing an increase in the amount of pre-existing IL-8 in vivo or by decreasing the amount of pre-existing IL-8 in vivo. In one embodiment, disclosure C provides anti-IL-8 antibodies that inhibit the accumulation of IL-8 in an individual, thereby inhibiting the accumulation of biologically active IL-8. As used herein, "IL-8 present in vivo" may refer to IL-8 complexed with an anti-IL-8 antibody or free IL-8; alternatively, it may refer to total IL-8 as the sum thereof. As used herein, "present in vivo" may mean "secreted in vivo to the outside of a cell". In one embodiment, disclosure C provides a method for inhibiting the accumulation of IL-8 having biological activity comprising the step of administering to a subject an effective amount of an anti-IL-8 antibody. In one embodiment, the disclosure C relates to a pharmaceutical composition for inhibiting the accumulation of biologically active IL-8 comprising an effective amount of an anti-IL-8 antibody. In one embodiment, disclosure C relates to the use of an anti-IL-8 antibody in the preparation of a pharmaceutical composition for inhibiting the accumulation of biologically active IL-8. In one embodiment, disclosure C relates to the use of an effective amount of an anti-IL-8 antibody to inhibit the accumulation of biologically active IL-8. In one embodiment, the anti-IL-8 antibody of disclosure C inhibits the accumulation of IL-8 compared to an anti-IL-8 antibody that does not have pH-dependent binding activity. In the above embodiments, the "individual" is preferably a human.

In an alternative embodiment, disclosure C provides anti-IL-8 antibodies for use in inhibiting angiogenesis (e.g., neovascularization). In one embodiment, disclosure C provides a method for inhibiting angiogenesis in an individual comprising administering to the individual an effective amount of an anti-IL-8 antibody, and also provides an anti-IL-8 antibody for use in the method. In one embodiment, disclosure C relates to a pharmaceutical composition for inhibiting neovascularization comprising an effective amount of an anti-IL-8 antibody. In one embodiment, disclosure C relates to the use of an anti-IL-8 antibody in the preparation of a pharmaceutical composition for inhibiting neovascularization. In one embodiment, disclosure C relates to the use of an effective amount of an anti-IL-8 antibody in inhibiting neovascularization. In the above embodiments, the "individual" is preferably a human.

In an alternative aspect, disclosure C provides anti-IL-8 antibodies for use in inhibiting the promotion of neutrophil migration. In one embodiment, disclosure C provides a method for inhibiting promotion of neutrophil migration in an individual comprising administering to the individual an effective amount of an anti-IL-8 antibody; and also provides anti-IL-8 antibodies for use in the methods. In one embodiment, disclosure C relates to a pharmaceutical composition for inhibiting the promotion of neutrophil migration in an individual comprising an effective amount of an anti-IL-8 antibody. In one embodiment, disclosure C relates to the use of an anti-IL-8 antibody in the preparation of a pharmaceutical composition for inhibiting the promotion of neutrophil migration in an individual. In one embodiment, disclosure C relates to the use of an effective amount of an anti-IL-8 antibody to inhibit the promotion of neutrophil migration in an individual. In the above embodiments, the "individual" is preferably a human.

In an alternative embodiment, the disclosure C provides a pharmaceutical composition comprising an anti-IL-8 antibody provided herein, e.g., for use in any of the above-described methods of treatment. In one embodiment, the pharmaceutical composition comprises an anti-IL-8 antibody provided in disclosure C and a pharmaceutically acceptable carrier.

The antibodies of disclosure C can be used in therapy, alone or in combination with other agents. For example, the antibody of disclosure C can be co-administered with at least one additional therapeutic agent.

The antibody of disclosure C (and any additional therapeutic agent) may be administered by any suitable means, including parenterally, intrapulmonary and intranasally, and if desired for local treatment, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing may be by any suitable route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules include, but are not limited to, single or multiple administrations at various time points, bolus administrations are contemplated herein, and pulse infusions.

The antibodies of disclosure C are preferably formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site at which the pharmaceutical composition is delivered, the method of administration, the schedule of administration, and other factors known to the medical practitioner. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the condition of interest.

The effective amount of the additional agent depends on the amount of antibody present in the agent, the type of disorder or treatment, and other factors discussed above. These may generally be used at the same dosages and via the same routes of administration as described within the ranges described for disclosure C herein, or 1 to 99% of the dosages described within the ranges described for disclosure C herein, or at any dosage and any route empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of the antibody of disclosure C (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody variant is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg to 10mg/kg) of the antibody may be an initial candidate dose for administration to the patient, e.g., by a single administration or multiple divided administrations, or by continuous infusion. A typical daily dosage may range from about 1mg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment can generally be continued until the desired inhibitory effect of the disease symptoms is observed. Typical antibody doses may fall, for example, within the range of about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of, for example, about 0.5mg/kg, e.g., 2.0mg/kg, e.g., 4.0mg/kg, or, e.g., 10mg/kg (or any combination thereof) may be administered to the patient. The dose may be administered intermittently, e.g., weekly or every three weeks (e.g., in such a way that the patient receives about two to about twenty doses, or about six doses of the antibody). It is possible to administer an initial higher loading dose followed by one or more lower doses; however, other dosage regimens may be useful. The progress of the therapy can be readily monitored by conventional techniques and assays.

F. Article of manufacture

In another aspect of disclosure C, the present disclosure provides an article of manufacture comprising a substance for treating, preventing and/or diagnosing the above-mentioned conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, intravenous solution bags. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition, either by itself or in combination with another composition effective in treating, preventing and/or diagnosing the condition, and may have a sterile interface (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of disclosure C. The label or package insert indicates that the composition is to be used for treating a selected condition.

Further, the article may comprise: (a) a first container comprising a composition comprising an antibody of disclosure C; and (b) a second container comprising a composition comprising an additional cytotoxic agent or a different therapeutic agent. The articles of manufacture in embodiments of disclosure C may also include package insert indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may also include, for example, a second (or third) container comprising a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. The article of manufacture may also include other materials as desired from a commercial or user standpoint, including other buffers, diluents, filters, needles and syringes.

Based on the knowledge of one of ordinary skill in the art, one of ordinary skill in the art will understand that the disclosure C also includes all combinations of all or portions of one or more of the overall embodiments described herein, except where technically contradictory.

Disclosure of A, B, or C

All technical background documents cited herein are incorporated herein by reference.

As used herein, the term "and/or" is understood to mean a combination of items preceding and following the term "and/or" including all combinations of items connected as appropriate by the term.

Where various elements are described herein using terms such as first, second, third, fourth, etc., it is to be understood that the elements are not limited by these terms. These terms are only used to distinguish an element from other elements, and it is to be understood that, for example, a first element can be referred to as a second element, and similarly, a second element can be referred to as a first element, without departing from the scope of the disclosure A, B, and C.

Any term expressed in the singular herein is intended to also include the plural unless specifically stated otherwise or unless there is an inconsistency in the context, and any term expressed in the plural herein is intended to also include the singular.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless otherwise defined differently, all terms (including technical and scientific terms) used herein are to be interpreted as having the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure relates and will not be interpreted in an idealized or overly formal sense.

As used herein, the term "comprising" is intended to specify the presence of the stated items (members, steps, elements, amounts, etc.) unless the context clearly indicates otherwise; and the term does not exclude the presence of other items (members, steps, elements, quantities, etc.).

Embodiments of the present disclosure a, B, and C are described with reference to schematic illustrations, which may be exaggerated for clarity.

Unless the context requires otherwise, the numerical values used herein are to be understood as indicating a range of values based on the ordinary skill of the art. For example, the expression "1 mg" is understood to describe "about 1 mg", with certain variations. For example, the expression "1 to 5" is understood to be specifically described and to be "1, 2, 3, 4, 5", respectively, unless the context is inconsistent.

Examples

Hereinafter, the disclosures a, B and C will be specifically described by examples 1 to 4 and 21 to 23, examples 5 to 7, 19 and 20, and examples 8 to 19, respectively, but they are not to be considered as being limited thereto. It is to be understood that various other embodiments may be practiced in view of the general description provided above.

Example 1

Production of human antibodies that bind human IL-6 receptor pH-dependently with increased pI

Fv4-IgG1 disclosed in WO2009/125825 is an antibody that binds to the human IL-6 receptor in a pH-dependent manner and comprises VH3-IgG1(SEQ ID NO: 24) as the heavy chain and VL3-CK (SEQ ID NO: 32) as the light chain. To increase the pI of Fv4-IgG1, amino acid substitutions that reduce the number of negatively charged amino acids (e.g., aspartic acid and glutamic acid) while increasing positively charged amino acids (e.g., arginine and lysine) were introduced into the variable region of Fv4-IgG 1. Specifically, by replacing the glutamic acid at position 16 with glutamine, the glutamic acid at position 43 with arginine, the glutamine at position 64 with lysine, and the glutamic acid at position 105 with glutamine in the heavy chain VH3-IgG1 (numbering according to Kabat), VH3 (high _ pI) -IgG1(SEQ ID NO: 25) is produced, which is a heavy chain with increased pI. Similarly, by replacing the serine at position 18 with arginine, the glutamine at position 24 with arginine, the glutamic acid at position 45 with lysine, the glutamic acid at position 79 with glutamine, and the glutamic acid at position 107 with lysine in light chain VL3-CK (numbering according to Kabat), VL3 (high _ pI) -CK (SEQ ID NO: 33), which is a light chain with increased pI, is produced. When a substitution was introduced at position 79 of VL3-CK, modifications involving the substitution of alanine with proline at position 80 and alanine with isoleucine at position 83 were introduced simultaneously, although not for the purpose of increasing pI.

The following antibodies were produced by the method of reference example 2: (a) low _ pI-IgG1 comprising VH3-IgG1 as the heavy chain and VL3-CK as the light chain; (b) medium _ pI-IgG1 comprising VH3-IgG1 as the heavy chain and VL3 (high _ pI) -CK as the light chain; and (c) high _ pI-IgG1 comprising VH3 (high _ pI) -IgG1 as the heavy chain and VL3 (high _ pI) -CK as the light chain.

Next, the theoretical pI of each antibody produced was calculated using methods known in the art using GENETYX-SV/RC Ver 9.1.0(GENETYX CORPORATION) (see, e.g., Skoog et al, Trends Analyt. chem.5 (4): 82-83 (1986)). The side chains of all cysteines in the antibody molecule are believed to form disulfide bonds, and the contribution of the cysteine side chains to pKa is excluded from the calculation.

The calculated theoretical pI values are shown in table 3. While the theoretical pI of the low _ pI-IgG1 was 6.39, those of the medium _ pI-IgG1 and high _ pI-IgG1 were 8.70 and 9.30, respectively, indicating that the theoretical pI values increased in a stepwise manner.

WO2011/122011 discloses Fv4-IgG1-F11 (hereinafter, referred to as low _ pI-F11) and Fv4-IgG1-F939 (hereinafter, referred to as low _ pI-F939), whose FcRn-mediated intracellular uptake is enhanced by introducing amino acid substitutions into the Fc region of Fv4-IgG1 and conferring FcRn-binding ability under neutral pH conditions. Further, WO2013/125667 discloses Fv4-IgG1-F1180 (hereinafter, referred to as low _ pI-F1180) whose Fc γ R-mediated intracellular uptake is enhanced by introducing an amino acid substitution into the Fc region of Fv4-IgG1 to increase its Fc γ R-binding ability under neutral pH conditions. Meanwhile, amino acid modifications for enhancing the plasma retention of antibodies by increasing their FcRn binding at acidic pH conditions in endosomes were introduced into Fv4-IgG 1-F1180. By increasing the pI of antibodies containing these novel Fc region variants, the antibodies shown below were generated.

Specifically, VH3-IgG1-F11(SEQ ID NO: 30) and VH3-IgG1-F939(SEQ ID NO: 26) in WO2011/122011, and VH3-IgG1-F1180(SEQ ID NO: 28) in WO2013/125667 were each subjected to a substitution of glutamic acid with glutamine at position 16, a substitution of glutamic acid with arginine at position 43, a substitution of glutamine with lysine at position 64, and a substitution of glutamic acid with glutamine at position 105 (numbering according to Kabat) to yield VH3 (high pI) -F11(SEQ ID NO: 31), VH3 (high pI) -F939(SEQ ID NO: 27), and VH3 (high pI) -F1180(SEQ ID NO: 29), respectively, which are heavy chains with increased pI.

The following antibodies were produced by the method of reference example 2 using these heavy chains: (1) low _ pI-F939 comprising VH3-IgG1-F939 as the heavy chain and VL3-CK as the light chain; (2) medium _ pI-F939 comprising VH3 (high _ pI) -F939 as the heavy chain and VL3-CK as the light chain; (3) high _ pI-F939 comprising VH3 (high _ pI) -F939 as the heavy chain and VL3 (high _ pI) -CK as the light chain; (4) low _ pI-F1180 comprising VH3-IgG1-F1180 as the heavy chain and VL3-CK as the light chain; (5) medium _ pI-F1180 comprising VH3-IgG1-F1180 as the heavy chain and VL3 (high _ pI) -CK as the light chain; (6) high _ pI-F1180 comprising VH3 (high _ pI) -F1180 as the heavy chain and VL3 (high _ pI) -CK as the light chain; (7) low _ pI-F11 comprising VH3-IgG1-F11 as the heavy chain and VL3-CK as the light chain; and (8) high _ pI-F11 comprising VH3 (high _ pI) -F11 as the heavy chain and VL3 (high _ pI) -CK as the light chain.

Next, for the theoretical pI of each antibody produced, it was calculated by a method similar to that described previously using GENETYX-SV/RC Ver 9.1.0(GENETYX CORPORATION). The calculated theoretical pI values are shown in table 3. In all new antibodies containing Fc region variants, the theoretical pI values increase in a stepwise manner in the order low _ pI, medium _ pI and high _ pI.

[ Table 3]

Example 2

Antigen removal effect of antibodies with increased pI showing pH-dependent binding

(2-1) in vivo assay of pI-modulated pH-dependent antibodies binding to human IL-6 receptor

The various pH-dependent antibodies binding to human IL-6 receptor generated in example 1 were used for in vivo assays as shown below: low _ pI-IgG1, high _ pI-IgG1, low _ pI-F939, medium _ pI-F939, high _ pI-F939, low _ pI-F1180, medium _ pI-F1180, and high _ pI-F1180.

Soluble human IL-6 receptor (also referred to as "hsIL-6R"), anti-human IL-6 receptor antibody, and human immunoglobulin preparation Sangropor prepared by the method of reference example 3 were administered simultaneously to human FcRn transgenic mice (B6.mFcRn-/-. hFcRn Tg strain 32+/+ mice, Jackson Laboratories; Methods mol. biol. 602: 93-104(2010)) and the in vivo kinetics of the subsequent soluble human IL-6 receptor was assessed. A mixed solution containing soluble human IL-6 receptor, anti-human IL-6 receptor antibody and Sanglapor (at concentrations of 5. mu.g/mL, 0.1mg/mL, and 100mg/mL, respectively) was administered once at 10mL/kg through the tail vein. Since anti-human IL-6 receptor antibodies are present well above soluble human IL-6 receptor, it is believed that almost all soluble human IL-6 receptor is bound by the antibody. Blood was collected 15 minutes, seven hours, one day, two days, three days, and seven days after administration. The collected blood was immediately centrifuged at 4 ℃ and 15,000rpm for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or below until measurement was performed.

(2-2) measurement of soluble human IL-6 receptor concentration in plasma by electrochemiluminescence method

The concentration of soluble human IL-6 receptor in mouse plasma was measured by the electrochemiluminescence method. Soluble human IL-6 receptor samples adjusted to concentrations of 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90pg/mL for calibration curves and mouse plasma assay samples diluted 50-fold or more were prepared, respectively. The sample was mixed with a monoclonal anti-human IL-6R antibody (R & D) labeled with SULFO-TAG NHS ester (Meso Scale Discovery) ruthenium, a biotinylated anti-human IL-6R antibody (R & D), and Tolizumab (Tocilizumab) (CAS No.: 375823-41-9), which is an antibody that binds to a human IL-6 receptor, and then they were reacted at 37 ℃ overnight. The final concentration of tositumumab was adjusted to 333. mu.g/mL. Then, the reaction solution was dispersed in a streptavidin Gold Multi-ARRAY plate (Meso Scale Discovery). After another one hour of reaction at room temperature, the reaction solution was washed. Then, after dispersing Read Buffer T (x4) (Meso Scale Discovery) into the plate, measurement was immediately performed using SECTOR Imager 2400(Meso Scale Discovery). The concentration of soluble human IL-6 receptor was calculated based on the response in the calibration curve using analytical software, SOFTMax PRO (Molecular Devices).

The observed changes in the concentration of soluble human IL-6 receptor in plasma of human FcRn transgenic mice following intravenous administration are shown in figures 1, 2 and 3. FIG. 1 shows the effect of enhancing antigen removal, where the pI of the variable region is increased in the case of the native IgG1 constant region. Fig. 2 shows the effect of enhancing antigen removal, in which the pI of the variable region is increased in an antibody (F939) endowed with the ability to bind FcRn under neutral pH conditions. Fig. 3 shows the effect of enhancing antigen removal, in which the pI of the variable region is increased in an antibody (F1180) whose Fc γ R-binding ability is enhanced under neutral pH conditions.

In all cases, it was shown that the rate of antigen removal by pH-dependent bound antibodies can be accelerated by increasing the pI of the antibody. It has also been shown that by further conferring an increase in binding capacity for FcRn or fcyr under neutral pH conditions, the antigen removal rate can be further accelerated compared to when only pI is increased in pH-dependent binding antibodies (compare fig. 1 and fig. 2 and 3).

(2-3) in vivo infusion assay of pI-modulated pH-dependent antibodies that bind to the human IL-6 receptor

The various pH-dependent antibodies binding to human IL-6 receptor generated in example 1 were used below: low _ pI-IgG1, high _ pI-IgG1, low _ pI-F11, and high _ pI-F11, for in vivo assays.

Infusion pumps containing soluble human IL-6 receptor (MINI-OSMOTIC Pump model 2004; alzet) were subcutaneously transplanted in the back of human FcRn transgenic mice (B6.mFcRn-/-. hFcRn Tg strain 32+/+ mice, Jackson Laboratories; Methods mol. biol. 602: 93-104(2010)) to generate model animals whose plasma concentration of soluble human IL-6 receptor remained constant. Anti-human IL-6 receptor antibody is administered to model animals, and the in vivo kinetics of the antibody after administration is assessed.

Specifically, the monoclonal anti-mouse CD4 antibody obtained by a method known in the art was administered once at 20mg/kg into the tail vein to inhibit the production of neutralizing antibodies against the soluble human IL-6 receptor that the mouse itself may produce. Then, containing 92.8 u g/ml soluble IL-6 receptor infusion pump subcutaneous transplantation in the mouse back. Three days after the implantation of the infusion pump, the anti-human IL-6 receptor antibody was administered at 1mg/kg into the tail vein once. Blood was collected from the mice 15 minutes, seven hours, one day, two days, three or four days, six or seven days, 13 or 14 days, 20 or 21 days, and 27 or 28 days after administration of the anti-human IL-6 receptor antibody. The collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator at-20 ℃ or below until measurement was performed.

(2-4) measurement of plasma hsIL-6R concentration by electrochemiluminescence method

The hsIL-6R concentration in the plasma of mice was measured by electrochemiluminescence. hsIL-6R samples for calibration curves adjusted to 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90pg/mL and mouse plasma assay samples diluted 50-fold or more were mixed with monoclonal anti-human IL-6R antibody (R & D) labeled with SULFO-TAG NHS ester (Meso Scale Discovery) ruthenium, biotinylated anti-human IL-6R antibody (R & D), and tollizumab and reacted at 37 ℃ overnight. The final concentration of tositumumab was adjusted to 333. mu.g/mL. Then, the reaction solution was dispersed in a streptavidin Gold Multi-ARRAY plate (Meso Scale Discovery). After another hour at room temperature, the reaction solution was washed. Then, after dispersing Read Buffer T (x4) (Meso Scale Discovery) into the plate, measurement was immediately performed using SECTOR Imager 2400(Meso Scale Discovery). Using the analytical software SOFT max PRO (Molecular Devices), hsIL-6R concentrations were calculated based on the responses in the calibration curves.

The measured changes in the concentration of human IL-6 receptor are shown in FIG. 4. For antibodies whose Fc region is that of native IgG1 (high _ pI-IgG1) and antibodies containing a novel Fc region variant with enhanced binding to FcRn under neutral pH conditions (high _ pI-F11), the plasma concentration of soluble human IL-6 receptor was reduced upon administration of the high-pI antibody (also referred to as "high _ pI") (compared to administration of the low-pI antibody (also referred to as "low _ pI")).

Without being bound by a particular theory, the results obtained from these experiments can also be explained as follows: when the Fc region of the administered antibody is that of a native IgG antibody, it is believed that uptake into the cells occurs primarily through non-specific uptake (pinocytosis). Herein, because the cell membrane is negatively charged, the higher the pI of the administered antibody-antigen complex (i.e., the charge of the molecule as a whole tends to be positive), the easier the complex can approach the cell membrane, and the more non-specific uptake can occur. When an antibody with an increased pI forms a complex with an antigen, the complex as a whole also has an increased pI (compared to the complex formed between the original antibody and antigen); thus, intracellular uptake may be increased. Thus, by increasing the pI of an antibody that exhibits pH-dependent antigen binding, the rate or speed of removal of antigen from plasma can be further accelerated, and the concentration of antigen in plasma can be kept at a lower level.

In these examples, the increase in the pI of the antibody is accomplished by introducing amino acid substitutions that reduce the number of negatively charged amino acids and/or increase the number of positively charged amino acids, which may be exposed on the surface of the antibody molecule (in the antibody variable region). It will be appreciated by those skilled in the art that the effect obtained by such an increase in pI is largely (or substantially) independent of the type of target antigen or the amino acid sequence constituting the antibody, but can be expected to be dependent on pI. For example, WO2007/114319 and WO2009/041643 describe the following in general terms.

Because the molecular weight of IgG antibodies is large enough, its primary metabolic pathway is not involved in renal secretion. IgG antibodies with Fc are known to have a long half-life because they are recycled through salvage pathways of FcRn expressed in cells (including endothelial cells of blood vessels), and IgG is thought to be metabolized mainly in endothelial cells. More specifically, IgG that is not specifically taken up into endothelial cells is thought to be recycled by binding to FcRn, and molecules that cannot bind to FcRn are metabolized. IgG, which has a reduced FcRn-binding ability, has a shorter blood half-life, and conversely, the blood half-life can be prolonged by increasing its binding ability to FcRn. In this way, previous methods for controlling IgG kinetics in the blood involve modifying Fc to alter FcRn binding ability; however, working examples of WO2007/114319 (mainly a technique of replacing amino acids in FR regions) and WO2009/041643 (mainly a technique of replacing amino acids in CDR regions) show that, regardless of the type of target antigen, the blood half-life thereof can be controlled without modifying Fc by modifying the pI of the variable region of an antibody. The rate of nonspecific uptake of IgG antibodies into endothelial cells is thought to depend on the physicochemical coulombic interaction of the negatively charged cell surface and IgG antibodies. Thus, it is believed that decreasing (increasing) the pI of IgG antibodies and thus decreasing (increasing) coulomb interactions decreases (increases) their uptake by non-specific endothelial cells and, thus, decreases (increases) their metabolism in endothelial cells, thereby enabling control of plasma pharmacokinetics. Since coulomb interaction between endothelial cells and negative charges on the cell surface is physicochemical interaction, it is considered that the interaction does not mainly depend on the amino acid sequence itself constituting the antibody. Thus, the methods of controlling plasma pharmacokinetics provided herein are not only applicable to particular antibodies, but they can be used broadly with any polypeptide containing antibody variable regions. Herein, a decrease (increase) of coulomb interaction means a decrease (increase) of coulomb force expressed as attractive force and/or an increase (decrease) of coulomb force expressed as repulsive force.

The amino acid substitutions used to accomplish the above may be single amino acid substitutions or a combination of multiple amino acid substitutions. In some embodiments, methods are provided for introducing a single amino acid substitution or a combination of multiple amino acid substitutions into one or more locations exposed on the surface of an antibody molecule. Alternatively, the introduced plurality of amino acid substitutions may be disposed conformationally close to one another. The present inventors have made an idea that, for example, when an amino acid that can be exposed on the surface of an antibody molecule is replaced with a positively charged amino acid (preferably arginine or lysine) or when a pre-existing positively charged amino acid (preferably arginine or lysine) is used, it may be preferable to further replace one or more amino acids that are conformationally close to those amino acids (in some cases, even one or more amino acids buried within the antibody molecule) with a positively charged amino acid, thereby generating a positive charge that is locally clustered at the conformationally close positions. Herein, the definition of "one or more positions in close conformation" is not particularly limited, but for example, it may mean a state in which a single amino acid substitution or a plurality of amino acid substitutions are introduced within 20 angstroms, preferably within 15 angstroms, or more preferably within 10 angstroms of each other. Whether an amino acid substitution of interest is at a position exposed on the surface of an antibody molecule, or whether an amino acid substitution is positionally close, can be determined by known methods such as X-ray crystallography.

Thus, by noting that pI is an index representing the overall charge of the molecule, and that the charge buried within the antibody molecule and the charge on the surface of the antibody molecule are handled without any distinction, the present inventors also considered that by generating antibody molecules by widely and deeply considering the influence from the charge, which includes not only pI but also surface charge and local clustering of charge on the antibody molecule, the removal rate of antigen from plasma can be further accelerated and the antigen concentration in plasma can be maintained at an even lower level.

Receptors such as FcRn or fcyr are expressed on cell membranes, and it is thought that antibodies with enhanced affinity for FcRn or fcyr under neutral pH conditions are taken into cells mainly through these Fc receptors. Because the cell membrane is negatively charged, when its pI is high (the charge of the molecule as a whole is changed to a positive charge), the administered antibody-antigen complex is more accessible to the cell membrane, and uptake by Fc receptors can occur more easily. Thus, antibodies with enhanced affinity for FcRn or fcyr under neutral pH conditions and increased pI also show increased intracellular uptake through Fc receptors when they form complexes with antigen. Thus, the rate of antigen removal from plasma by antibodies that bind antigen in a pH-dependent manner and have enhanced affinity for FcRn or fcyr under neutral pH conditions can be accelerated by increasing their pI, and plasma antigen concentrations can be kept at lower levels.

Example 3

Evaluation of extracellular matrix binding of pH-dependent binding antibodies with increased pI.

(3-1) evaluation of extracellular matrix-binding Capacity

The following experiments were performed to evaluate the effect of conferring pH-dependent antigen binding properties to antibodies and further modifying pI on their extracellular matrix binding capacity.

In a similar manner to the method of example 1, three types of antibodies with different pI were generated, which are antibodies showing pH-dependent binding to the IL-6 receptor: low _ pI-IgG1, medium _ pI-IgG1, and high _ pI-IgG 1. As a general antibody not showing pH-dependent binding to IL-6 receptor, low _ pI (NPH) -IgG1 comprising H54(SEQ ID NO: 34) and L28(SEQ ID NO: 35) and high _ pI (NPH) -IgG1 comprising H (WT) (SEQ ID NO: 36) and L (WT) (SEQ ID NO: 37) described in WO2009125825, respectively, were produced by the method of reference example 2.

In a similar manner to the method of example 1, the theoretical pI was calculated for these antibodies and is shown in table 4. Antibodies that do not exhibit pH-dependent binding to the IL-6 receptor also exhibit increased pI similar to antibodies that exhibit pH-dependent binding.

[ Table 4]

(3-2) evaluation of binding of antibody to extracellular matrix by electrochemical luminescence (ECL) method

The extracellular Matrix (BD Matrigel base Membrane Matrix; manufactured by BD) was diluted to 2mg/mL using TBS (Takara). The diluted extracellular matrix was dispersed in a MULTI-ARRAY 96-well plate, High binding (High bind), Bare (manufactured by Meso Scale Discovery: MSD) at 5. mu.L/well, and fixed at 4 ℃ overnight. Then, a solution containing 150mM NaCl, 0.05% Tween 20, 0.5% BSA, and 0.01% NaN, pH 7.4 was added₃Was dispersed in the plate for blocking. 20mM ACES buffer (ACES-T buffer) pH 7.4 (containing 150mM NaCl, 0.05% Tween 20, and 0.01% NaN was used₃) The antibodies to be evaluated were diluted to 30, 10, and 3. mu.g/mL, and then used containing 150mM NaCl, 0.01% Tween 20, 0.1% BSA, and 0.01% NaN₃Was further diluted with 20mM ACES buffer pH 7.4 (Dilution buffer) to give final concentrations of 10, 3.3, and 1. mu.g/mL, respectively. The diluted antibody solution was added to the plate with the blocking solution removed, and it was shaken at room temperature for one hour. The antibody solution was removed, ACES-T buffer containing 0.25% glutaraldehyde (glutamide) was added, and after that, it was allowed to stand for 10 minutes, and the plate was treated with DPBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween 20 Manufacturing) and washing. Antibodies for ECL detection were prepared by Sulfo-labeled goat anti-human IgG (γ) (manufactured by Zymed Laboratories) using Sulfo-labeled NHS ester (manufactured by MSD). The antibody for detection was diluted to 1 μ g/mL in dilution buffer, added to the plate, and then shaken at room temperature for one hour in the dark. Removing the antibody for detection, and adding a 2-fold diluted solution prepared by diluting MSD Read buffer T (4 ×) (manufactured by MSD) with ultrapure water; and then the amount of luminescence was measured on the SECTOR imager 2400 (manufactured by MSD).

The results are shown in fig. 5. Both antibodies that show pH-dependent binding and antibodies that do not show pH-dependent binding show increased binding to the extracellular matrix by increasing their pI. Furthermore, surprisingly, the effect of improving extracellular matrix binding by increasing pI was significant in antibodies with pH-dependent antigen binding. In other words, it was found that antibodies that bind antigen in a pH-dependent manner and have a high pI (high pI-IgG1) have the strongest affinity for extracellular matrix.

Without being limited to a particular theory, the results obtained from these experiments may also be explained as follows. Introduction of histidine modifications into antibody variable regions is known as one method of conferring pH-dependent antigen binding properties to antibodies (see, e.g., WO 2009/125825). Histidine has an imidazole group on its side chain and is uncharged at neutral to basic pH conditions, but is known to be positively charged at acidic pH conditions. Using this property of histidine, one can alter the charge environment and conformational environment at the site of antigen interaction between neutral and acidic pH conditions by introducing histidine into the antibody variable region, particularly in one or more CDRs adjacent to the site of antigen interaction. The antibody can be expected to have an antigen affinity that changes in a pH-dependent manner. The skilled person will understand that the effect obtained by said histidine introduction is mainly (essentially) independent of the type of target antigen or the amino acid sequence constituting the antibody, but rather dependent on the site of histidine introduction or the number of histidine residues introduced.

WO2009/041643 is described in its entirety as follows: protein-protein interactions consist of hydrophobic interactions, electrostatic interactions, and hydrogen bonding, and the strength of the binding can be generally expressed using a binding constant (affinity) or an apparent binding constant (avidity). pH-dependent binding, in which the binding strength varies between neutral pH conditions (e.g., pH 7.4) and acidic pH conditions (e.g., pH 5.5 to pH 6.0), depends on the naturally occurring protein-protein interaction. For example, the binding between the aforementioned IgG molecules and FcRn (which is known to be a rescue receptor for IgG molecules) shows strong binding under acidic pH conditions and very weak binding under neutral pH conditions. Histidine residues are involved in many protein-protein interactions that are altered in a pH-dependent manner. Since the pKa of histidine residues is close to 6.0 to 6.5, the state of proton dissociation in histidine residues changes between neutral and acidic pH conditions. More specifically, histidine residues are uncharged and neutral at neutral pH and function as hydrogen atom acceptors; and under acidic pH conditions it is positively charged and functions as a hydrogen atom donor. In addition, in the above-mentioned IgG molecule-FcRn interaction, it has been reported that histidine residues present on IgG molecules are involved in pH-dependent binding (Martin et al, mol. cell.7 (4): 867-877 (2001)).

Thus, substitution of amino acid residues involved in protein-protein interactions with histidine residues, or introduction of histidine at the site of interaction, may confer pH-dependence on protein-protein interactions. Similar processing is performed for protein-protein interactions between antibodies and antigens; and an antibody mutant having reduced affinity for antigen under acidic pH conditions was successfully obtained by introducing histidine into the CDR sequence of an anti-egg white lysozyme antibody (Ito et al, FEBS Lett.309 (1): 85-88 (1992)). In addition, an antibody that specifically binds to an antigen at low pH of cancer tissue due to introduction of histidine in CDR sequences and weakly binds to the corresponding antigen under neutral pH conditions has been reported (WO 2003/105757).

Meanwhile, the amino acid residue introduced to increase the pI is preferably lysine, arginine, or histidine having a positively charged side chain. The standard pKa of the side chains of these amino acids is 10.5 for lysine, 12.5 for arginine, and 6.0 for histidine (Skoog et al, Trends anal. chem.5 (4): 82-83 (1986)). Based on the theory of acid-base equilibrium known in the art, these pKa values mean that, in a solution at pH 10.5, 50% of the lysine side chains are positively charged and the remaining 50% are uncharged. As the pH of the solution increased, the proportion of the lysine side chains that were positively charged decreased, and in a solution at pH 11.5, which was 1 pH higher than the pKa of lysine, the proportion of positive charges became about 9%. On the other hand, as the pH of the solution decreased, the proportion of positive charges increased, and in a solution of pH 9.5, which was 1 pH value lower than the pKa of lysine, the proportion of positive charges became about 91%. This theory applies in a similar manner for arginine and histidine. More specifically, approximately 100% of the lysine or arginine in a neutral pH (e.g., pH 7.0) solution is positively charged, while approximately 9% of the histidine is positively charged. Therefore, in the case where histidine is positively charged at neutral pH, because the level is low compared to lysine or arginine, lysine and arginine are considered to be more favorable amino acids to be introduced for increasing pI. Furthermore, according to Holash et al (Proc. Natl. Acad. Sci.99 (17): 11393-11398(2002), while the introduction of a modification to increase the pI is considered to be effective as a modification to increase extracellular matrix binding, it has been considered that the introduction of a substitution of lysine or arginine is a more favorable amino acid modification than the introduction of a substitution of histidine for the reasons described above.

As first disclosed herein, by combining the introduction of histidine (to confer pH-dependent antigen binding properties) with the modification of the antibody to increase pI, an unexpected synergistic increase in the affinity of the antibody for the extracellular matrix is possible. In fact, while the pI of high _ pI-IgG1 is 9.30, the pI of high _ pI (nph) -IgG1 is 9.35, and thus the value of high _ pI-IgG1 is slightly lower. Nevertheless, the actual affinity for extracellular matrix was significantly stronger for the high pI-IgG 1. This indicates that the affinity for the extracellular matrix cannot be explained only by pI levels, and it can be said that introducing a combination of a modification that increases pI and a histidine modification shows a synergistic effect. This result is an unexpected and unexpected phenomenon.

However, the skilled person must note that, since the pKa of the amino acid side chains in proteins is greatly influenced by the surrounding environment, they will not always match the above theoretical pKa values. More specifically, the above description is provided herein based on general scientific theory; however, the skilled person can easily guess that there may be many exceptions in the actual protein. For example, in Hayes et al, j.biol.chem.250 (18): 7461-7472(1975), when the pKa of histidine contained in myosin was experimentally determined, although the value was centered around 6.0, they were also reported to vary between 5.37 and 8.05. Naturally, histidines with high pKa are predominantly positively charged at neutral pH. Therefore, the above theory does not deny the pI-increasing effect of introducing histidine in the actual three-dimensional structure of the protein. It is fully possible that amino acid modifications incorporating histidine, as observed with lysine or arginine, may also exert the pI increasing effect.

Example 4

Increasing pI by one amino acid substitution in the constant region

Methods have been described for increasing the pI of antibodies that bind antigen in a pH-dependent manner by introducing amino acid substitutions into the variable regions of the antibodies. Furthermore, methods for increasing the pI of an antibody can be performed by performing as few as one amino acid substitution in the antibody constant region.

The method of adding one amino acid substitution to the antibody constant region to increase the pI is not particularly limited, but, for example, it may be performed by the method described in WO 2014/145159. As in the case of the variable region, amino acid substitutions introduced into the constant region are preferably those that decrease the number of negatively charged amino acids (e.g., aspartic acid or glutamic acid) while increasing positively charged amino acids (e.g., arginine or lysine).

Without limitation, the position in the constant region at which an amino acid substitution is introduced is preferably a position in which an amino acid side chain can be exposed on the surface of an antibody molecule. Preferred embodiments include methods of introducing combinations of amino acid substitutions at such positions that can be exposed on the surface of an antibody molecule. Alternatively, the plurality of amino acid substitutions introduced are preferably positions that are conformationally close to each other. Further, without limitation, the introduced multiple amino acid substitutions are preferably substituted with positively charged amino acids, such that in some cases they result in a state in which multiple positive charges are present at conformationally close positions. The definition of "conformationally close position" is not particularly limited, but for example, it may mean a state in which a single amino acid substitution or a plurality of amino acid substitutions are introduced within 20 angstroms, preferably within 15 angstroms, or more preferably within 10 angstroms of each other. Whether the amino acid substitution of interest is at a position exposed on the surface of the antibody molecule, or whether multiple positions of the amino acid substitution are at adjacent positions, can be determined by known methods such as X-ray crystallography.

Further, in addition to the above methods, the method of imparting multiple positive charges at conformationally close positions includes a method using an amino acid initially positively charged in an IgG constant region. Examples of such positively charged amino acid positions include (a) arginine at position 255, 292, 301, 344, 355, or 416, according to EU numbering; and (b) a lysine according to EU numbering position 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, or 439. By substituting a positively charged amino acid at a position conformationally adjacent to these positively charged amino acids, it is possible to impart multiple positive charges at the conformationally adjacent positions.

(4-1) production of pH-dependent anti-IgE binding antibodies

The following three antibodies, which are pH-dependent anti-human IgE antibodies, were produced by the method of reference example 2: (1) ab1, which is a conventional antibody comprising Ab1H (SEQ ID NO: 38) as the heavy chain and Ab1L (SEQ ID NO: 39) as the light chain; (2) ab2, which is a conventional antibody comprising Ab2H (SEQ ID NO: 40) as the heavy chain and Ab2L (SEQ ID NO: 41) as the light chain; and (3) Ab3, which is a conventional antibody comprising Ab3H (SEQ ID NO: 42) as the heavy chain and Ab3L (SEQ ID NO: 43) as the light chain.

(4-2) evaluation of pH dependence of human IgE binding

Ab1 was evaluated for affinity for human IgE at pH 7.4 and pH 5.8 as follows. Kinetic analysis of human IgE and Ab1 was performed using BIACORE T100(GE Healthcare). The following two buffers were used as running buffersAnd (4) flushing liquid measurement: (1)1.2mM CaCl₂0.05% Tween 20, 20mM ACES, 150mM NaCl, pH 7.4; and (2)1.2mM CaCl₂0.05% Tween 20, 20mM ACES, 150mM NaCl, pH 5.8.

An appropriate amount of protein a/g (actigen) was immobilized on a sensor chip CM4(GE Healthcare) by an amide coupling method to capture the target antibody. Next, human IgE was interacted with the antibody captured on the sensor chip by injecting diluted IgE solution and running buffer (used as reference solution). For the running buffer, any of the above-described buffers (1) and (2) was used, and human IgE was diluted with each buffer. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 25 ℃. Kd (m) of human IgE was calculated for each antibody based on the binding rate constant ka (1/Ms) and dissociation rate constant kd (1/s), which is a kinetic parameter calculated from sensorgrams obtained by measurement. Each parameter was calculated using BIACORE T100 evaluation software (GE Healthcare).

The affinities of Ab2 and Ab3 for human IgE at pH 7.4 and pH 5.8 were calculated as follows. BIACORE T200(GE Healthcare) was used to evaluate the binding activity of anti-hIgE antibodies to hIgE (dissociation constant KD (M)). The following two buffers were used as running buffers for the measurements: (1)1.2mM CaCl₂0.05% Tween 20, 20mM ACES, 150mM NaCl, pH 7.4; and (2)1.2mM CaCl₂0.05% Tween 20, 20mM ACES, 150mM NaCl, pH 5.8.

By utilizing the affinity between streptavidin and biotin, an appropriate amount of a peptide produced by adding biotin to the C-terminal Lys present in chemically synthesized human glypican 3 (aka, GPC3) protein-derived peptide (having the amino acid sequence of (VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO: 44) ("biotinylated GPC3 peptide") was added to the sensor chip SA (GE Healthcare) and immobilized on the chip. injection of an appropriate concentration of hIgE and immobilization of biotinylated GPC3 peptide on the chip. injection of an appropriate concentration of anti-hIgE antibody as an analyte and interaction with hIgE on the sensor chip. then, for regeneration of the sensor chip, injection of 10mM glycine-HCl pH 1.5. all measurements were performed at 37 ℃ S) and calculating the dissociation constant kd (m) based on those values.

The results are provided in table 5. All antibodies, Ab1, Ab2 and Ab3, showed pH-dependent binding to human IgE and showed a significant weakening of their affinity at acidic pH conditions (pH 5.8) when compared to their affinity at neutral pH conditions (pH 7.4). Therefore, it is expected that administration of these antibodies to living animals shows an effect of accelerating the removal of human IgE, which is an antigen.

[ Table 5]

Theoretical pI values for Ab1-Ab3 (pIs) calculated in a similar manner to the method of example 1 are shown in table 6.

[ Table 6]

Name of antibody	Theoretical pI
		Ab1	6.77
Ab2	6.48
		Ab3	6.48

(4-3) production of peptides with increased content by Single amino acid modification in the constant regionAntibodies to the pI

Ab1 produced in example (4-1) was an antibody having natural human IgG1 as a constant region. Ab1H-P600 was produced by modifying the Fc region of Ab1H (which is the heavy chain of Ab 1) by substituting proline at position 238 according to EU numbering with aspartic acid and serine at position 298 according to EU numbering with alanine. In addition, various Fc variants were generated by introducing various single amino acid substitutions indicated in tables 7-1 and 7-2, respectively, into the Fc region of Ab1H-P600 by the method of reference example 2. For all Fc variants, Ab1L (SEQ ID NO: 39) was used as the light chain. The affinity of these antibodies to hfcyrii 2b was comparable to that of the P600 variant (data not shown).

[ Table 7-1]

Name of variants	Amino acid mutations added to P600	Biaeore	Imaging
				P600	None	1.00	1.00
P828	Q196K	1.27	0.98
				P829	S337R	0.17	0.89
P830	L358K	1.24	2.35
				P831	P387R	3.85	1.30
P836	E345Q	1.85	Without data
				P837	E345R	1.88	Without data
P838	D356Q	1.87	Without data
				P839	D356N	2.17	Without data
P840	T359K	2.25	Without data
				P841	N361R	1.86	Without data
P842	Q362K	2.37	Without data
				P843	E380R	-0.04	Without data
P844	E382Q	1.24	Without data
				P845	E382K	1.38	Without data
P846	Q386K	1.71	Without data
				P847	N389K	1.57	Without data
P848	S415R	1.38	Without data
				P849	Q418R	2.21	Without data
P850	Q419K	2.22	Without data
				P851	N421R	1.43	1.56
P852	S424K	1.40	Without data
				P854	L443R	1.93	Without data
P905	N384R	1.34	2.36
				P906	G385R	1.74	1.12
P907	H433R	0.09	3.55
				P908	N434R	0.42	1.88
P909	H435R	0.73	0.77
				P910	L309R	1.73	1.80
P912	T307R	0.24	1.30
				P914	D399R	1.72	3.78
P915	S400R	0.85	2.01
				P917	A327R	-0.05	2.49
P918	L328R	-0.06	0.00
				P919	P329R	-0.06	0.00

[ tables 7-2]

P920	A330R	0.01	1.67
				P921	P331R	-0.07	0.02
P923	Q311R	1.63	2.88
				P924	N315R	2.08	2.66
P925	Y296R	-0.05	0.34
				P926	Q295R	-0.06	0.04
P927	E294R	0.25	0.29
				P928	E293R	0.20	0.04
P929	P291R	0.47	0.53
				P930	A287R	-0.07	0.00
P931	N286R	0.68	1.27
				P932	H285R	1.38	1.96
P934	V282R	1.54	1.67
				P935	G281R	-0.06	2.14
P937	E272R	0.21	0.56
				P938	P271R	-0.06	0.04
P939	D270R	-0.07	0.01
				P940	E269R	-0.05	0.01
P941	H268R	0.47	0.44
				P942	E258R	Without data	3.07
P944	T256R	Without data	1.49
				P945	S254R	Without data	5.70
P946	I253R	Without data	1.05
				P947	M252R	Without data	0.94
P948	L251R	Without data	0.13

(4-4) human Fc. gamma. RIIb-binding assay by BIACORE Using novel Fc region variant-containing antibodies

Using BIACORE (registrar)Beacon) T200(GE Healthcare) antibody binding assays containing Fc region variants between soluble human Fc γ RIIb (also known as "hFc γ RIIb") and antigen-antibody complexes were performed. Soluble hFc γ RIIb was generated as a His-tag tagged molecule using methods known in the art. An appropriate amount of anti-His antibody was immobilized on the sensor chip CM5(GE Healthcare) by amine coupling method using a His capture kit (GE Healthcare) to capture hFc γ RIIb. Next, the antibody-antigen complex and running buffer (as reference solution) were injected and allowed to interact with the hfcyriib captured on the sensor chip. 20mM N- (2-acetamido) -2-aminoethanesulfonic acid, pH 7.4, 150mM NaCl, 1.2mM CaCl ₂And 0.05% (w/v) tween 20 was used as the running buffer, and each buffer was also used to dilute the soluble hFc γ RIIb. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 25 ℃. Analysis was performed based on binding (RU) calculated from a sensorgram obtained by measurement, and a relative value was displayed when the binding amount of P600 was defined as 1.00. For calculating the parameters, BIACORE (registered trademark) T100 evaluation software (GE Healthcare) was used. The results are shown in tables 7-1 and 7-2 (see column "BIACORE" in the tables) and in FIG. 6. Several Fc variants were shown to have enhanced affinity for hFc γ RIIb immobilized on BIACORE (registered trademark) sensor chips.

Without being bound to a particular theory, this result can be explained as follows. BIACORE (registered trademark) sensor chips are known to be negatively charged, and the charged state can be considered to resemble a cell membrane surface. More specifically, it is assumed that the binding of antigen-antibody complexes to hFc γ RIIb immobilized on a negatively charged BIACORE sensor chip is similar to the way in which antigen-antibody complexes bind to hFc γ RIIb present on the surface of negatively charged cell membranes.

Antibodies generated by introducing modifications that increase the pI into the Fc region are those in which the charge of the Fc region (constant region) is more positively charged when compared to those before the introduction of the modifications. Thus, it is believed that the coulombic interaction between the Fc region (positive charge) and the sensor chip surface (negative charge) is enhanced by amino acid modifications that increase the pI. Furthermore, the effect is expected to similarly occur on negatively charged cell membrane surfaces; thus, they are also expected to show the effect of accelerating the rate or rate of uptake of cells in vivo.

From the above results, a ratio of more than about 1.2-fold or more for the binding of variant to hfcyriib was considered to have a strong charge effect on the binding of antibody to hfcyriib on the sensor chip when compared to the binding of Ab1H-P600 to hfcyriib. Thus, modifications contemplated to result in charge effects include, for example, modifications according to EU numbering positions 196, 282, 285, 309, 311, 315, 345, 356, 358, 359, 361, 362, 382, 384, 385, 386, 387, 389, 399, 415, 418, 419, 421, 424, or 443. Preferably, the modification is at position 282, 309, 311, 315, 345, 356, 359, 361, 362, 385, 386, 387, 389, 399, 418, 419, or 443. The amino acid substitution introduced at said position is preferably arginine or lysine. Another example of a mutated amino acid position where the charge effect can be expected includes glutamic acid at position 430 according to EU numbering. Preferred amino acid substitutions to be introduced at position 430 are arginine or lysine, which are positively charged, or in uncharged residues, glycine or threonine are preferred.

(4-5) uptake of antibody containing Fc region variant by hFc γ RIIb-expressing cells

To evaluate the rate of intracellular uptake into cell lines expressing hfcyriib using the novel Fc region variant-containing antibodies generated, the following assay was performed.

MDCK (Madin-Darby canine kidney) cell lines constitutively expressing hFc γ RIIb were generated using known methods. Using these cells, intracellular uptake of antigen-antibody complexes was evaluated. Specifically, pHrodoRed (Life technologies) was used to label human IgE (antigen) according to an established protocol, and an antigen-antibody complex was formed in the culture solution, in which the antibody concentration was 10.8mg/mL and the antigen concentration was 12.5 mg/mL. The culture solution containing the antigen-antibody complex was added to a culture plate of the above-described MDCK cells constitutively expressing hFc γ RIIb and incubated for one hour, and then the fluorescence intensity of the antigen taken into the cells was quantified using cell Analyzer 6000(GE healthcare). The amount of antigen taken is expressed relative to the P600 value taken as 1.00.

Tables 7-1 and 7-2 (see "imaging" column in tables) and FIG. 7 show the results. Strong fluorescence from antigens in cells was observed in multiple Fc variants.

While not being limited to a particular theory, this result may be explained as follows: the antigen and the antibody added to the cell culture solution form an antigen-antibody complex in the culture solution. The antigen-antibody complex binds to hfcyriib expressed on the cell membrane via the antibody Fc region and is taken up into the cell in a receptor-dependent manner. Ab1 used in this experiment was an antibody that binds antigen in a pH-dependent manner; thus, the antibody can be dissociated from the antigen. Because the dissociated antigen is labeled with a pHrodoRed as described previously, it fluoresces in the endosome. Thus, a stronger fluorescence intensity inside the cell compared to the control is considered to indicate that the uptake of the antigen-antibody complex into the cell occurs faster or at a higher frequency.

Herein, a ratio of greater than about 1.05-fold or more of the fluorescence intensity of the antigen taken up into the cells of the variant compared to the fluorescence intensity of Ab1H-P600 is considered to have a charge effect on the antigen taken up into the cells. A ratio of the fluorescence intensity of the antigen taken up into the cells of the variant of more than about 1.5-fold or more as compared with the fluorescence intensity of Ab1H-P600 is considered to have a strong charge effect on the antigen taken up into the cells. Thus, the above results indicate that by introducing a modification to increase the pI into an appropriate position of the Fc region, uptake into cells can be accelerated compared to before the introduction of the modification. Amino acid position modifications that show such effects are, for example, according to EU numbering positions 253, 254, 256, 258, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 358, 384, 385, 387, 399, 400, 421, 433, or 434. Preferably, the modification is at position 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 358, 384, 399, 400, 421, 433, or 434 according to EU numbering. The amino acid substitution introduced at said position is preferably arginine or lysine. Without limitation, the position at which an amino acid substitution is introduced into the constant region in order to increase the pI of the antibody may be, for example, the amino acid residue at position 285 according to EU numbering. Alternatively, other examples may include amino acid substitutions of the amino acid residue at position 399 according to EU numbering.

Example 5

Production of Fc variants with enhanced FcRn binding at acidic pH for improved retention in plasma

Under acidic pH conditions in endosomes, IgG antibodies taken up into cells are known to return to plasma by binding to FcRn. Thus, IgG antibodies typically have a long plasma half-life compared to proteins that do not bind FcRn. Methods are known to exploit this property to enhance the plasma retention of antibodies by increasing their FcRn affinity under acidic pH conditions (by introducing amino acid modifications in the Fc region of the antibody). Specifically, methods are known for improving the plasma retention of antibodies by increasing their affinity for FcRn under acidic pH conditions (by amino acid modifications such as M252Y/S254T/T256E (YTE) modification) (Dall' Acqua et al, J.biol. chem.281: 23514-.

On the other hand, as described above, it is also known that an Fc variant having an increased affinity for FcRn under acidic pH conditions shows an undesirable affinity for Rheumatoid Factor (RF) (WO 2013/046704). Thus, the following studies were performed in order to generate Fc variants that can improve plasma retention (with reduced or substantially no binding to rheumatoid factor).

(5-1) production of novel antibody containing Fc region variant

As shown below, Fc variants with increased FcRn affinity under acidic pH conditions were generated, such as those including known modifications, YTE, LS, or N434H, as well as a number of novel Fc variants (F1847m, F1848m, F1886m, F1889m, F1927m, and F1168 m).

The sequences encoding the heavy chains (in which the amino acid modifications were introduced into the Fc region of the heavy chains (VH3-IgG1m) of Fv4-IgG1, which is an anti-human IL-6 receptor antibody) were generated by the method of reference example 1. These heavy chains were used to generate the following antibodies by the method of reference example 2: (a) fv4-IgG1 comprising VH3-IgG1m (SEQ ID NO: 46) as the heavy chain and VL3-CK as the light chain; (b) fv4-YTE comprising VH3-YTE (SEQ ID NO: 47) as the heavy chain and VL3-CK as the light chain; (c) fv4-LS comprising VH3-LS (SEQ ID NO: 48) as the heavy chain and VL3-CK as the light chain; (d) fv4-N434H comprising VH3-N434H (SEQ ID NO: 49) as the heavy chain and VL3-CK as the light chain; (e) fv4-F1847m which comprises VH3-F1847m (SEQ ID NO: 50) as the heavy chain and VL3-CK as the light chain; (f) fv4-F1848m which comprises VH3-F1848m (SEQ ID NO: 51) as the heavy chain and VL3-CK as the light chain; (g) fv4-F1886m comprising VH3-F1886m (SEQ ID NO: 52) as the heavy chain and VL3-CK as the light chain; (h) fv4-F1889m comprising VH3-F1889m (SEQ ID NO: 53) as the heavy chain and VL3-CK as the light chain; (i) fv4-F1927m comprising VH3-F1927m (SEQ ID NO: 54) as the heavy chain and VL3-CK as the light chain; and (j) Fv4-F1168m comprising VH3-F1168m (SEQ ID NO: 55) as the heavy chain and VL3-CK as the light chain.

(5-2) analysis of binding kinetics to human FcRn

An antibody containing VH3-IgG1m or the above-described variant as a heavy chain and L (WT) (SEQ ID NO: 37) as a light chain was produced by the method of reference example 2, and the binding activity to human FcRn was evaluated as follows.

Kinetic analysis of human FcRn and each of the antibodies was performed using BIACORE T100(GE Healthcare). An appropriate amount of protein l (actigen) was immobilized on a sensor chip CM4(GE Healthcare) by an amine coupling method to capture the target antibody. Subsequently, human FcRn was interacted with the antibody captured on the sensor chip by injecting diluted FcRn solution and running buffer (used as reference solution). For the running buffers, 50mM sodium phosphate, 150mM NaCl, and 0, 05% (w/v) tween 20(pH 6.0) were used, and each buffer was also used to dilute FcRn. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 25 ℃. Kd (m) for human FcRn was calculated for each antibody based on the association rate constant ka (1/Ms) and dissociation rate constant kd (1/s), which is a kinetic parameter calculated from sensorgrams obtained by measurement. BIACORE T100 evaluation software (GE Healthcare) was used to calculate each parameter.

The results are shown in Table 8.

[ Table 8]

Example 6

Evaluation of affinity of antibody containing Fc region variant with enhanced FcRn binding to rheumatoid factor under acidic pH conditions

Anti-drug antibodies (ADAs) affect the efficacy and pharmacokinetics of therapeutic antibodies and sometimes cause serious side effects; thus, the clinical utility and efficacy of therapeutic antibodies may be limited by the production of ADA. Many factors influence the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is a factor. Furthermore, the presence of ADA (also referred to as "pre-existing ADA") in a patient prior to administration of a therapeutic antibody may have similar problems. In particular, in the case of therapeutic antibodies in patients with self-diseases such as Rheumatoid Arthritis (RA), Rheumatoid Factor (RF), which is an autoantibody against human IgG, can cause "preexisting ADA" problems. Recently, it was reported that humanized anti-CD 4IgG1 antibodies with an N434H (Asn434His) mutation caused significant rheumatoid factor binding (Zheng et al, Clinical Pharmacology & Therapeutics 89 (2): 283-. Detailed studies confirmed that the N434H mutation in human IgG1 increased the binding of rheumatoid factor to the Fc region of the antibody compared to the parent human IgG 1.

Rheumatoid factor is a polyclonal autoantibody against human IgG and its epitope in human IgG varies from clone to clone and appears to be located at the CH2/CH3 junction and in the CH3 domain which may overlap with the FcRn-binding epitope. Thus, mutations that increase the binding activity (binding affinity) for FcRn may increase the binding activity (binding affinity) of rheumatoid factor to a particular clone.

Indeed, with regard to Fc having increased affinity for FcRn at acidic pH or neutral pH, it is also known that not only the N434H modification but also many other amino acid modifications similarly increase Fc binding to rheumatoid factor (WO 2013/046704).

On the other hand, various amino acid modifications that selectively inhibit the affinity for rheumatoid factor without affecting the affinity for FcRn are provided as examples in WO2013/046704, and among them, combinations of two amino acid mutations, i.e., Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D, have been indicated. Therefore, Q438R/S440E was introduced into the Fc first disclosed herein with a new increased affinity at acidic pH to investigate whether binding to rheumatoid factors could be reduced.

(6-1) determination of rheumatoid factor binding of antibody containing Fc region variant

Binding assays for rheumatoid factor were performed by using individual sera (Proteogenex) from 30 RA patients at pH 7.4 using Electrochemiluminescence (ECL). A50-fold dilution of the serum sample, biotinylated test antibody (1. mu.g/mL), and SULFO-TAG NHS ester (Meso Scale Discovery) -labeled test antibody (1. mu.g/mL) were each mixed and incubated at room temperature for three hours. Thereafter, the mixture was added to a streptavidin-coated MULTI-ARRAY 96-well plate (Meso Scale Discovery) and the plate was incubated at room temperature for two hours and subsequently washed. Immediately after adding read buffer T (x4) (Meso Scale Discovery) to each well, the plate was placed on the SECTOR imager 2400 reader (Meso Scale Discovery) and chemiluminescence was measured.

The results of this assay are shown in figures 8 to 17. Fv4-IgG1 (fig. 8) with native human IgG1 showed only weak binding to rheumatoid factor, whereas existing Fc variants with increased FcRn binding, Fv4-YTE (fig. 9), Fv4-LS (fig. 10), and Fv4-N434H (fig. 11), all showed significantly increased rheumatoid factor binding in many donors. On the other hand, all of the new Fc region variants with increased FcRn binding, Fv4-F1847m (fig. 12), Fv4-F1848m (fig. 13), Fv4-F1886m (fig. 14), Fv4-F1889m (fig. 15), Fv4-F1927m (fig. 16), and Fv4-F1168m (fig. 17), showed only weak rheumatoid factor binding, and this suggests that binding to rheumatoid factor is significantly inhibited as a result of the modification to increase FcRn binding.

Figure 18 shows the mean values of rheumatoid factor-binding affinity for each variant in the sera of 30 RA patients. All six new variants showed lower affinity than the three pre-existing variants (YTE, LS and N434H) and they also showed lower affinity for rheumatoid factors compared to native human IgG 1. Thus, when considering the clinical development of therapeutic antibodies with improved affinity for FcRn for autoimmune diseases such as rheumatoid arthritis and the like, the risks associated with rheumatoid factor, which are a problem with existing Fc variants, are suppressed in the Fc variants first disclosed herein, and thus they can be used more safely than the existing known Fc variants.

Example 7

PK assessment of Fc variants with increased FcRn binding in cynomolgus monkeys under acidic pH conditions

In example 7, the effect of improving plasma retention in cynomolgus monkeys was evaluated using the novel Fc region variant-containing antibodies provided herein, whose binding to rheumatoid factor was confirmed to be inhibited, by the following method.

(7-1) production of novel antibody containing Fc region variant

The following anti-human IgE antibodies were produced: (a) OHB-IgG1 comprising OHBH-IgG1(SEQ ID NO: 56) as the heavy chain and OHBL-CK (SEQ ID NO: 57) as the light chain; (b) OHB-LS comprising OHBH-LS (SEQ ID NO: 58) as the heavy chain and OHBL-CK as the light chain; (c) OHB-N434A comprising OHBH-N434A (SEQ ID NO: 59) as the heavy chain and OHBL-CK as the light chain;

(d) OHB-F1847m comprising OHBH-F1847m (SEQ ID NO: 60) as the heavy chain and OHBL-CK as the light chain; (e) OHB-F1848m comprising OHBH-F1848m (SEQ ID NO: 61) as the heavy chain and OHBL-CK as the light chain; (f) OHB-F1886m comprising OHBH-F1886m (SEQ ID NO: 62) as the heavy chain and OHBL-CK as the light chain; (g) OHB-F1889m comprising OHBH-F188 1889m (SEQ ID NO: 63) as the heavy chain and OHBL-CK as the light chain; and (h) OHB-F1927m comprising OHBH-F1927m (SEQ ID NO: 64) as the heavy chain and OHBL-CK as the light chain.

(7-2) monkey PK assay for novel antibodies containing Fc region variants

The in vivo kinetics of anti-human IgE antibodies in plasma after administration of anti-human IgE antibodies to cynomolgus monkeys was evaluated. The anti-human IgE antibody solution was administered once intravenously at 2 mg/kg. Blood collection was performed five minutes, (two hours), seven hours, one day, two days, three days, (four days), seven days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4 ℃ and 15,000rpm for 5 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-80 ℃ or lower until the measurement was performed. Eight types of anti-human IgE antibodies were used, namely OHB-IgG1, OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927 m.

(7-3) measurement of concentration of anti-human IgE antibody in plasma by ELISA

The concentration of anti-human IgE antibody in the plasma of cynomolgus monkeys was measured by ELISA. First, anti-human IgG kappa chain antibody (antibody solution) was dispersed in Nunc-Immuno plate, maxisorp (nalge Nunc international) and allowed to stand at 4 ℃ overnight to produce anti-human IgG kappa chain antibody-immobilized plate. Calibration curve samples were prepared at plasma concentrations of 640, 320, 160, 80, 40, 20 or 10ng/mL, and cynomolgus monkey plasma measurement samples diluted 100-fold or more. These calibration curve samples and plasma measurement samples were generated such that cynomolgus monkey IgE (product prepared in company) was added at a concentration of 1 μ g/mL. Subsequently, the sample was dispersed in an anti-human IgG kappa chain antibody-immobilized plate and allowed to stand at room temperature for two hours. Then, HRP-anti-human IgG γ chain antibody (Southern Biotech) was dispersed and allowed to stand at room temperature for one hour. Subsequently, a chromogenic reaction was performed using TMB Chromogen (Chromogen) solution (Life Technologies) as a substrate, and after the reaction was terminated by adding 1N sulfuric acid (Wako), absorbance at 450nm was measured by a microplate reader. The concentration of anti-human IgE antibody in monkey plasma was calculated from the absorbance of the calibration curve using analytical software SOFTmax PRO (Molecular Devices). The changes in the measured concentrations of anti-human IgE antibodies in monkey plasma are shown in fig. 19. From the changes in the measured concentration of anti-human IgE antibodies in monkey plasma, the removal clearance was calculated using a transient analysis of Phoenix WinNonlin ver.6.2(Pharsight Corporation). The calculated pharmacokinetic parameters are shown in table 9. Samples from individuals in plasma positive for the administered sample antibodies were excluded from the calculation of changes in anti-human IgE antibody concentration and clearance in monkey plasma.

[ Table 9]

Elimination of samples administered after administration of anti-human IgE antibodies

(7-4) measurement of antibodies against the administered sample in plasma by the electrochemiluminescence method

Antibodies in monkey plasma against the administered samples were measured by electrochemiluminescence methods. The applied sample was ruthenium-labeled with SULFO-TAG NHS ester (Meso Scale Discovery), the applied sample biotinylated with EZ-Link Micro SULFO-NHS-biotinylation kit (Pierce), and cynomolgus monkey plasma measurement samples were mixed in equal amounts and allowed to stand overnight at 4 ℃. The sample was added to a Multi-ARRAY 96-pore streptavidin Gold Plate (Meso Scale Discovery), then allowed to react at room temperature for two hours, and washed. Then, the reading buffer T (x4) (Meso Scale Discovery) was dispersed into the plate and immediately measured using the SECTOR imager 2400(Meso Scale Discovery).

As a result, it was confirmed that all the novel Fc variants showed greatly improved plasma retention compared to the Fc region of native IgG 1.

(7-5) mouse PK assay for Fc variants

The following experiment was performed to compare F1718, which is the Fc variant described in WO2013/046704, and F1848m, which is the newly discovered Fc variant of this time, which is an Fc variant that increases FcRn binding at acidic pH.

The sequence encoding the heavy chain in which amino acid modifications were introduced into the Fc region of the heavy chain (VH3-IgG1) of Fv4-IgG1 (anti-human IL-6 receptor antibody) was generated by the method of reference example 1. Using these heavy chains, the following antibodies were produced with reference to the method of example 2: (a) fv4-IgG1 comprising VH3-IgG1 as the heavy chain and VL3-CK as the light chain; and (b) Fv4-F1718 comprising VH3-F1718(SEQ ID NO: 65) as the heavy chain and VL3-CK as the light chain.

The above anti-human IL-6 receptor antibody was administered at 1mg/kg to the tail vein of human FcRn transgenic mice (B6.mFcRn-/-. hFcRn Tg line 32+/+ mice; Jackson Laboratories, Methods mol. biol. 602: 93-104 (2010). once, 15 minutes, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after the administration of the anti-human IL-6 receptor antibody, blood was collected, the collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to obtain plasma, and the separated plasma was stored in a refrigerator at-20 ℃ or below until measurement was performed.

(7-6) measurement of the concentration of anti-human IL-6 receptor antibody in plasma by ELISA

The concentration of anti-human IL-6 receptor antibody in the plasma of mice was measured by ELISA. First, an anti-human IgG (gamma-chain specificity) F (ab') of the antibody was introduced ₂Fragments (SIGMA) were dispersed in Nunc-Immuno plates, MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4 ℃ to produce plates immobilized with anti-human IgG. Calibration curve samples containing anti-human IL-6 receptor antibody at plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, or 0.0125. mu.g/mL and mouse plasma measurement samples diluted 100-fold or more were prepared, respectively. 200 μ L of 20ng/mL soluble human IL-6 receptor was added to 100 μ L of the calibration curve sample or plasma measurement sample, and the mixed solution was then allowed to stand at room temperature for one hour. Subsequently, the mixed solution was dispersed in each well of the plate on which the anti-human IgG was immobilized, and the plate was left to stand at room temperature for one hour. Then, biotinylated anti-human IL-6R antibody (R) was added&D) The reaction was carried out at room temperature for one hour. Subsequently, streptavidin-PolyHRP 80 (Stereospeicic Detection Technologies) was added and reacted at room temperature for One hour, and the color reaction of the reaction solution was performed using TMB One-Component HRP Microwell Substrate (TMB One Component HRP Microwell Substrate) (BioFX Laboratories) as a Substrate. After the reaction was terminated by adding 1N sulfuric acid (Showa Chemical), the absorbance of the reaction solution at 450nm in each well was measured on a microplate reader. Antibodies in mouse plasma Bulk concentration was calculated from the absorbance of the calibration curve using analytical software SOFTmax PRO (Molecular Devices).

The results are shown in FIG. 20. F1718, which is an Fc variant that increases FcRn binding at acidic pH as described in WO2013/046704, did not show any effect of prolonging antibody PK, but showed comparable plasma retention to native IgG 1.

F1718 has four mutations introduced into the Fc region, namely N434Y/Y436V/Q438R/S440E. In contrast, F1848m, first disclosed herein, introduced four mutations, N434A/Y436V/Q438R/S440E. The only difference in the amino acid mutations introduced into these two types of Fc was that the amino acid mutation introduced at position 434 according to EU numbering was Y (tyrosine) in F1718 and a (alanine) in F1848 m. In example (7-2), F1848m showed improved plasma retention compared to native IgG1, whereas F1718 did not show any improvement in plasma retention. Therefore, without limitation, this suggests that a (alanine) is preferred as the amino acid mutation to be introduced at position 434 for improving plasma retention.

(7-7) predicted immunogenicity scores for Fc variants

The production of anti-drug antibodies (ADA) affects the efficacy and pharmacokinetics of therapeutic antibodies and in some cases carries serious side effects; and thus, clinical utility and efficacy of therapeutic antibodies may be limited by the production of ADA. The immunogenicity of therapeutic antibodies is known to be influenced by many factors and, in particular, the importance of the effector T cell epitopes carried by therapeutic antibodies has been reported many times.

Computational tools such as epibase (lonza), iTope/tced (antitope), and epimatrix (epivax) have been developed for predicting T cell epitopes. Using these computational tools, T cell epitopes in each amino acid sequence can be predicted (Walle et al, Expert Opin. biol. Ther.7 (3): 405-418(2007)) and the potential immunogenicity of therapeutic antibodies can be evaluated.

EpiMatrix was used to calculate the immunogenicity of the Fc variants evaluated. EpiMatrix is a system for predicting the immunogenicity of proteins studied by automatically designing sequences of peptide fragments in nine amino acid segments by the amino acid sequence of the protein whose immunogenicity is to be predicted, and then calculating their ability to bind eight major MHC class II alleles (DRB1 x 0101, DRB1 x 0301, DRB1 x 0401, DRB1 x 0701, DRB1 x 0801, DRB1 x 1101, DRB1 x 1301, and DRB1 x 1501), a system for predicting the immunogenicity of the protein studied (clin. immunol.131 (2): 189-.

F1718 and F1756 described in WO2013/046704 (N434Y/Y436T/Q438R/S440E) contain the N434Y mutation. In contrast, the newly disclosed F1848m and F1847m contained the N434A mutation.

The immunogenicity scores for these four variants, i.e., F1718, F1848m, F1756, and F1847m, were calculated as described above and are shown in table 31 under the "EpiMatrix score" column. In addition, with respect to EpiMatrix scores, immunogenicity scores calibrated for Tregitope content are shown in the column "tReg adjusted Epx score". Tregitope is a peptide fragment sequence that is largely found in naturally occurring antibody sequences and is a sequence thought to suppress immunogenicity by activating regulatory T cells (tregs).

[ Table 31]

Based on these results, both the "EpiMatrix score" and the "tReg-modulated Epx score" showed a reduction in the immunogenicity scores of N434A variants F1848m and F1847m compared to N434Y variant. This suggests that a (alanine) is preferred as the amino acid mutation to be introduced at position 434 for a lower immunogenicity score.

Example 8

Production of humanized anti-human IL-8 antibodies

(8-1) Generation of humanized anti-human IL-8 antibody hWS-4

The humanized anti-IL-8 antibodies disclosed in U.S. Pat. No. 6,245,894 bind to human IL-8(hIL-8) and block its physiological function. Modified humanized anti-IL-8 antibodies can be generated by combining the heavy and light chain variable region sequences disclosed in U.S. Pat. No. 6,245,894 with virtually any of a variety of known human antibody constant region sequences. Thus, the human antibody constant region sequences of these modified antibodies are not particularly limited, but a natural human IgG1 sequence or a natural human IgG4 sequence may be used as the heavy chain constant region, and a natural human kappa sequence may be used as the light chain constant region sequence.

From the humanized IL-8 antibody disclosed in U.S. Pat. No. 6,245,894, the coding sequence of hWS4H-IgG1 (SEQ ID NO: 83) combined with the heavy chain variable region RVHg and the native human anti-IgG 1 sequence for the heavy chain constant region was generated by the method of reference example 1. In addition, the coding sequence of hWS4L-k0MT (SEQ ID NO: 84), which combines the light chain variable region RVLa with the native human kappa sequence for the light chain constant region, was generated by the method of reference example 1. An antibody combining the above heavy and light chains was generated and designated humanized WS-4 antibody (below, hWS-4).

(8-2) production of humanized anti-human IL-8 antibody Hr9

A novel humanized antibody was generated using human consensus framework region sequences different from the FR used in hWS-4.

Specifically, the hybridizing sequence of VH3-23 and VH3-64 was used as heavy chain FR1, the sequence seen in VH3-15 and VH3-49 was used as FR2, the sequence seen in VH3-72 was used as FR3 (provided that 82a according to Kabat numbering is not included), and the sequence seen in JH1 was used as FR 4. These sequences were ligated to the CDR sequences of the hWS-4 heavy chain to generate Hr9-IgG1(SEQ ID NO: 85), a novel humanized antibody heavy chain.

Next, two types of antibodies were produced, namely, hWS-4 having hWS4H-IgG1 as the heavy chain and hWS4L-k0MT as the light chain, and Hr9 having Hr9-IgG1 as the heavy chain and hWS4L-k0MT as the light chain. Within the scope of the disclosure C described herein, especially with reference to light chains, Hr9 is written Hr9/hWS 4L. Antibodies were expressed using FreeStyle 293F cells (Invitrogen) according to the protocol attached to the product. The antibody was purified from the culture supernatant by the method of reference example 2. As a result, antibodies were obtained in the amounts shown in table 11. Surprisingly, the expression level of Hr9 was approximately 8-fold higher than the expression level of hWS-4.

[ Table 11]

	Antibody yield/1 mL Medium (μ g)
		hWS-4	6.4
Hr9	50

(8-3) human IL-8-binding Activity of hWS-4 and Hr9

The binding affinity of hWS-4 and Hr9 to human IL-8 was determined using BIACORE T200(GE Healthcare) as follows.

A running buffer having a composition of 0.05% Tween 20, 20mM ACES, and 150mM NaCl (pH 7.4) was used. An appropriate amount of protein a/g (pierce) was immobilized on a sensor chip CM4(GE Healthcare) by an amine coupling method and a target antibody was captured. Next, the human IL-8 was allowed to interact with the antibody captured on the sensor chip by injecting a diluted human IL-8 solution and a running buffer (used as a reference solution). For the running buffer, a solution having the above composition was used, and the buffer was also used to dilute human IL-8. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 37 ℃. KD (M) for human IL-8 is calculated for each antibody based on the association rate constant kon (1/Ms) and the dissociation rate constant koff (1/s), which are kinetic parameters calculated from sensorgrams obtained by measurement. BIACORE T200 evaluation software (GE Healthcare) was used to calculate each parameter.

The results are shown in table 12. hWS-4 and Hr9 were confirmed to have comparable binding affinity for human IL-8.

[ Table 12]

Name of antibody	kon(1/Ms)	koff(1/s)	KD(M)
				hWS-4	9.74E+05	2.03E-04	2.09E-10
Hr9	1.11E+06	2.17E-04	1.95E-10

For the development of antibody drugs, the production level of antibody molecules is an important factor, and in general, a high production level is desired. Of particular note from the above assays, a more appropriate human consensus framework-derived sequence was selected for combination with the HVR sequence of hWS-4 and yielded Hr9 with improved production levels while maintaining binding affinity for human IL-8.

Example 9

Production of antibodies with pH-dependent IL-8 affinity

(9-1) production of Hr 9-modified antibody conferring pH dependence

To impart pH-dependent IL-8 affinity to the Hr9 antibody obtained in example 8, a study was conducted.

Without being bound by a particular theory, an antibody with pH-dependent affinity for IL-8 may exhibit the following in vivo behavior. Antibodies administered to living organisms can strongly bind to IL-8 and block its function while maintaining a neutral pH environment (e.g., in plasma). A portion of the IL-8/antibody complex is taken up into the cell by non-specific interactions with the cell membrane (pinocytosis), hereinafter referred to as non-specific uptake. Under acidic pH conditions of endosomes, the binding affinity of the aforementioned antibodies to IL-8 becomes weak, and thus the antibodies dissociate from IL-8. The antibody dissociated from IL-8 can then be returned extracellularly via FcRn. The aforementioned antibodies that return extracellularly (into the plasma) in this way can bind another IL-8 again and block its function. It is thought that antibodies with pH-dependent affinity for IL-8 are able to bind IL-8 multiple times by the above mechanism.

In contrast, in the case of an antibody that does not have the properties of the aforementioned antibody, the antibody molecule can neutralize the antigen only once, but not many times. Typically, because IgG antibodies have two Fabs, a single antibody molecule can neutralize two molecules of IL-8. On the other hand, antibodies capable of binding IL-8 multiple times can bind IL-8 any number of times as long as they remain in vivo. For example, up to 20 molecules of IL-8 can be neutralized by a single molecule of a pH-dependent IL-8-binding antibody that is administered until removed, taken up ten times into the cell. Thus, antibodies that can bind IL-8 multiple times have the advantage of being able to neutralize multiple IL-8 molecules even with small amounts of antibody. From another perspective, antibodies that can bind IL-8 multiple times have the advantage of being able to remain in a state that is capable of neutralizing IL-8 for a longer period of time than when the same amount of antibody that does not have the properties that it possesses is administered. From another perspective, antibodies that can bind IL-8 multiple times have the advantage of being able to block the biological activity of IL-8 more strongly than when the same amount of antibody is administered that does not have the properties possessed.

To achieve these advantages, amino acid modifications (mainly histidine) were introduced into the variable regions of Hr9-IgG1 and WS4L-k0MT, with the aim of generating antibodies capable of multiple binding to IL-8. Specifically, the variants shown in table 13 were produced by the methods of reference examples 1 and 2.

The markers shown in table 13 are as "Y97H" showing the position where the mutation was introduced, as defined by Kabat numbering, the amino acid before the mutation was introduced, and the amino acid after the mutation was introduced. Specifically, when denoted as "Y97H", it denotes the substitution of the amino acid residue at position 97 from Y (tyrosine) to H (histidine) according to Kabat numbering. Furthermore, when combinations of multiple amino acid substitutions are introduced, they are described as "N50H/L54H".

[ Table 13]

Name of antibody	Mutations introduced into the heavy chain	Introduction of mutations in the light chain
			Hr9/WS4L	Is free of	Is free of
Hr9/L16	Is free of	L54H
			H89/WS4L	Y97H	Is free of
H89/L12	Y97H	N50H
			H89/L16	Y97H	L54H

(9-2) pH-dependent affinity for IL-8

The human IL-8-binding affinity of the antibody produced in example 9-1 was determined using BIACORE T200(GE Healthcare), as described below. The following two running buffers were used: (1) 0.05% Tween 20, 20mM ACES, 150mM NaCl, pH 7.4; and (2) 0.05% Tween 20, 20mM ACES, 150mM NaCl, pH 5.8.

An appropriate amount of protein a/g (pierce) was immobilized on a sensor chip CM4(GE Healthcare) by an amine coupling method and a target antibody was captured. Next, the human IL-8 was interacted with the captured antibody on the sensor chip by injecting diluted human IL-8 solution and running buffer (used as a reference solution). For the running buffer, use the above solution in any kind, and each buffer also used for dilution of IL-8. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 37 ℃. KD (M) for human IL-8 is calculated for each antibody based on the association rate constant kon (1/Ms) and the dissociation rate constant koff (1/s), which are kinetic parameters calculated from sensorgrams obtained by measurement. BIACORE T200 evaluation software (GE Healthcare) was used to calculate each parameter.

The results are shown in Table 14-1. First, Hr9/L16, which contained L54H modifications in the light chain, had slightly enhanced human IL-8-binding affinity at neutral pH (pH 7.4), but had reduced human IL-8-binding affinity at acidic pH (pH 5.8), compared to Hr 9. On the other hand, anti-IL-8 antibodies (H89/WS4L, H89/L12, and H89/L16) produced by combining various light chains with H89 containing Y97H modifications in the heavy chain all showed human IL-8-binding affinity decreased at acidic pH and human IL-8-binding affinity decreased at neutral pH.

[ Table 14-1]

(9-3) production and evaluation of modified antibody for imparting pH dependence

Combinations of promising modifications and new amino acid mutations found in 9-2 were evaluated, and the following combinations were found as a result.

[ tables 14-2]

Name of antibody	Introduction of one or more mutations in the heavy chain	Introduction of one or more mutations in the light chain
			H89/L63	Y97H	N50H/L54H
H89/L118	Y97H	N50H/L54H/Q89K

Variants were generated by the methods of reference examples 1 and 2, and the binding affinity to human IL-8 was assessed by methods similar to those of example 9-2.

The results are also shown in table 14. H89/L63, having H89-IgG1(SEQ ID NO: 86) as the heavy chain and L63-k0MT (SEQ ID NO: 87) as the light chain, showed comparable human IL-8-binding affinity to Hr9 at neutral pH (pH 7.4) and reduced human IL-8-binding affinity at acidic pH (pH 5.8). Specifically, both koff (dissociation rate constant) and KD (dissociation constant) of H89/L63 at ph5.8 were higher than those of Hr 9. This means that H89/L63 has the property of readily releasing human IL-8 under acidic pH conditions in endosomes.

It was surprisingly found that H89/L118 (which has H89-IgG1 as the heavy chain and L118-k0MT (SEQ ID NO: 88) as the light chain) has an enhanced human IL-8-binding affinity (KD) at neutral pH conditions compared to Hr9, but a reduced human IL-8-binding affinity (KD) at acidic pH conditions compared to Hr 9. Without particular limitation, in general, when antibodies that can bind to an antigen multiple times are used as a pharmaceutical product, pH-dependent antigen-binding antibodies preferably have a strong binding affinity (small KD) so that they can strongly neutralize the antigen under neutral pH conditions (such as in plasma). On the other hand, antibodies preferably have a large dissociation rate constant (koff) and/or a weak binding affinity (large KD) so that they can rapidly release antigen under acidic pH conditions, such as in endosomes. In comparison to Hr9, H89/L118 has the advantageous properties that can be obtained under both these neutral and acidic pH conditions.

Thus, useful amino acid modifications were identified for Hr9, such as Y97H for its heavy chain and N50H/L54H/Q89K for its light chain. Without being limited thereto, it has been shown that pH-dependent IL-8-binding antibodies which are preferred as medicaments can be produced by a combination of single or multiple amino acid modifications selected from these modifications.

Without being bound by a particular theory, it is considered that an important factor when using pH-dependent antigen-binding antibodies as a drug is whether the antibody administered to the body can release the antigen in the endosome. In this regard, sufficiently weak binding (large dissociation constant (KD)) or sufficiently fast dissociation rate (large dissociation rate constant (koff)) under acidic pH conditions is considered important. Therefore, it was investigated in the following experiments whether the KD or koff of H89/L118 obtained by BIACORE was sufficient to dissociate the antigen in vivo in endosomes.

Example 10

Generation of high-affinity antibodies for mouse PK assays

The method for confirming the effect of the antibody on the rate of removal of human IL-8 in mice is not particularly limited. In one instance, the method comprises administering the antibody to a mouse in admixture with human IL-8, and then comparing the rate of removal of human IL-8 from mouse plasma.

Herein, it is desirable that the reference antibody to be used in the mouse PK assay has a sufficiently strong binding affinity both at neutral pH and acidic pH conditions. Then, a modification conferring high-affinity to Hr9 was searched, and thereby, H998/L63 having H998-IgG1(SEQ ID NO: 89) as a heavy chain and L63-k0MT as a light chain was produced.

H998/L63 was used to evaluate human IL-8-binding affinity by a method similar to that of example 9-2. The resulting sensorgram is shown in fig. 21.

H998/L63 showed an unexpectedly slow dissociation rate in both neutral pH and acidic pH conditions, and it was shown to have a stronger IL-8-binding affinity than Hr 9. However, it is known that analytical values such as the dissociation rate constant (koff) and dissociation constant (KD) cannot be accurately calculated in the case of protein-protein interactions having a low dissociation rate due to the mechanical limitations of BIACORE. Accurate analytical values could not be obtained for H998/L63, which are shown herein. However, from the experimental results, it was confirmed that H998/L63 has very strong binding affinity at both neutral pH and acidic pH and is suitable as a comparative antibody to be used for mouse PK assay.

Example 11

Mouse PK assay using pH-dependent IL-8-binding antibody H89/L118

(11-1) mouse PK assay Using H89/L118

The in vivo removal rate of human IL-8 was evaluated using H89/L118 produced in example 9 and H998/L63 produced in example 10.

The pharmacokinetics of human IL-8 was assessed following the simultaneous administration of human IL-8 and anti-human IL-8 antibodies to mice (C57BL/6J, Charles river). A mixed solution of human IL-8 and anti-human IL-8 antibody (10. mu.g/mL and 200. mu.g/mL, respectively) was administered to the tail vein in a single dose of 10 mL/kg. At this time, since there is a sufficient excess of anti-human IL-8 antibody with respect to human IL-8, it is considered that almost all human IL-8 binds to the antibody. Blood was collected five minutes, two hours, four hours, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or below until measurement was performed.

(11-2) measurement of human IL-8 concentration in plasma

The concentration of human IL-8 in the plasma of mice was determined by the electrochemiluminescence method. First, an anti-human IL-8 antibody containing a mouse IgG constant region (prepared internally) was dispersed into a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and allowed to stand at room temperature for one hour. PBS-Tween solution containing 5% BSA (w/v) was then used for blocking at room temperature for two hours to prepare plates immobilized with anti-human IL-8 antibody. Calibration curve samples containing human IL-8 at plasma concentrations of 275, 91.7, 30.6, 10.2, 3.40, 1.13, or 0.377ng/mL and mouse plasma measurement samples diluted 25-fold or more were prepared. The sample was mixed with hWS-4 and allowed to react overnight at 37 ℃. Subsequently, 50. mu.L of the mixed solution was dispersed in each well of the plate on which the anti-human IL-8 antibody was immobilized, and the solution was stirred at room temperature for one hour. The final concentration of hWS-4 was adjusted to 25. mu.g/mL. Then, after reacting with biotin mouse anti-human Ig κ light chain (BD Pharmingen) for one hour at room temperature and then with SULFO-TAG labeled streptavidin (Meso Scale Discovery) for one hour at room temperature, reading buffer T (x1) (Meso Scale Discovery) was dispersed and measured immediately using SECTOR imager 2400(Meso Scale Discovery). Human IL-8 concentration was calculated based on the response of the calibration curve using analytical software, SOFT MaxPRO (Molecular Devices).

The resulting data for the concentration of human IL-8 in plasma are shown in FIG. 22, and the values for the Clearance (CL) of human IL-8 from mouse plasma are shown in Table 15.

[ Table 15]

As seen in FIG. 22, human IL-8 administered concurrently with H89/L118 showed surprisingly rapid removal from mouse plasma as compared to human IL-8 administered concurrently with H998/L63. Furthermore, the CL values, which quantitatively represent the removal rate of human IL-8 from mouse plasma, indicate that the removal rate of human IL-8 is increased by about 19-fold for H89/L118 compared to H998/L63.

Without being bound by a particular theory, the following can be guessed from the data obtained. Most of the human IL-8 administered simultaneously with the antibody binds to the antibody in plasma and is present in complexed form. Due to the strong affinity of antibodies, human IL-8 bound to H998/L63 can exist in an antibody bound state, even under acidic pH conditions in endosomes. Thereafter, H998/L63 can be returned to plasma via FcRn while still in human IL-8-complexed form; thus, when this occurs, human IL-8 is also returned to the plasma. Therefore, most of the human IL-8 taken up into the cells can be returned to the plasma again. That is, the rate of removal of human IL-8 from plasma was significantly reduced when H998/L63 was administered simultaneously. On the other hand, as described previously, with H89/L118 (pH-dependent IL-8-binding antibody) complex form uptake of cells of human IL-8, can in endosome in acidic pH conditions and antibody dissociation. Human IL-8 dissociated from the antibody is degraded after translocation to lysosomes. Thus, pH-dependent IL-8-binding antibodies can significantly accelerate the removal of human IL-8 compared to IL-8-binding antibodies such as H998/L63, which have strong binding affinity at both acidic and neutral pH.

(11-3) mouse PK assay with increasing dose of H89/L118

Next, experiments to verify the effect of varying the dose of H89/L118 were performed as follows. After simultaneous administration of human IL-8 and H89/L118(2mg/kg or 8mg/kg) to mice (C57BL/6J, Charles river), the pharmacokinetics of human IL-8 was assessed. A mixed solution of human IL-8 (2.5. mu.g/mL) and anti-human IL-8 antibody (200. mu.g/mL or 800. mu.g/mL) was administered to the tail vein in a single dose of 10 mL/kg. At this time, since there is a sufficient excess of anti-human IL-8 antibody compared to human IL-8, it is considered that almost all human IL-8 binds to the antibody. Blood was collected five minutes, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000rpm and 4 ℃ for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or below until measurement was performed.

Measurement of human IL-8 concentration in mouse plasma was carried out by a similar method to that of example 11-2. The obtained data on the concentration of human IL-8 in plasma are shown in FIG. 23, and the values of the Clearance (CL) of human IL-8 from mouse plasma are shown in Table 16.

[ Table 16]

Thus, it was confirmed that the group administered with 8mg/kg of antibody had an approximately 2-fold slower removal rate of human IL-8 compared with the group administered with 2mg/kg of H89/L118.

Hereinafter, the present inventors describe contents summarized as one of possible factors bringing about the aforementioned results based on scientific background, but the contents of the disclosure C are not limited to the contents discussed below.

Among the antibodies that return to plasma from the inside of endosomes via FcRn, it is preferable that the proportion of antibodies that bind to human IL-8 is low. With respect to human IL-8 present in endosomes, it is desirable to have a high proportion of free form that is not bound by the antibody. When human IL-8 and not pH-dependent IL-8-affinity antibody together with application, thought the majority of endosome (approximately 100%) human IL-8 with antibody complex form exists, and a small amount (approximately 0%) is considered free. On the other hand, when administered with a pH-dependent IL-8-binding antibody (e.g., H89/L118), a specific portion of human IL-8 should be present in free form in endosomes. In this case, the proportions of the free form can be understood, supposedly, as follows: [ proportion (%) of free human IL-8 in endosome ] - [ concentration of free human IL-8 in endosome ]/[ concentration of total human IL-8 in endosome ] x 100.

Ideally, the proportion of free human IL-8 in endosomes as understood by the above equation is higher and is, for example, 20% more preferred than 0%, 40% more preferred than 20%, 60% more preferred than 40%, 80% more preferred than 60%, and 100% more preferred than 80%.

Thus, there is a correlation between the proportion of free human IL-8 in the above endosomes and the binding affinity (KD) and/or the dissociation rate constant (koff) for human IL-8 at acidic pH. That is, the weaker the binding affinity and/or the stronger the off-rate of human IL-8 at acidic pH, the higher the proportion of free human IL-8 in endosomes. However, in the case of a pH-dependent IL-8-binding antibody that can bring the proportion of free human IL-8 close to 100% in endosomes, further weakening of the binding affinity and/or increasing the dissociation rate at acidic pH does not necessarily result in an effective increase in the proportion of free human IL-8. The skilled artisan will readily appreciate that, for example, even if the proportion of free human IL-8 is improved from 99.9% to 99.99%, the degree of improvement may not be significant.

Furthermore, according to the general chemical equilibrium theory, when an anti-IL-8 antibody and human IL-8 coexist and their binding and dissociation reactions reach equilibrium, the proportion of free human IL-8 is unambiguously determined by three parameters: antibody concentration, antigen concentration, and dissociation constant (KD). Herein, when the antibody concentration is high, when the antigen concentration is high, or when the dissociation constant (KD) is small, the complex is easily formed and the proportion of free human IL-8 is reduced. On the other hand, when the antibody concentration is low, when the antigen concentration is low, or when the dissociation constant (KD) is large, complex formation becomes difficult, and the proportion of free human IL-8 increases.

Also, in this experiment, the rate of removal of human IL-8 when H89/L118 was administered at 8mg/kg was lower than when the antibody was administered at 2 mg/kg. This therefore suggests that in endosomes, the proportion of free human IL-8 is reduced when the antibody is administered at 8mg/kg compared to when the antibody is administered at 2 mg/kg. The reason for this decrease may be that increasing the antibody dose four-fold increases the antibody concentration in the endosomes and thereby promotes the formation of IL-8-antibody complexes in the endosomes. That is, in the group administered with the antibody at an increased dose, the proportion of free human IL-8 in endosomes was decreased, and thus the rate of removal of human IL-8 was decreased. This also suggests that the degree of dissociation constant (KD) of H89/L118 under acidic pH conditions is not sufficient to bring free human IL-8 close to 100% when the antibody is administered at 8 mg/kg. More specifically, if it is an antibody with a large dissociation constant (KD) (weaker binding) under acidic pH conditions, it can achieve a state close to 100% free IL-8 even when the antibody is administered at 8mg/kg, and the rate of human IL-8 removal is equal to the rate when the antibody is administered at 2 mg/kg.

Based on the above, in order to confirm whether the investigated pH-dependent IL-8-binding antibody can achieve a ratio close to 100% free human IL-8 in vivo, the skilled person is not particularly limited, and can verify whether there is room for increasing the degree of the in vivo antigen-removing effect. For example, one method compares the rate of human IL-8 removal when using a new pH-dependent IL-8-binding antibody with that when using H89/L118, in which case the new antibody has a weaker binding affinity at acidic pH and/or an increased off-rate at acidic pH compared to H89/L118. In the case of the aforementioned novel pH-dependent IL-8-binding antibodies that exhibit a human IL-8 removal rate comparable to H89/L118, this suggests that the binding affinity and/or off-rate of H89/L118 at acidic pH is already at a level sufficient to achieve a proportion of free human IL-8 in endosomes approaching 100%. On the other hand, in the case where the aforementioned novel pH-dependent IL-8-binding antibody shows a higher removal rate of human IL-8, this suggests that H89/L118 has room for improvement in binding affinity and/or dissociation rate at acidic pH.

Example 12

production and evaluation of the pH-dependent IL-8-binding antibody H553/L118

(12-1) production of antibody H553/L118 having pH-dependent IL-8 binding ability

Herein, the inventors aimed to generate antibodies with even weaker human IL-8-binding affinity and/or higher off-rate under acidic pH conditions compared to H89/L118.

The modified antibodies shown in table 17 were produced by a method similar to example 9 using H89/L118 as a basis to introduce amino acid modifications (mainly involving histidine). In addition, by the example 9-2 similar method to determine the antibodies of human IL-8-binding affinity.

Some of the results are shown in table 17. It was shown that the antibody H553/L118 comprising H553-IgG1(SEQ ID NO: 90) as the heavy chain and L118-k0MT as the light chain, and the antibody H496/L118 comprising H496-IgG1(SEQ ID NO: 101) as the heavy chain and L118-k0MT as the light chain had a further increased pH dependence than H89/L118.

[ Table 17]

In the obtained H553/L118, two amino acid modifications, Y55H and R57P, were introduced into the heavy chain of H89/L118. On the other hand, H496/L118, in which R57P was introduced only into the heavy chain of H89/L118, had an enhanced binding affinity to human IL-8 at neutral pH but a hardly altered human IL-8-binding affinity at acidic pH compared to H89/L118. More specifically, the R57P modification introduced into H89/L118 is a modification that only enhances human IL-8-binding affinity at neutral pH but does not alter binding affinity at acidic pH. Furthermore, H553/L118 produced by introducing a Y55H modification into the heavy chain of H496/L118 had retained or slightly enhanced binding affinity at neutral pH, but on the other hand, reduced binding affinity at acidic pH, compared to H89/L118. That is, the introduction of a combination of two amino acid modifications (Y55H and R57P) into H89/L118 allowed for further enhancement of the property of reducing binding affinity at acidic pH while maintaining or slightly enhancing binding affinity at neutral pH.

(12-2) mouse PK assay Using H553/L118

Evaluation of the removal rate of human IL-8 in mice was carried out by a method similar to that of example 11-2 using H553/L118. The resulting data on the concentration of human IL-8 in plasma are shown in FIG. 24, and the values of the Clearance (CL) of human IL-8 from mouse plasma are shown in Table 18.

[ Table 18]

Thus, when comparing the data for mice administered with 2mg/kg antibody, no large difference was observed between H553/L118 and H89/L118; however, it was confirmed that H553/L118 accelerated the removal of human IL-8 by a 2.5-fold effect compared to H89/L118 when comparing the data for mice administered with 8mg/kg antibody. From another perspective, H553/L118 did not show a difference in the removal rate of human IL-8 between 2mg/kg and 8mg/kg, and no decrease in the removal rate of antigen due to increased antibody dose was observed with respect to H89/L118.

Without particular limitation, one reason for obtaining this result may be discussed as follows. H533/L118 showed comparable removal rates of human IL-8 when the antibody was administered at 2mg/kg and at 8 mg/kg. This may indicate that the proportion of free IL-8 in the endosomes can reach levels close to 100%, since the binding of H553/L118 to IL-8 at acidic pH is sufficiently weak, even under conditions of 8 mg/kg-administration. In other words, this suggests that although H89/L118 can achieve the greatest human IL-8 removal effect at a dose of 2mg/kg, its effect may be weakened at high doses of about 8 mg/kg. On the other hand, H553/L118 can achieve the greatest effect of removing human IL-8 even at high doses of 8 mg/kg.

(12-3) evaluation of stability Using H553/L118

H553/L118 was shown to be an antibody that can accelerate the removal of human IL-8 in mice more significantly than H89/L118. However, for the antibody to continue the inhibitory effect on human IL-8 in vivo for a long period of time, it is also important that IL-8-neutralizing activity (stability of IL-8-neutralizing activity of the antibody) be stably maintained during the time that the administered antibody is present in vivo (e.g., in plasma). Therefore, the stability of these antibodies in mouse plasma was evaluated by the following method.

Mouse plasma was collected from blood at C57BL/6J (Charles river) by methods known in the art. 200 μ L of 200mM PBS (Sigma, P4417) was added to 800 μ L of mouse plasma to give 1 mL. In addition, sodium azide was added as a preservative at a final concentration of 0.1%. Then, each antibody (Hr9, H89/L118 and H553/L118) was added to the above mouse plasma to give a final concentration of 0.2 mg/mL. At this point, a portion of the sample was collected as the initial sample. The remaining samples were stored at 40 ℃. One and two weeks after storage, a portion of each sample was collected and used as samples for one week of storage and samples for two weeks of storage. All samples were frozen at-80 ℃ and stored until each analysis was performed.

Next, the anti-IL-8 antibody contained in the mouse plasma was evaluated for its human IL-8-neutralizing activity as follows: CXCR1 and CXCR2 are known receptors for human IL-8. The PathHunter (registered trademark) CHO-K1CXCR2 β -repressor white cell line (DiscovexRx Co., Cat. #93-0202C2) expresses human CXCR2 and is an artificially generated cell line that emits chemiluminescence when a human IL-8-mediated signal is transmitted. It is not particularly limited, and the human IL-8-neutralizing activity of an anti-human IL-8 antibody can be evaluated using the cell. When human IL-8 was added to a culture solution of cells, a specific amount of chemiluminescence was exhibited in a manner dependent on the concentration of human IL-8 added. When human IL-8 and anti-human IL-8 antibody are added to the culture solution together, human IL-8 signaling can be blocked after the anti-human IL-8 antibody binds to human IL-8. Thus, chemiluminescence induced by the addition of human IL-8 will be inhibited by anti-human IL-8 antibodies, and will be weaker than without the addition of antibodies, or will not be present at all. Thus, as the antibody has more neutralizing activity against human IL-8, the degree of chemiluminescence becomes weaker; and as the antibody has less human IL-8 neutralizing activity, the degree of chemiluminescence becomes stronger.

This is also true for antibodies that have been added to mouse plasma and stored for a period of time. If the neutralizing activity of the antibody is not altered by storage in the plasma of mice, the degree of chemiluminescence described above should not be altered before and after storage. On the other hand, in the case of an antibody in which the neutralizing activity is decreased due to storage in mouse plasma, the degree of chemiluminescence by using the stored antibody will be increased as compared to before storage.

Subsequently, the above cell line was used to examine whether the neutralizing activity of the antibody stored in the plasma of the mouse was maintained. First, Cell lines were suspended in assaycomplete (tm) Cell Plating 0 reagent and subsequently seeded at 5000 cells/well in 384-well plates. One day after starting the cell culture, the following experiment was performed to determine the concentration of human IL-8 to be added. Serial dilutions of human IL-8 solution (containing a final concentration of human IL-8 of 45nM (400ng/mL) to 0.098nM (0.1 ng/mL)) were added to the cell culture solution. Next, a detection reagent is added according to the protocol of the product, and the relative chemiluminescence level is detected using a chemiluminescence detector. From the results, the reactivity of the cells to human IL-8 was confirmed, and the concentration of human IL-8 suitable for confirming the neutralizing activity of the anti-human IL-8 antibody was determined. Herein, the concentration of human IL-8 was set at 2 nM.

Next, the aforementioned mouse plasma to which the anti-human IL-8 antibody was added was used to evaluate the neutralizing activity of the antibody contained therein. Human IL-8 and the aforementioned mouse plasma containing anti-human IL-8 antibodies at the concentrations determined above were added to the cell culture. The amount of mouse plasma to be added was determined to contain a step concentration of anti-human IL-8 antibody ranging from 2. mu.g/mL (13.3nM) to 0.016. mu.g/mL (0.1 nM). Next, a detection reagent is added according to the product protocol, and the relative chemiluminescence levels are detected using a chemiluminescence detector.

Herein, the relative value of the relative chemiluminescence level at each antibody concentration was calculated by defining the average relative chemiluminescence level in wells to which human IL-8 and antibody were not added as 0%, and defining the average relative chemiluminescence level in wells to which only human IL-8 was added but no antibody was added as 100%.

The results of the human IL-8 inhibition assay using cells expressing human CXCR2 are shown in fig. 25A (which shows results from the initial sample (without preservative treatment in mouse plasma), fig. 25B (which shows results for samples stored at 40 ℃ for one week), and fig. 25C (which shows results for samples stored at 40 ℃ for two weeks).

Thus, no difference in human IL-8-neutralizing activity was observed for Hr9 and H89/L118 before and after storage in mouse plasma. On the other hand, H553/L118 showed a decrease in human IL-8-neutralizing activity after two weeks of storage. Therefore, the human IL-8-neutralizing activity of H553/L118 was easily decreased in mouse plasma compared to Hr9 and H89/L118, and H553/L118 was shown to be an antibody having unstable properties with respect to the IL-8-neutralizing activity.

Example 13

Production of antibodies with reduced predicted immunogenicity scores using a computer system

(13-1) predicted immunogenicity scores for various IL-8-binding antibodies

The production of anti-drug antibodies (ADA) affects the efficacy and pharmacokinetics of therapeutic antibodies and in some cases brings with it a variety of side effects; thus, clinical utility and drug efficacy of therapeutic antibodies may be limited by ADA production. The immunogenicity of therapeutic antibodies is known to be influenced by many factors and there are many reports describing the importance of effector T cell epitopes in therapeutic antibodies.

Herein, EpiMatrix was used to calculate the immunogenicity score for each anti-IL-8 antibody. EpiMatrix is a system for predicting the immunogenicity of proteins studied by automatically designing sequences of peptide fragments in nine amino acid segments by the amino acid sequence of the protein whose immunogenicity is to be predicted, and then calculating their ability to bind eight major MHC class II alleles (DRB1 x 0101, DRB1 x 0301, DRB1 x 0401, DRB1 x 0701, DRB1 x 0801, DRB1 x 1101, DRB1 x 1301, and DRB1 x 1501), a system for predicting the immunogenicity of the protein studied (clin. immunol.131 (2): 189-.

The immunogenicity scores (calculated as described above) for the heavy and light chains of each anti-IL-8 antibody are shown in Table 19 in the "EpiMatrix score" column. In addition, with respect to EpiMatrix scores, immunogenicity scores calibrated for Tregitope content are shown in the column "tReg adjusted Epx score". Tregitope is a peptide fragment sequence that is largely found in the native antibody sequence and is a sequence thought to suppress immunogenicity by activating regulatory T cells (tregs).

Further, with respect to these scores, the sum of the scores for the heavy and light chains is shown in the "total" column.

[ Table 19]

Based on these results, both the "EpiMatrix score" and the "tReg-modulated Epx score" showed a reduction in the immunogenicity scores of H89/L118, H496/L118 and H553/L118 compared to hWS-4, which is a known humanized anti-human IL-8 antibody.

Furthermore, with EpiMatrix, it is possible to compare the frequency of ADA production predicted for the antibody molecule as a whole by considering the heavy and light chain scores with the actual frequency of ADA production caused by various commercially available antibodies. The results of performing the analysis are shown in fig. 26. Due to system limitations, the notation used in FIG. 26 is "WS 4" for hWS-4, "HR 9" for Hr9, "H89L 118" for H89/L118, "H496L 118" for H496/L118, and "H553L 118" for H553/L118.

As shown in fig. 26, the frequency of ADA production in humans by various commercially available antibodies is known to be 45% for Campath (Alemtuzumab), 27% for Rituximab (Rituximab), and 14% for Zenapax (Daclizumab). On the other hand, while the frequency of ADA production predicted from the amino acid sequence was 10.42% for hWS-4 (which is a known humanized anti-human IL-8 antibody), the frequency of H89/L118 (5.52%), H496/L118 (4.67%), or H553/L118 (3.45%) newly identified herein was significantly lower compared to hWS-4.

(13-2) production of modified antibodies with reduced predicted immunogenicity scores

As described above, the immunogenicity scores were lower for H89/L118, H496/L118, and H553/L118 compared to hWS-4; however, as is apparent from table 19, the immunogenicity score for the heavy chain is higher than the immunogenicity score for the light chain, suggesting that there is still room for improvement in the amino acid sequence of the heavy chain (especially from an immunogenicity perspective). Then, a search for amino acid modifications that can reduce the immunogenicity score was performed in the heavy chain variable region of H496. As a result of a close search, three variants were found, H496v1 (in which the alanine at position 52c was replaced with aspartic acid according to Kabat numbering), H496v2 (in which the glutamine at position 81 was replaced with threonine), and H496v3 (in which the serine at position 82b was replaced with aspartic acid). In addition, H1004s was produced containing all three modifications.

The results of the immunogenicity scores calculated by a method similar to example 13-1 are shown in table 20.

[ Table 20]

The three heavy chains, H496v1, H496v2, and H496v3 (all of which contained a single modification), showed reduced immunogenicity scores compared to H496. Furthermore, H1004 (a combination containing three modifications) achieved significant improvements in immunogenicity scores.

Herein, L395 was identified as a light chain suitable for combination with H1004, in addition to L118. Thus, in the calculation of the immunogenicity score, L118 combinations and L395 combinations were used. As shown in table 20, H1004/L118 and H1004/L395 (which are combinations of heavy and light chains) also showed very low immunogenicity scores.

Next, the frequency of ADA production was predicted for these combinations in a similar manner to example 13-1. The results are shown in FIG. 27. The symbols used in FIG. 27 are "V1" for H496V1/L118, "V2" for H496V2/L118, "V3" for H496V3/L118, "H1004L 118" for H1004/L118, and "H1004L 395" for H1004/L395.

Surprisingly, H1004/L118 and H1004/L395 (which have significantly reduced immunogenicity scores) also show an improvement in the value predicted for ADA production frequency and show a predicted value of 0%.

(13-3) measurement of IL-8-binding affinity of H1004/L395

H1004/L395 was produced, which is an antibody comprising H1004-IgG1m (SEQ ID NO: 91) as the heavy chain and L395-k0MT (SEQ ID NO: 82) as the light chain. BIACORE T200(GE Healthcare) was used to measure the binding affinity of H1004/L395 to human IL-8 as described below.

The following two running buffers were used and measurements were taken at each temperature: (1) 0.05% Tween 20, 40mM ACES, 150mM NaCl, pH 7.4, 40 ℃; and (2) 0.05% Tween 20, 40mM ACES, 150mM NaCl, pH 5.8, 37 ℃.

An appropriate amount of protein a/g (pierce) was immobilized on a sensor chip CM4(GE Healthcare) by an amine coupling method and a target antibody was captured. Next, a diluted human IL-8 solution or running buffer (used as a reference solution) was injected to allow the interaction of the antibody captured on the sensor chip with human IL-8. For the running buffer, use the above solution in any kind, and each buffer also used for dilution of IL-8. To regenerate the sensor chip, 25mM NaOH and 10mM glycine-HCl (pH 1.5) were used. KD (M) for human IL-8 is calculated for each antibody based on the association rate constant kon (1/Ms) and the dissociation rate constant koff (1/s), which are kinetic parameters calculated from sensorgrams obtained by measurement. BIACORE T200 evaluation software (GE Healthcare) was used to calculate each parameter.

The measurement results are shown in table 21. H1004/L395 (with reduced immunogenicity score) has comparable KD for human IL-8 at neutral pH, but increased KD and koff at acidic pH compared to H89/L118; and shows that it has the property of readily dissociating from IL-8 in endosomes.

[ Table 21-1]

Example 14

production and evaluation of pH-dependent IL-8-binding antibody H1009/L395

(14-1) production of various pH-dependent IL-8-binding antibodies

H1004/L395 (which has a pH-dependent IL-8 binding capacity and a reduced immunogenicity score) was obtained by the evaluation in example 13. Subsequently, extensive studies were performed to generate variants with these advantageous properties as well as stability in mouse plasma.

The following modified antibodies were generated by introducing various modifications based on H1004/L395.

[ tables 21-2]

Heavy chain

H1004	A52cD/R57P/Q81T/S82bD/Y97H
		H0932	A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H
H1000	D31E/A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H
		H1009	A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H
H1022	A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H
		H1023	A52cD/T56H/R57P/Q81T/S82bD/Y97H
H1028	A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H
		H1029	S30D/D31K/A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H
H1031	S30D/D31K/A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H
		H1032	S30D/D31K/A52cD/T56H/R57P/Q81T/S82bD/Y97H
H1037	S30D/D31K/A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H
		H1040	D31E/A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H
H1041	D31E/A52cD/T56H/R57P/Q81T/S82bD/Y97H
		H1046	D31E/A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H
H1047	S30D/D31K/A52cD/R57P/Q81T/S82bD/Y97H
		H1048	D31E/A52cD/R57P/Q81T/882bD/Y97H
H1049	S30D/D31K/A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H
		H1050	D31E/A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H

[ tables 21 to 3]

L395	N50K/L54H/Q89K
		L442	S31E/N50K/L54H/Q89K

A total of 36 types of antibodies were produced by combining the above 18 types of heavy chains and two types of light chains. These antibodies were evaluated variously as described below.

Human IL-8-binding affinity under neutral and acidic pH conditions was measured in a similar manner to the method of example 13-3. Among the results obtained, the KD at pH 7.4, and the KD and koff at pH 5.8 are shown in table 22.

Next, the stability with respect to IL-8 binding after storage of the antibody in PBS was evaluated by the following method.

Each antibody was dialyzed overnight against DPBS (Sigma-Aldrich), and then the concentration of each antibody was adjusted to 0.1 mg/mL. At this point, some antibody sample was collected as a starting sample. The remaining samples were stored at 50 ℃ for one week and subsequently collected as samples for the heat accelerated test.

Next, BIACORE measurements of IL-8-binding affinity were performed as follows using the initial samples and the samples used for the thermal acceleration test.

BIACORE T200(GE Healthcare) was used to analyze the level of binding of human IL-8 to the modified antibody. Measurement was performed at 40 ℃ by using 0.05% Tween 20, 40mM ACES, and 150mM NaCl (pH 7.4) as a running buffer.

An appropriate amount of protein a/g (pierce) was immobilized on a sensor chip CM4(GE Healthcare) by an amine coupling method and a target antibody was captured. Next, a diluted human IL-8 solution or a running buffer (used as a reference solution) was injected to allow the antibody captured on the sensor chip to interact with human IL-8. Running buffer was also used to dilute human IL-8. To regenerate the sensor chip, 25mM NaOH and 10mM glycine-HCl (pH 1.5) were used. The measured binding levels of human IL-8 and the amount of antibody captured at that binding level were extracted using BIACORE T200 evaluation software (GE Healthcare).

The amount of human IL-8-binding per 1000RU of captured antibody was calculated for the initial sample and the sample used for the thermal acceleration test. In addition, the ratio of the human IL-8-binding levels for the initial sample to the sample used for the thermal acceleration test was calculated.

The resulting ratio of IL-8-binding levels of the initial sample to the sample used for the thermal acceleration test is also shown in Table 22.

[ Table 22]

Through the above studies, H1009/L395 was obtained, which is an antibody comprising H1009-IgG1m (SEQ ID NO: 92) as the heavy chain and L395-k0MT as the light chain.

As shown in Table 22, H1009/L395 compared with H89/L118 at neutral pH slightly enhanced human IL-8-binding affinity, but on the other hand, at acidic pH with reduced binding affinity, i.e., pH-dependence further enhanced. Furthermore, H1009/L395 has slightly enhanced IL-8 binding stability compared to H89/L118 when exposed to stringent conditions such as in PBS at 50 ℃.

Therefore, H1009/L395 as its neutralizing activity in mouse plasma can stably maintain, while maintaining its pH-dependent IL-8 binding ability of the antibody.

(14-2) evaluation of stability of H1009/L395

Next, in a manner similar to the method of example 12-3, it was evaluated whether the IL-8 neutralizing activity of H1009/L395 was stably maintained in the plasma of mice. Herein, H1009/L395-F1886s are used, which will be described in detail later in example 19. The antibody has the same variable regions as H1009/L395, and constant regions with modifications that enhance FcRn binding under acidic pH conditions and modifications to reduce its binding to Fc γ r(s) compared to native human IgG 1. The variable regions of H1009/L395, especially the regions surrounding the HVRs, are responsible for the human IL-8-binding and IL-8-neutralizing activity of the antibody, and modifications introduced into the constant regions are not believed to affect these properties.

Evaluation of stability of mouse plasma was performed as follows. mu.L of 200mM phosphate buffer (pH 6.7) was added to 585. mu.L of mouse plasma. Then, sodium azide was added as a preservative at a final concentration of 0.1%. Each antibody (Hr9, H89/L118, or H1009/L395-F1886s) was added to the above mouse plasma at a final concentration of 0.4 mg/mL. At this point, a portion of the sample was collected as the initial sample. The remaining samples were stored at 40 ℃. One and two weeks after the start of storage, a portion of each sample was collected and used as samples for one week of storage and samples for two weeks of storage. All samples were frozen at-80 ℃ and stored until analysis.

Measurement of human IL-8-neutralizing activity was carried out by a method similar to that of example 12-3 using cells expressing human CXCR 2. However, this time for confirming the anti human IL-8 antibody neutralization activity of human IL-8 concentration is 1.2 nM.

The results of the human IL-8 inhibition assay obtained using the above antibody with cells expressing human CXCR2 are shown in fig. 28A (which shows the results for the initial sample (without storage treatment in mouse plasma), fig. 28B (which shows the results for the sample stored at 40 ℃ for one week), and fig. 28C (which shows the results for the sample stored at 40 ℃ for two weeks).

Thus, surprisingly, even after storing it in mouse plasma at 40 ℃ for two weeks, human IL-8-neutralizing activity was maintained in H1009/L395-F1886s, and IL-8-neutralizing activity was more stably maintained than in the case of H553/L118.

(14-3) mouse PK assay Using H1009/L395

The rate of removal of human IL-8 in mice by H1009/H395 was evaluated by the following method. H1009/L395, H553/L118, and H998/L63 were used as antibodies. Administration to mice and blood collection, as well as measurement of human IL-8 concentration in mouse plasma, were performed by the methods shown in example 11.

The resulting data for the concentration of human IL-8 in plasma are shown in FIG. 29, and the values for the Clearance (CL) of human IL-8 from mouse plasma are shown in Table 23.

[ Table 23]

Thus, when H1009/L395 at 2mg/kg application, in mice in human IL-8 removal rate and H553/L118 equivalent, show, H1009/L395 to achieve endosome near 100% of the free IL-8. Shows that the quantitative representation of mouse plasma removal rate of IL-8 Clearance (CL) value is H998/L63 about 30-times higher.

Without particular limitation, the effect of increasing the removal rate of human IL-8 can be understood as follows. Generally, in a living body that holds an antigen at an almost constant concentration, the production rate and removal rate of the antigen will also be kept at almost constant values. When antibodies are administered under such conditions, the rate of antigen removal can be altered due to the formation of complexes of antigen and antibody, even where the rate of antigen production is not affected. Generally, since the antigen-removal rate is greater than the antibody-removal rate, in this case, the removal rate of the antigen that has formed a complex with the antibody is reduced. The concentration of antigen in plasma increases when the antigen removal rate decreases, but the degree of increase in this case can also be defined by the ratio of the removal rate when the antigen is present alone to the removal rate when the antigen forms a complex. That is, if the removal rate at the time of complex formation is reduced to one tenth compared to the removal rate when the antigen is present alone, the antigen concentration in the plasma of an organism to which the antibody is administered can be increased by about ten times as compared to before the antibody is administered. Herein, Clearance (CL) may be used as the removal rate. More specifically, the increase in antigen concentration (antigen accumulation) that occurs after administration of an antibody to an organism can be defined by the antigen CL under each condition before and after antibody administration.

Here, the presence of an approximately 30-fold difference in CL for human IL-8 when H998/L63 and H1009/L395 were administered suggests that there may be an approximately 30-fold difference between the elevated levels of human IL-8 concentration in plasma when these antibodies are administered to humans. Furthermore, the generation of a 30-fold difference in plasma human IL-8 concentration indicates that there will be an approximately 30-fold difference in the amount of antibody required to completely block the biological activity of human IL-8 under each condition. That is, H1009/L395 can block the biological activity of IL-8 in plasma in an amount of about 1/30, which is a very small amount of antibody, compared to H998/L63. Furthermore, when H1009/L395 and H998/L63 are administered separately to humans at the same dose, H1009/L395 will be able to block the biological activity of IL-8 at higher intensities over a long period of time. In order to block the biological activity of IL-8 for a long period of time, it is necessary to stably maintain IL-8-neutralizing activity. As shown in example 14, using mouse plasma experiments demonstrated that H1009/L395 can maintain its human IL-8-neutralizing activity for a long period of time. It was also shown that H1009/L39, having these significant properties, is a better antibody from the point of view of neutralizing the efficacy of IL-8 in vivo.

Example 15

Evaluation of extracellular matrix binding Using pH-dependent IL-8-binding antibody H1009/L395

The excellent 30-fold better effect of H1009/L395 in eliminating human IL-8 as shown in example 14 is an unexpected effect. It is known that the antigen removal rate when a pH-dependent antigen-binding antibody is administered depends on the rate of uptake of the antibody-antigen complex into cells. That is, if the rate of uptake of the pH-dependent antigen-binding antibody into cells is increased when an antigen-antibody complex is formed (compared to when no complex is formed), the antigen-removing effect of the pH-dependent antibody can be increased. Known methods for increasing the rate of uptake of an antibody into a cell include a method for imparting an antibody with the ability to bind FcRn under neutral pH conditions (WO 2011/122011), a method for enhancing the binding ability of an antibody to fcyr(s) (WO 2013/047752), and a method for promoting the formation of a complex containing a multivalent antibody and a multivalent antigen using (WO 2013/081143).

However, the above technique is not used in the constant region of H1009/L395. Furthermore, while IL-8 is known to form homodimers, human IL-8 bound by H1009/L395 was found to exist as a monomer, since H1009/L395 recognizes the homodimer-forming surface of human IL-8. Thus, the antibody will not form a multivalent complex.

More specifically, when the above-described technique is not applied to H1009/L395, H1009/L395 shows a 30-fold higher effect of human IL-8-removal.

The inventors then conducted the following discussion of possible factors that may contribute to the above-described properties of pH-dependent IL-8-binding antibodies, represented by H1009/L395. However, the following is only one possibility that the present inventors speculate on the technical background, and the content of the disclosure C is not limited to the content discussed below.

Human IL-8 is a protein with a high isoelectric point (pI) and the theoretical isoelectric point calculated by known methods is about 10. That is, human IL-8 is a protein whose charge is shifted positively under neutral pH conditions. The pH-dependent IL-8-binding antibody represented by H1009/L395 is also a protein with a positive charge transition, and the theoretical isoelectric point of H1009/L395 is about 9. That is, the isoelectric point of the complex produced by H1009/L395 (a protein that has a high isoelectric point and is initially rich in positive charges) in combination with human IL-8 having a high isoelectric point will be higher than H1009/L395 alone.

As shown in example 3, increasing the isoelectric point of an antibody (which includes increasing the number of positive charges on the antibody and/or decreasing the number of negative charges of the antibody) can be considered to increase the non-specific uptake of the antibody-antigen complex into the cell. The isoelectric point of the complex formed between the anti-IL-8 antibody and human IL-8 having a high isoelectric point is higher and the complex can be more easily taken up into cells than the anti-IL-8 antibody alone.

As previously described, affinity for the extracellular matrix is also a factor that may influence uptake into cells. Then, whether there is a difference in extracellular matrix binding between the antibody alone and the complex with the human IL-8-antibody was examined.

Evaluation of the amount of antibody binding to the extracellular matrix by the ECL (electrochemiluminescence) method

Extracellular matrix (BD Matrigel basement membrane matrix/manufactured by BD) was diluted to 2mg/mL using TBS (Takara, T903). The diluted extracellular matrix was dispersed at 5. mu.L/well in a MULTI-ARRAY 96-well plate, high binding, Bare (manufactured by Meso Scale Discovery: MSD), and fixed at 4 ℃ overnight. Then, a solution containing 150mM NaCl, 0.05% Tween 20, 0.5% BSA, and 0.01% NaN was used₃20mM ACES buffer (pH 7.4)And (6) sealing.

The antibody to be evaluated was prepared as follows. Antibody samples to be added separately were prepared by using buffer 1 (containing 150mM NaCl, 0.05% Tween 20, and 0.01% NaN)₃20mM ACES buffer, pH 7.4) to 9. mu.g/mL, and then buffer 2 (containing 150mM NaCl, 0.05% Tween 20, 0.1% BSA, and 0.01% NaN) was used₃20mM ACES buffer, pH 7.4) to a final concentration of 3. mu.g/mL.

On the other hand, as with IL-8 complex with the addition of antibody samples as follows preparation: human IL-8 was added to the antibody samples at a molar concentration ten times that of the antibodies, and then each antibody was diluted with buffer-1 so that the antibody concentration became 9. mu.g/mL, respectively, and then each of them was further diluted with buffer-2 to a final antibody concentration of 3. mu.g/mL. At this time, the concentration of human IL-8 was about 0.6. mu.g/mL. It was shaken for one hour at room temperature for complex formation.

Next, the blocking solution was removed from the plate, a solution of the antibody alone or as a complex was added to the plate, and this was shaken at room temperature for one hour. Then, after removing the individual antibody solution or the complex solution, buffer-1 containing 0.25% glutaraldehyde was added. Then, after the plate was allowed to stand for 10 minutes, it was washed with DPBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween 20. Antibodies for ECL detection were prepared by using Sulfo-labeled NHS ester (manufactured by MSD) Sulfo-labeled goat anti-human IgG (γ) (manufactured by Zymed Laboratories). The antibody for ECL detection was diluted to 1 μ g/mL with buffer-2, added to the plate, and then shaken for one hour at room temperature in the dark. The antibody for ECL detection was removed, a solution produced by diluting MSD reading buffer T (4 ×) (manufactured by MSD) 2-fold using ultrapure water was added, and then the light emission amount was measured by SECTOR imager 2400 (manufactured by MSD).

The results are shown in fig. 30. Interestingly, all anti-IL-8 antibodies like H1009/L395 showed hardly any binding to the extracellular matrix like the antibody alone (-IL8), but bound to the extracellular matrix after forming a complex with human IL-8 (+ hIL 8).

As described above, the nature of anti-IL-8 antibodies to gain affinity for extracellular matrix by binding to human IL-8 has not been elucidated. Furthermore, without limitation, combining this property with a pH-dependent IL-8-binding antibody may more effectively increase the rate of IL-8 removal.

Example 16

Mouse PK assay using non-FcRn-binding antibodies

The following method was used to determine whether a complex between human IL-8 and a pH-dependent IL-8-binding antibody was formed and whether uptake of the complex into cells was increased in mice.

First, antibody variants were generated comprising the variable region of H1009/L395 and an Fc region lacking binding affinity for various Fc receptors. Specifically, as modifications for deletion of binding ability to human FcRn under acidic pH conditions, heavy chain H1009-IgG1 replaced alanine to isoleucine at position 253 and aspartate to serine at position 254 (according to EU numbering). Furthermore, as modifications for the deletion of binding to mouse Fc γ r(s), the leucine at position 235 was replaced with arginine, the glycine at position 236 was replaced with arginine, and the serine at position 239 was replaced with lysine. H1009-F1942m (SEQ ID NO: 93) were generated as heavy chains containing four of these modifications. In addition, H1009/L395-F1942m was produced comprising H1009-F1942m as the heavy chain and L395-k0MT as the light chain.

Since the antibody having this Fc region lacks FcRn binding affinity under acidic pH conditions, it does not transfer from endosomes to plasma. Thus, the antibody is rapidly removed from plasma in vivo, as compared to an antibody comprising a native Fc region. In this case, after the antibody containing the native Fc region is taken into the cell, only a part of them, which is not rescued by FcRn, is degraded after transfer to lysosomes, but in the case of an antibody containing an Fc region having no FcRn-binding affinity, all the antibody taken into the cell is degraded in lysosomes. More specifically, in the case of an antibody comprising the modified Fc region, the rate of removal of the administered antibody from plasma may be comparable to the rate of binding into cells. That is, the rate of intracellular uptake of antibodies whose FcRn-binding affinity is deleted can also be confirmed by measuring the rate of removal of these antibodies from plasma.

Then, it was tested whether the intracellular uptake of the complex formed between H1009/L395-F1942m and human IL-8 was increased compared to the uptake of H1009/L395-F1942m alone. In particular, it was tested whether the rate of antibody removal from plasma will change when the antibody is administered alone and when the antibody is administered after forming a complex with human IL-8.

The individual biokinetics (biokinetics) of anti-human IL-8 antibodies were evaluated when anti-human IL-8 antibodies were administered to human FcRn transgenic mice alone (B6.mFcRn-/-. hFcRn Tg strain 32+/+ mice; Jackson Laboratories; Methods mol. biol. 602: 93-104(2010)) and human IL-8 and anti-human IL-8 antibodies were administered to human FcRn transgenic mice simultaneously. An anti-human IL-8 antibody solution (200. mu.g/mL), and a mixed solution of human IL-8 (10. mu.g/mL) and an anti-human IL-8 antibody (200. mu.g/mL) were administered to the tail vein once at 10mL/kg, respectively. In this case, because anti-human IL-8 antibody is present in an amount sufficiently exceeding human IL-8, so think almost all human IL-8 binding antibody. Blood was collected five minutes, two hours, seven hours, one day, and two days after administration. The collected blood was immediately centrifuged at 4 ℃ and 15,000rpm for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or below until measurement was performed.

The concentration of anti-human IL-8 antibody in the plasma of mice was measured by the electrochemiluminescence method. First, to a streptavidin Gold Multi-ARRAY plate (Meso Scale Discovery) blocked overnight at room temperature using a PBS-Tween solution containing 5% BSA (w/v), an anti-human kappa light chain goat IgG Biotin (IBL) was reacted at room temperature for one hour to produce a plate on which an anti-human antibody was immobilized. Samples for calibration curve and samples for mouse plasma measurement diluted 100-fold or more containing anti-human IL-8 antibody at concentrations of 3.20, 1.60, 0.800, 0.400, 0.200, 0.100, and 0.0500 μ g/mL in plasma were prepared. Each sample was mixed with human IL-8, and then dispersed at 50. mu.L/well in a plate on which an anti-human antibody was immobilized, and then stirred at room temperature for one hour. Human IL-8 was adjusted to a final concentration of 333 ng/mL.

Then, an anti-human IL-8 antibody (prepared internally) containing a mouse IgG constant region was added to the plate and allowed to react at room temperature for one hour. In addition, ruthenium-labeled anti-mouse IgG (BECKMAN COULTER) with SULFO-TAG NHS ester (Meso Scale Discovery) was added to the plate and allowed to react for one hour. Then, immediately after the reading buffer T (x1) (Meso Scale Discovery) was dispersed into the plate, measurement was performed using the SECTOR imager 2400(Meso Scale Discovery). Anti-human IL-8 antibody concentrations were calculated based on the response of the calibration curve using analytical software, SOFTMax PRO (Molecular Devices).

The antibody concentrations in the plasma of the mice thus obtained are shown in fig. 31, and the antibody clearance under the respective conditions are shown in table 24.

[ Table 24]

Shows that the intracellular uptake rate of the complex of H1009/L395-F1942m and human IL-8 is increased at least 2.2 fold compared to the uptake rate of H1009/L395-F1942 m. Herein, labeled "at least 2.2-fold" for the following reasons included as one possibility of a value that may actually be 5-fold, 10-fold, or 30-fold. Because the rate of removal of human IL-8 from mouse plasma is very fast compared to the rate of removal of H1009/L395-F1942m, the proportion of human IL-8 bound H1009/L395-F1942m in plasma is rapidly reduced after administration. More specifically, even when administered simultaneously with human IL-8, not all of the H1009/L395-F1942m present in plasma is in human IL-8-bound form, and indeed, approximately seven hours after administration, most of them are already present in free form. Because the uptake rates were evaluated under this condition, even though the intracellular uptake rate of the complex of H1009/L395-F1942m and human IL-8 actually increased five-fold, ten-fold, or 30-fold compared to the uptake rate of H1009/L395-F1942m, the results of this experimental system were only partially reflected; thus, the effect may be expressed as an approximately 2.2-fold increase. Thus, from these obtained results, although the in vivo H1009/L395 and IL-8 complex intracellular uptake rate compared, showed that H1009/L395 and IL-8 complex intracellular uptake rate increase, but the effect is not limited to the obtained 2.2-fold increase value.

Without particular limitation, the following explanation can be made from the research results obtained at present. When H1009/L395 (which is a pH-dependent IL-8-binding antibody) forms a complex with human IL-8, the complex has a higher isoelectric point and is more converted to positive charge than when the antibody is present alone. At the same time, the affinity of the complex for the extracellular matrix is increased compared to the affinity of the antibody alone. Properties such as an increase in isoelectric point and enhancement of extracellular matrix binding can be considered as factors that promote antibody uptake into cells in vivo. Furthermore, according to mouse experiments, it was shown that the intracellular uptake rate of the complex of H1009/L395 and human IL-8 was increased by 2.2-fold or more compared to the uptake rate of H1009/L395. Thus, the theoretical explanation and in vitro properties and in vivo phenomena consistently support the following hypothesis: H1009/L395 and human IL-8 form a complex, promote complex uptake into cells, and lead to a significant increase in the removal of human IL-8.

A variety of antibodies against IL-8 have been reported so far, but there has been no report so far on an increase in binding affinity to an extracellular matrix and an increase in uptake of a complex into cells when forming a complex with IL-8.

Furthermore, based on the results of studies in which an increase in intracellular uptake of anti-IL-8 antibodies was observed when the antibodies form complexes with IL-8, the skilled person may consider that anti-IL-8 antibodies that form complexes with IL-8 rapidly take up cells in plasma, while free antibodies that do not form complexes with IL-8 tend to remain in plasma without being taken up by cells. In this case, when the anti-IL-8 antibody is pH-dependent, the anti-IL-8 antibody taken into the cell releases an IL-8 molecule in the cell and then returns to the outside of the cell, and then it is capable of binding another IL-8 molecule; and therefore, an increase in intracellular uptake upon complex formation may have the further effect of more strongly removing IL-8. That is, selecting an anti-IL-8 antibody with increased binding to the extracellular matrix or an anti-IL-8 antibody with increased intracellular uptake can also be another embodiment of disclosure C.

Example 17

Prediction of pH-dependent IL-8-binding antibody H1009/L395 using computer System immunogenicity

Next, immunogenicity scores and ADA production frequencies were predicted for H1009/L395 by a method similar to example 13-1. The results are shown in table 25 and fig. 32. In FIG. 32, H1009/L395 is referred to as "H1009L 395".

[ Table 25]

The results in Table 25 show that H1009/L395 has the same low immunogenicity score level as H1004/L395. Further, according to the results in fig. 32, the predicted ADA generation frequency for H1009/L395 is 0%, and this is also similar to that of H1004/L395.

Thus, the predicted immunogenicity for H1009/L395 is greatly reduced compared to the known anti-human IL-8 antibody hWS-4. Therefore, H1009/L395 in human has very low immunogenicity, and can long-term stable maintenance of anti-IL-8-neutralizing activity.

Example 18

Cynomolgus monkey PK assay using H89/L118 variants with enhanced FcRn-binding capacity under acidic pH conditions

As described in the above examples, in the case of antibodies with native IgG1 as their constant region, the pH-dependent IL-8-binding antibody H1009/L395 is an antibody with better properties. However, the antibodies may also be used as antibodies containing amino acid substitutions in the constant region, e.g., those containing an Fc region with enhanced FcRn binding at acidic pH, as exemplified in example 5. Thus, the use of H89/L118 to confirm that an Fc region with enhanced FcRn binding at acidic pH may also function in pH-dependent IL-8-binding antibodies.

(18-1) production of H89/L118 Fc region-modified antibodies with enhanced FcRn binding at acidic pH

Various modifications described in example 5-1 for enhancing FcRn binding were introduced into the Fc region of H89/L118. Specifically, the following variants were generated by introducing modifications for F1847m, F1848m, F1886m, F1889m, F1927m, and F1168m into the Fc region of H89-IgG 1: (a) H89/L118-IgG1 comprising H89-IgG1m (SEQ ID NO: 94) as the heavy chain and L118-K0MT as the light chain; (b) H89/L118-F1168m comprising H89-F1168m (SEQ ID NO: 95) as the heavy chain and L118-K0MT as the light chain; (c) H89/L118-F1847m comprising H89-F1847m (SEQ ID NO: 96) as the heavy chain and L118-K0MT as the light chain; (d) H89/L118-F1848m comprising H89-F1848m (SEQ ID NO: 97) as the heavy chain and L118-K0MT as the light chain; (e) H89/L118-F1886m comprising H89-F1886m (SEQ ID NO: 98) as the heavy chain and L118-K0MT as the light chain; (f) H89/L118-F1889m comprising H89-F1889m (SEQ ID NO: 99) as the heavy chain and L118-K0MT as the light chain; and (g) H89/L118-F1927m comprising H89-F1927m (SEQ ID NO: 100) as the heavy chain and L118-K0MT as the light chain. Cynomolgus monkey PK assay was performed by the following method using these antibodies.

(18-2) cynomolgus monkey PK assay for antibodies containing novel Fc region variants

Following administration of anti-human IL-8 antibodies to cynomolgus monkeys, the biokinetics of the anti-human IL-8 antibodies were evaluated. Anti-human IL-8 antibody solution at 2mg/kg intravenous administration. Blood was collected five minutes, four hours, one day, two days, three days, seven days, ten days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4 ℃ and 15,000rpm for ten minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-60 ℃ or below until measurement was performed.

The concentration of anti-human IL-8 antibody in cynomolgus monkey plasma was measured by electrochemiluminescence methods. First, anti-h κ capture Ab (antibody solution) was dispersed in a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and stirred at room temperature for one hour. Then, a PBS-Tween solution containing 5% BSA (w/v) was used for blocking at room temperature for two hours, and a plate immobilized with an anti-human antibody was prepared. Samples for calibration curve and samples for cynomolgus monkey plasma measurement diluted 500-fold or more containing anti-human IL-8 antibodies at concentrations of 40.0, 13.3, 4.44, 1.48, 0.494, 0.165, and 0.0549 μ g/mL in plasma were prepared, 50 μ L of the solution was dispersed in each well of the anti-human antibody-immobilized plate, and the solution was stirred at room temperature for one hour. Then, an anti-h κ reporter Ab, biotin conjugate (antibody solution) was added to the aforementioned plate and allowed to react at room temperature for one hour. After further adding SULFO-TAG labeled streptavidin (Meso Scale Discovery) and allowing it to react at room temperature for one hour, the reading buffer T (x1) (Meso Scale Discovery) was dispersed in the plate and immediately measured using the ECTOR imager 2400(Meso Scale Discovery). Anti-human IL-8 antibody concentrations were calculated based on the response of the calibration curve using analytical software, SOFTMax PRO (Molecular Devices).

The results obtained for the half-life (t1/2) and Clearance (CL) of each antibody are shown in table 26, and the changes in antibody concentration in cynomolgus monkey plasma are shown in fig. 33.

[ Table 26]

The above results confirm that all Fc region variants show prolonged retention in plasma compared to antibodies with native IgG1 Fc regions. In particular, H89/L118-F1886m showed the most ideal hemodynamics.

Example 19

Fc region with reduced binding ability to Fc γ R

The Fc region of native human IgG1 is known to bind to one or more fey receptors (hereinafter, referred to as fcyr (s)) of various cells of the immune system and exhibit effector functions such as ADCC and ADCP to target cells.

On the other hand, IL-8 is a soluble cytokine, and mainly intended as a drug of anti-IL-8 antibody through IL-8 in excess existing sites of IL-8 and neutral IL-8 function and show pharmaceutical effects. The site where the IL-8 is present in excess is not particularly limited, and may be, for example, a site of inflammation. It is generally known that at the site of such inflammation, various immune cells aggregate and are activated. It is generally undesirable to transmit unwanted activation signals to these cells via Fc receptors and to induce activities such as ADCC and ADCP in unwanted cells. Therefore, without particular limitation, from the viewpoint of safety, it may be preferable that the anti-IL-8 antibody administered in vivo has low affinity for Fc γ r(s).

(19-1) production of modified antibodies with reduced binding to Fc γ R

Amino acid modifications were further introduced into the Fc region of H1009/L395-F1886m in order to reduce the binding capacity to various human and cynomolgus monkey Fc γ Rs. Specifically, H1009-F1886s (SEQ ID NO: 81) was generated by subjecting the H1009-F1886m heavy chain to each of the following substitutions: r is replaced by L at position 235, R is replaced by G at position 236, and K is replaced by S (according to EU numbering) at position 239. Similarly, H1009-F1974m (SEQ ID NO: 80) was generated by substituting H1009-F1886m for R at position 235 and R at position 236 for G (according to EU numbering), and substituting the region from position 327 to position 331 according to EU numbering with that of the native human IgG4 sequence. H1009/L395-F1886s and H1009/L395-F1974m were produced as antibodies with these heavy chains and L395-k0MT as the light chain.

(19-2) confirmation of affinity for various human Fc. gamma.R

Next, the affinity of H1009/L395-F1886s or H1009/L395-F1974m for Fc γ RIa or Fc γ RIIIa in a soluble form in humans or cynomolgus monkeys was confirmed by the following method.

Binding of H1009/L395-F1886s or H1009/L395-F1974m to soluble forms of Fc γ RIA or Fc γ RIIIa in humans or cynomolgus monkeys was determined using BIACORE T200(GE Healthcare). Soluble Fc γ RIa and Fc γ RIIIa in humans and cynomolgus monkeys were produced as His-tagged molecules by methods known to those skilled in the art. An appropriate amount of rProtein L (BioVision) was immobilized on the sensor chip CM4(GE Healthcare) by an amine coupling method and a target antibody was captured. Next, soluble Fc γ RIa or Fc γ RIIIa was injected with a running buffer (used as a reference solution) and allowed to interact with the antibody captured on the sensor chip. HBS-EP + (GE Healthcare) was used as the running buffer, and HBS-EP + was also used to dilute soluble Fc γ RIa or Fc γ RIIIa. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 20 ℃.

The results are shown in fig. 34. Herein, the designations for human Fc γ RIa, human Fc γ RIIIa, cynomolgus monkey Fc γ RIa, and cynomolgus monkey Fc γ RIIIa are, respectively, in the same order: hfcyria, hfcyriiia, cynoFc γ RIa, and cynoFc γ RIIIa. H1009/L395-F1886m was shown to bind all Fc γ Rs, while on the other hand, H1009/L395-F1886s and H1009/L395-F1974m were confirmed not to bind any of the Fc γ Rs.

(19-3) mouse IL-8 depletion assay for Fc variants

Next, for H1009/L395-F1886s and H1009/L395-F1974m, the human IL-8 removal rate and retention of antibodies in mouse plasma was confirmed by the following experiments. Herein, three doses of H1009/L395-F1886s, 2mg/kg, 5mg/kg, and 10mg/kg were used for evaluation, so that the effect of increasing the antibody dose can also be evaluated for H1009/L395-F1886 s.

The biokinetics of human IL-8 was assessed following the simultaneous administration of human IL-8 and anti-human IL-8 antibodies to human FcRn transgenic mice (B6.mFcRn-/-. hFcRn Tg strain 32 +/+ mice; Jackson Laboratories; Methods mol. biol. 602: 93-104 (2010)). A mixed solution of human IL-8 (10. mu.g/mL) and an anti-human IL-8 antibody (200. mu.g/mL, 500. mu.g/mL, or 1000. mu.g/mL) was administered once at 10mL/kg via the tail vein. In this case, because anti-human IL-8 antibody is present in a sufficient excess of human IL-8, almost all human IL-8 is considered to bind the antibody. Blood was collected five minutes, two hours, four hours, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 4 ℃ and 15,000rpm for 15 minutes to obtain plasma. The separated plasma was stored in a refrigerator set at-20 ℃ or below until measurement was performed.

The concentration of human IL-8 in the plasma of mice was measured by a method similar to that of example 11. The obtained data on the concentration of human IL-8 in plasma are shown in FIG. 35, and the values of the Clearance (CL) of human IL-8 from mouse plasma are shown in Table 27.

First, H1009/L395 comprising the Fc region of native IgG1 and H1009/L395-F1886s comprising the modified Fc region were shown to have comparable human IL-8-removal effects when compared to the 2 mg/kg-administration group.

Next, when the dose of H1009/L395-F1886s antibody was changed, no significant difference in the values of clearance of human IL-8 was observed between the 2mg/kg and 10mg/kg doses, whereas there was a slight difference in plasma IL-8 concentration one day after administration. This strongly suggests that antibodies comprising the variable region of H1009/L395 show sufficient IL-8-removal effect even when the antibodies are administered at high doses.

[ Table 27]

Name of antibody	Dosage form	Human IL-8 CL (mL/d/kg)
			H1009/L395	2mg/kg	740
H1009/L395-F1886s	2mg/kg	628
			H1009/L395-F1886s	5mg/kg	458
H1009/L395-F1886s	10mg/kg	560

(19-4) cynomolgus monkey PK assay for Fc variantsStator

Next, plasma retention of the antibody in cynomolgus monkeys was verified by the following method using H1009/L395-F1886s or H1009/L395-F1974 m.

The biokinetics of anti-human IL-8 antibodies were evaluated in the case of administering anti-human IL-8 antibodies to cynomolgus monkeys alone or in the case of administering human IL-8 and anti-human IL-8 antibodies to cynomolgus monkeys simultaneously. An anti-human IL-8 antibody solution (2mg/mL), or a mixed solution of human IL-8 (100. mu.g/kg) and an anti-human IL-8 antibody (2mg/kg), was administered once intravenously at 1 mL/kg. Blood was collected five minutes, four hours, one day, two days, three days, seven days, ten days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4 ℃ and 15,000rpm for a minute to obtain plasma. The separated plasma was stored in a refrigerator set at-60 ℃ or below until measurement was performed.

The concentration of anti-human IL-8 antibody in cynomolgus monkey plasma was measured by the method of example 18. The resulting data for the concentration of anti-human IL-8 antibody in plasma are shown in fig. 36, and the values for the half-life (t1/2) and Clearance (CL) of the anti-human IL-8 antibody from cynomolgus monkey plasma are shown in table 28.

First, H1009/L395-F1886s, which has an Fc region with improved function, showed significantly prolonged plasma retention compared to Hr9 and H89/L118, which have the Fc region of native human IgG 1.

Furthermore, when H1009/L395-F1886s was administered simultaneously with human IL-8, the change in plasma concentration was comparable to that of the antibody administered alone. Without particular limitation, the following discussion is possible based on the results of this study. As described above, it was shown that the intracellular uptake of the complex of H1009/L395 and human IL-8 was increased compared to the uptake of H1009/L395 alone. Generally, it is believed that high molecular weight proteins bind to cells either non-specifically or in a receptor-dependent manner, then migrate to lysosomes, and are degraded by various degrading enzymes present in the lysosome. Thus, if the rate of uptake of a protein into a cell is increased, the plasma retention of the protein may also deteriorate. However, in the case of antibodies, it has the property of returning plasma through FcRn in endosomes; and therefore, as long as adequate rescue is achieved by FcRn function, plasma retention may not be affected, even if the rate of intracellular uptake is accelerated. Herein, even when H1009/L395-F1886s was administered to cynomolgus monkeys at the same time as human IL-8, plasma remained unaffected. This indicates the following possibilities: while the rate of antibody uptake into cells was increased for H1009/L395-F1886s, the antibody was sufficiently rescued by FcRn that it could be returned to plasma.

In addition, another Fc variant, H1009/L395-F1974m, also showed comparable plasma retention to H1009/L395-F1886 s. While these Fc variants incorporate different modifications as described above with reduced binding capacity to various fcyrs, it was shown that they do not affect the plasma retention of the antibody itself. Thus, it was shown that the plasma retention in cynomolgus monkeys for H1009/L395-F1886s and H1009/L395-F1974m was significantly prolonged and very satisfactory compared to antibodies with native IgG1 Fc region.

[ Table 28]

	t1/2	CL
				Day(s)	mL/d/kg
Hr9	20.26	3.72
			H89/L118	11.88	2.95
H1009/L395-F1886s	35.75	1.64
			H1009/L395-F1886s +hIL-8	72.24	1.11
H1009/L395-F1974m +hIL-8	43.78	1.60

As demonstrated in the above examples, H1009/L395 was first obtained by including a pH-dependent IL-8 binding capacity (characteristic of rapid uptake into cells as a complex with IL-8) as an antibody that significantly increases the rate of in vivo removal of human IL-8. Furthermore, the IL-8-binding affinity of the antibody is also increased at neutral pH conditions compared to the known hWS-4 antibody, and the antibody can neutralize human IL-8 more strongly at neutral pH conditions, such as in plasma. Furthermore, it is an antibody which has excellent stability under plasma conditions and whose IL-8 neutralizing activity is not reduced after in vivo administration. Further, H1009/L395 (which was constructed based on Hr9 having a greatly improved production level compared to hWS-4) is an antibody suitable for manufacturing from the viewpoint of production level. Furthermore, in the computer immunogenicity prediction, the antibody showed a very low score for its immunogenicity, and this score was significantly lower than that of the known hWS-4 antibody and some other known commercially available antibodies. That is, it is expected that H1009/L395 will produce little ADA in humans and will be safe for long term use. Therefore, compared with known anti-human IL-8 antibody, H1009/L395 shows various improvements, and as a drug very useful.

H1009/L395, with native IgG Fc region as described above, is sufficiently useful; however, variants of H1009/L395 that contain a functionally-improved Fc region may also be suitable for use as antibodies with enhanced utility. In particular, it is possible to increase FcRn binding under acidic pH conditions to prolong plasma retention and long-term maintenance effects. Furthermore, variants comprising Fc regions incorporating one or more modifications that reduce binding to fcyr(s) may be used as high safety therapeutic antibodies to avoid undesired immune cell activation and production of cytotoxic activity in the administered organism. As such Fc variants, the use of F1886s or F1974m as developed herein is particularly advantageous, but it is not limited to these Fc variants; and therapeutic antibodies comprising other modified Fc regions are useful as embodiments of disclosure C, as long as the Fc variants have similar functions.

Thus, the present inventors have conducted intensive studies to generate antibodies of publication C (including H1009/L395-F1886s and H1009/L395-F1974m) which can maintain the situation in which the biological activity of human IL-8 is safely and chronically inhibited. Herein, levels that could not be achieved by known anti-IL-8 antibodies have been achieved, and these antibodies of disclosure C are expected to be useful as high quality refined anti-IL-8 antibody drugs.

Example 20

Anti-factor IXa/factor X bispecific antibodies

The humanized anti-factor IXa/factor X bispecific antibody disclosed in WO2012/067176 binds human factor IXa and factor X and induces a co-aggregation activity of blood. The humanized anti-factor IXa/factor X bispecific antibody F8M described in WO2012/067176 (Q499-z121/J327-z119/L404-k H chain (SEQ ID NO: 330)/H chain (SEQ ID NO: 331)/consensus L chain (SEQ ID NO: 332)) was used in this example and F8M comprises two different H chains and two identical consensus L chains. F8M was produced by the method described in the examples of WO 2012/067176.

(20-1) production of anti-factor IXa/factor X bispecific antibody

The following three antibodies were generated as F8M-based anti-factor IXa/factor X bispecific antibodies by the method of reference example 2: (a) F8M-F1847mv, which is a conventional antibody comprising F8M-F1847mv1(SEQ ID NO: 323) and F8M-F1847mv2(SEQ ID NO: 324) as heavy chains and F8ML (SEQ ID NO: 325) as light chains; (b) F8M-F1868mv, which is a conventional antibody comprising F8M-F1868mv1(SEQ ID NO: 326) and F8M-F1868mv2(SEQ ID NO: 327) as the heavy chain and F8ML (SEQ ID NO: 325) as the light chain; and (c) F8M-F1927mv, which is a conventional antibody comprising F8M-F1927mv1(SEQ ID NO: 328) and F8M-F1927mv2(SEQ ID NO: 329) as heavy chains and F8ML (SEQ ID NO: 325) as light chains.

The heavy chain sequences include the same Fc variant sequences as mentioned in example 5 below for enhanced FcRn binding and reduced rheumatoid factor binding:

[ Table 29]

Sequence name	Name in example 5
		F8M-F1847mv1(SEQ ID NO：323)	F1847m
F8M-F1847mv2(SEQ ID NO：324)	F1847m
		F8M-F1868mv1(SEQ ID NO：326)	F1868m
F8M-F1868mv2(SEQ ID NO：327)	F1868m
		F8M-F1927mv1(SEQ ID NO：328)	F1927m
F8M-F1927mv2(SEQ ID NO：329)	F1927m

(20-2) use of monoclonal antibody, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv in cynomolgus monkey Pharmacokinetic study

The pharmacokinetics of the monoclonal antibodies, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv, respectively, were evaluated after a single intravenous bolus administration to male cynomolgus monkeys at a dose of 0.6 mg/kg. Plasma concentrations of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were determined by sandwich ELISA. Pharmacokinetic parameters were calculated using WinNonlin ver 6.4 software. As shown in table 30, the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were 29.3 days, 54.5 days, and 35.0 days, respectively. The F8M PK study using cynomolgus monkeys was performed at a dose of 6mg/kg on different days and revealed a half-life of 19.4 days. Specifically, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv have longer half-lives than F8M. This suggests that the half-life of the anti-factor IXa/X bispecific antibody can be extended by the same modifications mentioned above in example 5 with respect to the sequence of the Fc region.

[ Table 30]

Half-life of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv and F8M following intravenous administration to male cynomolgus monkeys

	F8M-F1847mv	F8M-F1868mv	F8M-F1927mv	F8M
					Half-life period (Tian)	29.3	54.5	35.0	19.4

Example 21

Evaluation of IgE clearance from plasma Using pI-enhanced Fab variants

To enhance clearance of human IgE, replacement of the Fab of the antibody that increases pI is evaluated in this example using a pH-dependent antigen binding antibody. A method of adding an amino acid substitution to the antibody variable region to increase the pI is not particularly limited, but for example, it may be performed by a method described in WO2007/114319 or WO 2009/041643. Amino acid substitutions that introduce the variable region are preferably those that reduce the number of negatively charged amino acids (e.g., aspartic acid and glutamic acid) while increasing the number of positively charged amino acids (e.g., arginine and lysine). Furthermore, amino acid substitutions may be introduced at any position in the antibody variable region. Without particular limitation, the site of introducing an amino acid substitution is preferably at a position where the amino acid side chain can be exposed on the surface of the antibody molecule.

(21-1) production of antibody having an increased pI by modifying amino acids in the variable region

The antibodies tested are summarized in tables 32 and 33.

The heavy chain, Ab1H003 (also referred to as H003, SEQ ID NO: 144), was prepared by introducing a pI increasing substitution H32R in Ab1H (SEQ ID NO: 38). Other heavy chain variants were also prepared according to the method shown in reference example 1 by introducing the individual substitutions shown in table 32 in Ab 1H. All heavy chain variants were expressed with Ab1L (SEQ ID NO: 39) as the light chain. The pH-dependent binding properties of this antibody are summarized in table 5(Ab 1).

Similarly, we also evaluated substitutions in the light chain that increase pI.

The light chain, Ab1L001T (also called L001, SEQ ID NO: 164), was prepared by introducing the pI increasing substitution G16K into Ab 1L. Other light chain variants were also prepared according to the method shown in reference example 1 by introducing the individual substitutions shown in table 33 into Ab 1L. All light chain variants were expressed with Ab1H as the heavy chain.

[ Table 32]

Heavy chain variants of Ab1H evaluated in this example

[ Table 33]

Light chain variants of Ab1L evaluated in this example

(21-2) human Fc γ RIIb-binding assay by BIACORE Using pI-enhanced variants

For the antibodies produced containing Fc region variants, binding assays between soluble human Fc γ RIIb and antigen-antibody complexes were performed using BIACORE T200(GE Healthcare). Soluble human Fc γ RIIb (NCBI accession No. NM — 004001.3) was generated as a His-tagged molecule by methods known in the art. An appropriate amount of anti-His antibody was immobilized on the sensor chip CM5(GE Healthcare) by amine coupling method using a His capture kit (GE Healthcare) to capture human Fc γ RIIb. Next, the antibody-antigen complex and the running buffer (as a reference solution) were injected, and interaction with human Fc γ RIIb captured on the sensor chip was allowed to occur. 20mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150mM NaCl, 1.2mM CaCl ₂And 0.05% (w/v) tween 20(pH 7.4) was used as a running buffer, and each buffer was also used to dilute soluble human Fc γ RIIb. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 25 ℃. Analysis was performed based on the binding (RU) calculated from the sensorgram obtained by measurement, and it was shown that when the amount of binding of Ab1H/Ab1L (Ab 1 initially) was defined as 1Relative value at 00. For the calculation of the parameters, BIACORE T100 evaluation software (GE Healthcare) was used.

The SPR analysis results are summarized in tables 32 and 33. Some variants were shown to have enhanced binding to human Fc γ RIIb immobilized on BIACORE sensor chips.

Antibodies produced by introducing one or more pI-increasing modifications into the variable region are those in which the variable region is more positively charged than those before the modification. Thus, it is believed that the coulombic interaction between the variable region (positive charge) and the sensor chip surface (negative charge) is enhanced by amino acid modifications that increase the pI. Furthermore, this effect is expected to occur similarly at the same negatively charged cell membrane surface; therefore, they are also expected to show an effect of accelerating the rate of uptake of cells in vivo.

Herein, it is believed that about 1.2-fold or more of the binding of variant to hFc γ RIIb compared to the original Ab1 binding to hFc γ RIIb is for the binding of the antibody to hFc γ RIIb on the sensor chip to have a strong charge effect.

In pI-increased heavy chain variants, antibodies with Q13K, G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G or Q105R substitutions (numbering according to Kabat), alone or in combination, showed higher binding to hfcyriib. Single amino acid substitutions in the heavy chain or combinations of these substitutions are presumed to have strong charge effects on binding of hFc γ RIIb on the sensor chip. Thus, one or more positions that are expected to show an effect of accelerating the rate or rate of uptake into cells in vivo by introducing a modification that increases the pI into the heavy chain variable region of an antibody may include, for example, positions 13, 15, 64, 77, 82a, 82b, 85 and 105 according to Kabat numbering. The amino acid substitution introduced at the position may be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

In pI-increased light chain variants, antibodies with G16K, Q24R, a25R, S26R, E27R, Q37R, G41R, Q42K, S52K, S52R, S56K, S56R, S65R, T69R, T74K, S76R, S77R, Q79K substitutions (numbering according to Kabat K), alone or in combination, show higher binding to human Fc γ RIIb. It is postulated that a single amino acid substitution or a combination of these substitutions in the light chain has a strong charge effect on binding of human Fc γ RIIb on the sensor chip. Thus, one or more positions that are expected to show an effect of accelerating the rate or rate of uptake into cells in vivo by introducing a modification that increases the pI into the light chain variable region of an antibody may include, for example, positions 16, 24, 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77, and 79, according to Kabat numbering. The amino acid substitution introduced at the position may be arginine or lysine.

(21-3) cellular uptake of antibody containing variant Fab region with increased pI-value

To evaluate the rate of intracellular uptake of the hfcyriib-expressing cell line using the resulting antibodies containing Fab region variants, assays similar to those described above (4-5) were performed, provided that the amount of antigen taken up was provided as a relative value taking the Ab1H/Ab1L (initial Ab1) value of 1.00.

The results of quantification of cellular uptake are summarized in tables 32 and 33. Strong fluorescence from antigens in cells was observed in various Fc variants. Herein, a fluorescence intensity of about 1.5 times or more that of the antigen of the cell that ingested the variant compared to the original Ab1 is considered to have a strong charge effect on the antigen that was ingested into the cell.

In pI-increased heavy chain variants, antibodies with P41R, G44R, T77R, D82aN, D82aG, D82aS, S82bR or E85G substitutions (numbering according to Kabat), alone or in combination, showed stronger intracellular antigen uptake. It is presumed that a single amino acid substitution or a combination of these substitutions in the heavy chain has a strong charge effect on the intracellular antigen-antibody complex uptake. Thus, one or more positions that are expected to cause more rapid or more frequent uptake of the antigen-antibody complex into the cell by introducing a modification that increases the pI into the heavy chain variable region of the antibody may include, for example, positions 41, 44, 77, 82a, 82b or 85 according to Kabat numbering. The amino acid substitution introduced at the position may be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

In pI-increased light chain variants, antibodies with G16K, Q24R, a25R, a25K, S26R, S26K, E27R, E27Q, E27K, Q37R, G41R, Q42K, S52K, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (numbering according to Kabat) alone or in combination show stronger intracellular antigen uptake. It is presumed that a single amino acid substitution or a combination of these substitutions in the light chain has a strong charge effect on the intracellular antigen-antibody complex uptake. Variants with four or more amino acid substitutions tend to show stronger charge effects than those with fewer amino acid substitutions. One or more positions that are expected to cause more rapid or more frequent uptake of the antigen-antibody complex into the cell by introducing a modification that increases the pI into the light chain variable region of the antibody may include, for example, positions 16, 24, 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77 or 79 according to Kabat numbering. The amino acid substitution introduced at the position may be glutamine, arginine or lysine, and preferably arginine or lysine.

Without being bound to a particular theory, this result can be explained as follows: the antigen and the antibody added to the cell culture solution form an antigen-antibody complex in the culture solution. The antigen-antibody complex binds to human Fc γ RIIb expressed on the cell membrane via the antibody Fc region and is taken up into the cell in a receptor-dependent manner. The antibodies used in this experiment bind antigen in a pH-dependent manner; thus, the antibody can dissociate from the antigen in endosomes (acidic pH conditions) within the cell. As the dissociated antigen is transported to lysosomes and accumulates, it fluoresces within the cell. Thus, it is believed that a strong intracellular fluorescence intensity indicates that uptake of the antigen-antibody complex into the cell occurs more rapidly or more frequently.

(21-4) evaluation of human IgE clearance in the mouse Co-injection model

Some anti-IgE antibodies with pH-dependent antigen binding (initially Ab1, Ab1H/Ab1L013, Ab1H/Ab1L014, Ab1H/Ab1L007) were tested in a mouse co-injection model to evaluate their ability to accelerate IgE clearance from plasma. In the co-injection model, C57BL6J mice (Jackson Laboratories) were administered separately by a single intravenous injection of IgE pre-mixed with anti-IgE antibodies. All groups received 0.2mg/kg IgE along with 1.0mg/kg anti-IgE antibody. Total IgE plasma concentrations were determined by anti-IgE ELISA. First, anti-human IgE (clone 107, MABTECH) was dispersed in a microplate (Nalge nunc International) and left to stand at room temperature for two hours or overnight at 4 ℃ to prepare a plate on which an anti-human IgE antibody was immobilized. The standard curve samples and samples were mixed with excess anti-IgE antibody (prepared internally) to form a homogeneous structure of immune complexes. These samples were added to the plates immobilized with anti-human IgE antibody and left overnight at 4 ℃. These samples were then reacted sequentially with human GPC3 nucleoprotein (prepared internally), biotinylated anti-GPC 3 antibody (prepared internally), streptavidin Poly HRP80 conjugate (Stereospecific Detection Technologies) for one hour. Thereafter, SuperSignal (registered trademark) ELISA Pico chemiluminescent substrate (Thermo Fisher Scientific) was added. Chemiluminescence was read using SpectraMax M2(Molecular Devices). Concentrations of human IgE were calculated using SOFTmax PRO (Molecular Devices). Fig. 37 depicts plasma concentration time profiles of IgE in C57BL6J mice.

After administration of pI-elevated Fab variants with pH-dependent antigen binding, the plasma total IgE concentration was lower than that of the original Ab 1. These results indicate that antigen-antibody immune complexes with high pI variants of pH-dependent antigen binding are able to bind more strongly to cytoplasmic membrane receptors such as Fc γ Rs, which increases cellular uptake of the antigen-antibody immune complexes. The antigen taken up into the cell can be efficiently released from the antibody in the endosome, resulting in accelerated IgE removal. Mice treated with Ab1H/Ab1L007 (which showed weak efficacy in vitro studies) had higher IgE concentrations than other antibodies containing the pI-enhanced Fab variant. These results also suggest that the sensitivity of the in vitro system using fluorescence intensity by the above-mentioned InCell Analyzer 6000 can be higher than that of the above-mentioned in vitro BIACORE system in order to estimate the in vivo antigen clearance from plasma.

Example 22

Evaluation of the clearance of C5 from plasma using pI-increased Fab variants

To enhance clearance of human IgE, the replacement in the Fab portion of the antibody that increases pI is evaluated in this example using a pH-dependent antigen binding antibody.

(22-1) preparation of C5 [ Table of recombinant human C5Purification and purification]

Recombinant human C5(NCBI GenBank accession No.: NP-001726.2, SEQ ID NO: 207) was transiently expressed using FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned medium expressing human C5 was diluted with an equal volume of milliQ water and then applied to a Q-Sepharose FF or Q-Sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden) followed by a gradient elution with NaCl. Fractions containing human C5 were pooled, and the salt concentration and pH were then adjusted to 80mM NaCl and pH6.4, respectively. The resulting samples were applied to an SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were pooled and subjected to a CHT ceramic hydroxyapatite column (Bio-Rad Laboratories, Hercules, Calif., USA). The human C5 eluate was then applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). The fractions containing human C5 were pooled and stored at-150 ℃. Internally prepared recombinant human C5 or plasma derived human C5(CALBIOCHEM, Cat #204888) were used for the study.

Expression and purification of recombinant cynomolgus monkey C5(NCBI GenBank accession # XP-005580972, SEQ ID NO: 208) was performed in exactly the same manner as the human equivalent.

(22-2) preparation of synthetic calcium library

The gene library used as antibody heavy chain variable region of the synthetic human heavy chain library consisted of 10 heavy chain libraries. Germline framework regions VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59, VH4-61, VH4-B, VH5-51, and VH6-1 were selected for the library based on germline frequency and biophysical properties of the V-gene family in the human B-cell repertoire (reptotoires). A library of human B-cell antibodies was simulated and the synthetic human heavy chain library was diversified at the antibody-binding site.

The gene library of antibody light chain variable regions was designed with calcium binding motifs and with reference to the human B cell antibody repertoire, was diversified in positions that would promote antigen recognition. The design of a gene library of antibody light chain variable regions with properties resulting from calcium-dependent binding to an antigen is described in WO 2012/073992.

The combination of the heavy chain variable region library and the light chain variable region library was inserted into a phagemid vector, and a phage library was constructed with reference (de Heard et al, meth.mol.biol.178: 87-100 (2002)). The trypsin-cleavage site was introduced into the phagemid vector at the linker region between the Fab and pIII proteins. A modified M13KO7 helper phage with a trypsin cleavage site between the N2 and CT domains of gene III was used for phage preparation for Fab display.

(22-3) isolation of calcium-dependent anti-C5 antibody

Phage display libraries were supplemented with BSA and CaCl at final concentrations of 4% and 1.2mM, respectively₂TBS dilution of (a). As a method of panning, with reference to the general protocol (Junutula et al, J.Immunol.methods 332 (1-2): 41-52(2008), D' Mello et al, J.Immunol.methods 247 (1-2): 191-203(2001), Yeung et al, Biotechnol.Prog.18 (2): 212-220(2002), Jensen et al, mol.cell Proteimics 2 (2): 61-69 (2003)), conventional magnetic beads were used for selection, and NeutrAvidin-coated beads (Sera-Mag SpeedBeads Neutradin-coated) or streptavidin-coated (Dynabeads M-streptavidin) human C5 (BICOCHEM, Cat #204888) were used as magnetic beads labeled with EZ-Lin-NHS-4-Biotin (Cat. No. 3921329).

In a primary round of phage selection, the phage display library was incubated with biotinylated human C5(312.5nM) for 60 minutes at room temperature. The magnetic beads were then used to capture phage displaying the bound Fab variants.

After incubation with the beads for 15 minutes at room temperature, the beads were incubated with 1mL of 1.2mM CaCl₂And 0.1% Tween 20 TBS three times washing, and the beads with 1mL containing 1.2mM CaCl₂TBS of (a) was washed twice. Phage were eluted by resuspending the beads for 15 minutes with TBS containing 1mg/mL trypsin. Eluted phage were infected with ER2738 and rescued by helper phage. Rescued phage were precipitated with polyethylene glycol, supplemented with BSA and CaCl to final concentrations of 4% and 1.2mM, respectively ₂TBS resuspended and used for the next round of panning.

After the first round of screening, phages were selected for their calcium dependence, where the antibodies bound C5 more strongly in the presence of calcium ions. In the second and third rounds panning was performed in the same way as the first round, except that 50nM (second round) or 12.5nM (third round) of biotinylated antigen was used and finally eluted with 0.1mL of elution buffer (50mM MES, 2mM EDTA, 150mM NaCl, ph5.5) and contacted with 1 μ L of 100mg/mL trypsin to select for its calcium dependence. After selection, the selected phage clones were converted to the IgG format.

The binding capacity of the converted IgG antibodies to human C5 was evaluated at 30 ℃ using the Octet RED384 system (Pall Life Sciences) under two different conditions: at 1.2mM CaCl₂-pH 7.4(20mM MES，150mM NaCl，1.2mM CaCl₂) And binding and dissociation at 1.2mM CaCl₂-pH 7.4(20mM MES，150mM NaCl，1.2mM CaCl₂) Binding and CaCl at 3. mu.M₂-pH 5.8(20mM MES，150mM NaCl，3μM CaCl₂) Dissociation of (3). 25 out of the clones bound by the pH-calcium dependent antigen were isolated. The sensorgram for these antibodies is shown in fig. 38.

(22-4) identification of anti-C5 bispecific antibodies

From the clones isolated in example B-3, nine pH or calcium dependent anti-C5 antibody clones were selected for further analysis (CFP0008, 0011, 0015, 0016, 0017, 0018, 0019, 0020, 0021). Some amino acid substitutions are introduced into the CFP0016 heavy chain variable region by methods generally known to those of ordinary skill in the art to improve properties of the antibody, such as physicochemical properties. This CFP0016 variant, CFP0016H019, was used for further analysis instead of CFP 0016. The amino acid sequences of the VH and VL regions of these nine antibodies are described in table 34. In the table, names described in parentheses represent the names of abbreviations.

[ Table 34]

Clone name and amino acid sequence of selected antibody

Full-length genes having nucleotide sequences encoding the heavy and light chains of antibodies were synthesized and prepared by methods generally known to those of ordinary skill in the art. Heavy and light chain expression vectors are prepared by inserting the obtained plasmid fragments into a vector for expression in mammalian cells. The obtained expression vector is sequenced by methods generally known to those of ordinary skill in the art. To express the antibody, the prepared plasmid was transiently transfected into FreeStyle293-F cell line (Thermo Fisher Scientific). Purification from the conditioned medium expressing the antibody was performed by a method generally known to those of ordinary skill in the art using rProtein a sepharose Fast Flow (GE Healthcare).

(22-5) production and characterization of pH-dependent anti-C5 bispecific antibodies

Bispecific antibodies (which recognize two different epitopes of C5) were generated by combining CFP0020 and CFP 0018. Bispecific antibodies (which have two different Fab clones in each binding site of the antibody) are prepared in IgG format and prepared using methods generally known to those of ordinary skill in the art. In this bispecific IgG antibody, the two heavy chains comprise heavy chain constant regions (G1dP1, SEQ ID NO: 227 and G1dN1, SEQ ID NO: 228) that are different from each other, thereby effectively forming heterodimers of the two heavy chains. An anti-C5 bispecific antibody comprising a binding site for an anti-C5 MAb "X" and an anti-C5 MAb "Y" is denoted as "X// Y".

By introducing some amino acid substitutions into the heavy and light chain CDRs by methods generally known to those of ordinary skill in the art, we obtained a 20//18 light chain consensus variant named 'optimized 20// 18' (consisting of two heavy chains: CFP0020H0261-G1dP1, SEQ ID NO: 229 and CFP0018H0012-G1dN1, SEQ ID NO: 230 and a consensus light chain: CFP0020L233-k0, SEQ ID NO: 231).

The kinetic parameters of the optimized 20//18 pair of recombinant human C5 were evaluated at 37 ℃ using the BIACORE T200 instrument (GE Healthcare) under two different conditions (e.g., (A) binding and dissociation at pH 7.4 and (B) binding and dissociation at pH 7.4 and pH 5.8). Protein A/G (Pierce, Cat #21186) or anti-human IgG (Fc) antibody (in a human antibody capture kit; GE Healthcare, Cat No. BR-1008-39) was immobilized by amine coupling method in the series S CM4(GE Healthcare,cat No. BR-1005-34). The anti-C5 antibody was captured on an immobilized molecule and subsequently injected with human C5. The running buffers used were ACES pH 7.4 and pH 5.8(20mM ACES, 150mM NaCl, 1.2mM CaCl)₂0.05% tween 20). Kinetic parameters were determined at both pH conditions by matching sensorgrams to a 1: 1 binding-RI (no body effect adjustment) model using BIACORE T200 evaluation software, version 2.0 (GE Healthcare). Kinetic parameters at pH 7.4, the association rate (ka), the dissociation rate (KD), and the binding affinity (KD), as well as the dissociation rate (KD), determined by calculating only the dissociation phase at each pH condition, are described in table 35. The optimized 20//18 showed faster dissociation at pH 5.8 for human C5 compared to the dissociation rate at pH 7.4.

[ Table 35]

Kinetic parameters of the 20//18 variant for human C5 under two different conditions

(22-6) production of antibody with increased pI by modification of amino acids in variable region

The antibodies tested are summarized in tables 36 and 37.

The heavy chain, CFP0020H0261-001-G1dP1 (also referred to as 20H001, SEQ ID NO: 232), was prepared by introducing the pI raising substitution P41R/G44R into CFP0020H0261-G1dP1(SEQ ID NO: 229). Similarly, the heavy chain, CFP0018H0012-002-G1dN1 (also known as 18H002, SEQ ID NO: 251), was prepared by introducing the pI raising substitution T77R/E85R into CFP0018H0012-G1dN1(SEQ ID NO: 230). Additional heavy chain variants were also prepared by introducing the individual substitutions shown in Table 36 into CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1, respectively, according to the method shown in reference example 1. The heavy chain variants of both the CFP0020H0261-G1dP1 variant and the CFP0018H0012-G1dN1 variant were expressed together with CFP0020L233-k0(SEQ ID NO: 231) as the light chain to obtain bispecific antibodies.

Similarly, we also evaluated substitutions in the light chain that increase pI. The pI-increasing substitution G16K was introduced into CFP0020L233-k0 to produce the light chain, CFP0020L233-001-k0 (also known as 20L233-001, SEQ ID NO: 271). Other light chain variants were also prepared by introducing the individual substitutions shown in table 37 into CFP0020L233-k0 according to the method shown in reference example 1. All light chain variants were expressed with CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1 as heavy chains to obtain bispecific antibodies.

[ Table 36]

Heavy chain variants of CFP0020H0261-001-G1dP1 and CFP0018H0012-001-G1dN1 evaluated in this example

[ Table 37]

Light chain variants of CFP0020L233 evaluated in this example

(22-7) determination of human Fc γ RIIb-binding by BIACORE Using pI-enhanced variants

For the antibodies produced containing Fc region variants, binding assays between soluble hFc γ RIIb and antigen-antibody complexes were performed using BIACORE T200(GE Healthcare). Soluble hfcyrlib was generated as a His-tagged molecule by methods known in the art. Coupling by amine Using His Capture kit (GE Healthcare)Method an appropriate amount of anti-His antibody was immobilized on a sensor chip CM5(GE Healthcare) to capture the hfcyriib. Next, the antibody-antigen complex and running buffer (as reference solution) were injected and the interaction with the hfcyriib captured on the sensor chip occurred. 20mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150mM NaCl, 1.2mM CaCl₂And 0.05% (w/v) tween 20(pH 7.4) was used as running buffer, and each buffer was also used to dilute the soluble hFc γ RIIb. To regenerate the sensor chip, 10mM glycine-HCl pH 1.5 was used. All measurements were performed at 25 ℃. Analysis was performed based on the binding (RU) calculated from the sensorgram obtained by the measurement, and it was shown that the relative value when the amount of binding of CFP0020H0261-G1dP1/CFP0018H0012-G1dN1/CFP0020L233-k0 (original Ab2) was defined as 1.00. For the calculation of the parameters, BIACORE T100 evaluation software (GE Healthcare) was used.

The SPR analysis results are summarized in tables 36 and 37. Some variants were shown to have enhanced binding to hfcyriib immobilized on BIACORE sensor chips. Herein, about 1.2-fold or more of the binding of the variant to hfcyriib compared to the binding of the original Ab2 to hfcyriib is considered to have a strong charge effect on the binding of the antibody to hfcyriib on the sensor chip.

In pI-increased heavy chain variants, antibodies with L63R, F63R, L82K or S82bR substitutions (numbering according to Kabat) showed higher binding to hfcyriib. Single amino acid substitutions in the heavy chain or combinations of these substitutions are presumed to have strong charge effects on binding of hFc γ RIIb on the sensor chip. Thus, it is contemplated that one or more of the positions that show an effect of accelerating the rate or rate of uptake into cells in vivo by introducing a modification to increase the pI into the heavy chain variable region of an antibody may include, for example, position 63, 82 or 82b, according to Kabat numbering. The amino acid substitution introduced at the one or more positions may be arginine or lysine.

In pI-increased light chain variants, antibodies with G16K, Q27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (numbering according to Kabat) showed higher binding to hfcyriib. Single amino acid substitutions in the light chain or combinations of these substitutions are presumed to have strong charge effects on binding of human Fc γ RIIb on the sensor chip. Thus, it is contemplated that one or more of the positions that show an effect of accelerating the rate or rate of uptake into cells in vivo by introducing a modification that increases the pI into the light chain variable region of an antibody may include, for example, position 16, 27, 41, 52, 56, 65, 69, 74, 76, 77 or 79 according to Kabat numbering. The amino acid substitution introduced at the one or more positions may be arginine or lysine. Variants with four or more amino acid substitutions tend to show stronger charge effects than those with fewer amino acid substitutions.

(22-8) cellular uptake of antibody containing variant Fab region with increased pI-value

To evaluate the rate of intracellular uptake of a cell line expressing hfcyriib using the generated antibodies containing Fab region variants, the following assay was performed.

MDCK (Madin-Darby canine kidney) cell lines constitutively expressing hfcyriib were generated by known methods. Using these cells, intracellular uptake of antigen-antibody complexes was evaluated. Specifically, Alexa555(Life technologies) was used to label human C5 according to an established protocol, and an antigen-antibody complex was formed in a culture solution having an antibody concentration of 10mg/mL and an antigen concentration of 10 mg/mL. The culture solution containing the antigen-antibody complex was added to a culture plate of the above-described MDCK cells constitutively expressing hFc γ RIIb and incubated for one hour, and then the fluorescence intensity of the antigen taken into the cells was quantified using cell Analyzer 6000(GE healthcare). The amount of ingested antigen is provided as a relative value to the initial Ab2 value taken as 1.00.

The results of quantification of cellular uptake are summarized in tables 36 and 37. Strong fluorescence from antigens in cells was observed in several heavy and light chain variants. Herein, a fluorescence intensity of about 1.5 or more times that of the antigen taken up in the cells of the variant compared to the fluorescence intensity of the original Ab2 is considered to have a strong charge effect on the antigen taken up in the cells.

In pI-increased heavy chain variants, antibodies with G8R, L18R, Q39K, P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN, S82bR, T83R, a85R or E85G substitutions (numbering according to Kabat) show stronger intracellular antigen uptake. It is speculated that single amino acid substitutions in the heavy chain or combinations of these substitutions have a strong charge effect on intracellular antigen-antibody complex uptake. Thus, it is contemplated that one or more positions that more rapidly or more frequently cause uptake of an antigen-antibody complex into a cell by introducing a modification that increases the pI into the heavy chain variable region of an antibody may include, for example, position 8, 18, 39, 41, 44, 63, 64, 77, 82, 82a, 82b, 83, or 85 according to Kabat numbering. The amino acid substitution introduced at said one or more positions may be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

In pI-increased light chain variants, antibodies with G16K, Q27R, S27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (numbering according to Kabat) showed stronger intracellular antigen uptake. Single amino acid substitutions in the light chain or combinations of these substitutions are presumed to have a strong charge effect on intracellular antigen-antibody complex uptake. Variants with four or more amino acid substitutions tend to show stronger charge effects than those with fewer amino acid substitutions. As shown in example 21, (21-3), the combination of 42K and 76R substitutions was effective in IgE antibodies. However, in the case of the C5 antibody, the Kabat numbered 42 amino acid is already lysine, so we can observe the charge effect of 42K/76R by simply substituting 76R. The fact that the variant with the 76R substitution also showed a strong charge effect in the C5 antibody shows that the 42K/76R combination has a strong charge effect regardless of the antigen. Thus, it is contemplated that one or more positions that more rapidly or more frequently cause uptake of an antigen-antibody complex into a cell by introducing a modification that increases the pI into the light chain variable region of an antibody may include, for example, positions 16, 27, 41, 52, 56, 65, 69, 74, 76, 77, or 79, according to Kabat numbering. The amino acid substitution introduced at the one or more positions may be arginine or lysine.

(22-9) evaluation of C5 scavenging in mouse Co-injection model

Some anti-C5 bispecific antibodies (naive Ab2, 20L233-005, 20L233-006, and 20L233-009) were tested in a mouse co-injection model to evaluate their ability to accelerate C5 clearance from plasma. C57BL6J mice (Jackson Laboratories) were administered with a single intravenous injection of C5, premixed with an anti-C5 bispecific antibody, respectively, in a co-injection model. All groups received 0.1mg/kg C5 along with 1.0mg/kg of anti-C5 bispecific antibody. Total C5 plasma concentrations were determined by anti-C5 ECLIA. First, anti-human C5 mouse IgG was dispersed in ECL plates and left overnight at 5 ℃ to prepare plates immobilized with anti-human C5 mouse IgG. Samples and samples for the standard curve were mixed with anti-human C5 rabbit IgG. These samples were added to plates immobilized with anti-human C5 mouse IgG and left at room temperature for one hour. These samples were then reacted with HRP-conjugated anti-rabbit igg (jackson Immuno research). After incubating the plates for one hour at room temperature, a sulfo-tag conjugated anti-HR antibody was added. ECL signals were read with a Sector imager 2400(Meso Scale discovery). From the ECL signal, the concentration of human C5 was calculated in a standard curve using SOFTmax PRO (Molecular Devices). Fig. 39 depicts the C5 plasma concentration time curve in C57BL6J mice.

All bispecific antibodies tested in this study with one or more substitutions that increased pI compared to the original Ab2 demonstrated rapid clearance of C5 from plasma. Thus, amino acid substitutions in the light chain of T74K/S77R, S76R/Q79K and Q37R were suggested to accelerate the removal of C5-antibody immune complexes in vivo. In addition, C5 removal was faster for 20L233-005 and 20L233-006 than for 20L233-009, consistent with in vitro imaging and BIACORE analysis. These results suggest that it may not even seem possible to promote in vivo antigen clearance from plasma at one or more locations (examined under in vitro systems using fluorescence intensity by the above-mentioned InCell Analyzer 6000 or in vitro BIACORE systems), which may be found to be promoted by using more sensitive in vivo systems. These results also suggest that the sensitivity of the in vitro system using fluorescence intensity by the above-described InCell Analyzer 6000 can be higher than that of the above-described in vitro BIACORE system for the evaluation of the presumption of the in vivo clearance of antigens from plasma.

Example 23

Evaluation of IgE clearance from plasma Using pI-enhanced Fc variants

To enhance clearance of human IgE or human C5, displacement in the Fc portion of the antibody that increases pI was evaluated using pH-dependent antibodies. The method of adding amino acid substitutions to the antibody constant region to increase the pI is not particularly limited, but for example, it may be performed by the method described in WO 2014/145159.

(23-1) Generation of antibodies with increased pI by Single amino acid modification in the constant region

The antibodies tested are summarized in table 38. The heavy chain, Ab1H-P1394m (SEQ ID NO: 307), was prepared by introducing the pI increasing substitution Q311K into Ab 1H. Other heavy chain variants were also prepared by introducing the individual substitutions shown in table 38 into Ab1H according to the method shown in reference example 1. All heavy chain variants were expressed with Ab1L as the light chain.

[ Table 38]

(23-2) determination of human Fc γ RIIb-binding by BIACORE Using antibodies containing pI-enhanced Fc region variants

To evaluate the charge effects on FcR γ RIIb-binding of antigen-antibody complexes formed by using the antibodies described in table 38, an FcR γ RIIb-binding assay was performed in a similar manner to that described in example 21, (21-2). The measurement results are shown in table 38. Herein, the binding of about 1.2-fold or more of the variant to hfcyriib compared to the binding of the original Ab1 to hfcyriib is considered to have a strong charge effect on the binding of the antibody to hfcyriib on the sensor chip.

Among the pI-enhanced variants with a single amino acid substitution compared to the original Ab1, the antigen-antibody complexes generated by several variants such as P1398m, P1466m, P1482m, P1512m, P1513m, and P1514m showed the highest binding to hfcyriib. It is assumed that a single amino acid substitution with respect to D413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K, or G402K has a strong charge effect on binding of hfcyriib on the sensor chip. Thus, a single location expected to exhibit the effect of accelerating the rate or rate of intracellular uptake in vivo by introducing a modification that increases the pI into the constant or Fc region of an antibody may include, for example, location 311, 343, 384, 401, 402, or 413 according to EU numbering. The amino acid substitution introduced at the position may be arginine or lysine.

(23-3) cellular uptake of antibody containing pI-increased Fc region variant

To evaluate the intracellular uptake of antigen-antibody complexes formed by the antibodies described in table 38, cellular imaging assays were performed in a similar manner as described in example 21, (21-3). The measurement results are shown in table 38. Herein, the fluorescence intensity of the antigen taken up in the cells of the variant about 1.5 times or more as compared to the fluorescence intensity of the original Ab1 is considered to have a strong charge effect on the antigen taken up in the cells.

Among the pI-enhanced variants with a single amino acid substitution compared to the original Ab1, antigen-antibody complexes generated by several variants such as P1398m, P1466m, P1469m, P1470m, P1481m, P1482m, P1483m, P1512m, P1513m and P1653m showed stronger intracellular antigen uptake. It is hypothesized that a single amino acid substitution for D413K, Q311R, N315K, N384R, Q342K, P343R, P343K, D401R, D401K or D413R has a strong charge effect on antigen-antibody complex uptake in cells. Thus, a single location that is expected to cause faster or more frequent uptake of antigen-antibody complexes into cells by introducing a modification that increases the pI into the constant or Fc region of an antibody may include, for example, positions 311, 315, 342, 343, 384, 401, or 413 according to EU numbering. The amino acid substitution introduced at this position may be arginine or lysine.

(23-4) evaluation of human IgE clearance in mouse Co-injection model

Some anti-IgE antibodies with pH-dependent antigen binding (original Ab1, P1466m, P1469m, P1470m, P1480m, P1482m, P1512m, P1653m) were tested in a mouse co-injection model to evaluate their ability to accelerate IgE clearance from plasma. The measurement was carried out in a similar manner to that of example 21, (21-4). Fig. 40 depicts plasma concentration time curves in C57BL6J mice.

After administration of high pI variants with pH-dependent antigen binding (only a single amino acid substitution), the plasma total IgE concentration was lower than that of the original Ab1, except for P1480 m. P1480m (showing weak efficacy in vitro studies) did not accelerate IgE removal. Furthermore, the plasma total IgE concentration in mice treated with the high pI variant without pH-dependent antigen binding was significantly higher than that with the high pI variant with pH-dependent antigen binding (data not shown). These results indicate that cellular uptake of antigen-antibody immune complexes is increased by introducing modifications that increase pI. The antigen complexed with the pH-dependent antigen-binding antibody in the ingested cells can be efficiently released from the antibody inside the endosome, resulting in acceleration of IgE removal. These results suggest that even sites where it seems unlikely to promote clearance of antigen from plasma in vivo (examined under the in vitro BIACORE system described above) can be found to be promoted by the use of more sensitive in vivo systems. These results also suggest that the sensitivity of the in vitro system using fluorescence intensity by the above-mentioned InCell Analyzer 6000 can be higher than that of the above-mentioned in vitro BIACORE system for evaluation of presumption of in vivo antigen clearance from plasma.

Reference example 1

Construction of expression vector for amino acid-substituted IgG antibody

Mutants were prepared by the method described in the attached instruction manual using a Rapid Change Site-Directed Mutagenesis kit (Stratagene). The plasmid fragment containing the mutant is inserted into an animal cell expression vector to construct desired H-chain and L-chain expression vectors. The nucleotide sequence of the obtained expression vector is determined by methods known in the art.

Reference example 2

Expression and purification of IgG antibodies

Antibodies were expressed using the following method. Human embryonic kidney cancer cell-derived HEK293H cell line (Invitrogen) was suspended in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). At 5 to 6x10⁵Cell Density of Individual cells/mL cells were plated in dishes at 10 mL/dishFor adhering cells (10 cm diameter; CORNING) and in CO₂Incubator (37 ℃, 5% CO)₂) Culturing for one day. Then, the medium was removed by aspiration and 6.9mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid was introduced into cells by the lipofection method. The resulting culture supernatant was collected, centrifuged (about 2,000g, 5 minutes, room temperature) to remove cells, and sterilized by filtration through a 0.22- μm filter MILLEX (registered trademark) -GV (Millipore) to obtain a supernatant. By methods known in the art, rProtein A agarose gels were used ^TMAntibodies were purified from the obtained culture supernatant by Fast Flow (Amersham Biosciences). To determine the concentration of purified antibody, absorbance was measured at 280nm using a spectrophotometer. From the use of the Protein by Pace et al, Protein Science 4: 2411-2423(1995), calculating the antibody concentration.

Reference example 3

Preparation of soluble human IL-6 receptor

Recombinant soluble human IL-6 receptor (which is an antigen) was prepared in the following manner. Methods known in the art were used to construct e.g. Mullberg et al, j.immunol.152: 4958 and 4968(1994), CHO cell lines constitutively expressing a soluble human IL-6 receptor consisting of an amino acid sequence from the 1 st to the 357 th amino acids from the N-terminus. Soluble human IL-6 receptor was expressed by culturing the CHO line. Soluble human IL-6 receptor was purified from the culture supernatant of the CHO line obtained in the following two steps: blue Sepharose 6 FF column chromatography and gel filtration column chromatography. The fraction eluting as the main peak in the last step was used as the final purified product.

Claims

1. An isolated anti-IL-8 antibody, said isolated anti-IL-8 antibody comprising

(a) HVR-H1, the amino acid sequence of which is shown as SEQ ID NO:67,

(b) HVR-H2, the amino acid sequence of which is shown as SEQ ID NO:73,

(c) HVR-H3, the amino acid sequence of which is shown in SEQ ID NO:74,

(d) HVR-L1, whose amino acid sequence is shown in SEQ ID NO:70,

(e) HVR-L2 having an amino acid sequence of SEQ ID NO:75, and

(f) HVR-L3, whose amino acid sequence is shown in SEQ ID NO: 76.

2. An isolated anti-IL-8 antibody, said isolated anti-IL-8 antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:78 and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 79.

3. An isolated anti-IL-8 antibody, said isolated anti-IL-8 antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of said heavy chain is SEQ ID NO: 80 or SEQ ID NO: 81, and the amino acid sequence of the light chain is SEQ ID NO: 82.

4. An isolated nucleic acid encoding the anti-IL-8 antibody of any one of claims 1 to 3.

5. A vector comprising the nucleic acid of claim 4.

6. A host cell comprising the vector of claim 5.

7. A method for producing an anti-IL-8 antibody comprising culturing the host cell of claim 6.

8. The method of claim 7, further comprising isolating the antibody from the host cell culture.

9. A pharmaceutical composition comprising the anti-IL-8 antibody of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

10. Use of an anti-IL-8 antibody according to any one of claims 1 to 3 in the preparation of a pharmaceutical composition for the treatment of a disorder in which an excess of IL-8 is present.

11. Use of an anti-IL-8 antibody of any one of claims 1 to 3 in the preparation of a pharmaceutical composition for inhibiting angiogenesis.

12. Use of an anti-IL-8 antibody according to any one of claims 1 to 3 for the preparation of a pharmaceutical composition for inhibiting the promotion of neutrophil migration.