TW202342532A - Il-8-binding antibodies and uses thereof - Google Patents

Abstract

One nonexclusive aspect provides novel IL-8 antibodies that are superior as pharmaceuticals.

Description

IL-8 binding antibodies and their uses

Cross-reference with related applications This application relates to and claims priority to Japanese Priority Patent Application No. 2015-185254 filed in Japan on September 18, 2015, the contents of which are fully incorporated by reference.

In a 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. Also provided are methods and uses of IL-8 antibodies and pharmaceutical compositions for treating, for example, IL-8 related disorders.

Antibodies are highly stable in plasma and have few side effects, so they are valued as pharmaceuticals. Among them, most IgG-type therapeutic antibodies have been marketed, and many therapeutic antibodies are currently under development (Reichert et al., Nat. Biotechnol. 23:1073-1078 (2005); Pavlou et al., Eur. J. Pharm . Biopharm. 59(3):389-396 (2005)). On the other hand, various technologies have been developed for second-generation therapeutic antibodies, including those used to improve effector function, antigen-binding capacity, pharmacokinetics or stability, and to reduce the risk of immunogenicity. Technology (Kim et al., Mol. Cells. 20 (1):17-29 (2005)). The dosage of therapeutic antibodies is usually very high, so the development of therapeutic antibodies encounters difficulties such as preparation of subcutaneous administration preparations and high manufacturing costs. Methods for improving the pharmacokinetics, pharmacodynamics, and antigen-binding properties of therapeutic antibodies provide avenues for reducing dosage and manufacturing costs associated with therapeutic antibodies.

Substitution of amino acid residues in constant regions provides a means to improve antibody pharmacokinetics (Hinton et al., J. Immunol. 176 (1):346-356 (2006); Ghetie et al., Nat. Biotechnol. 15(7):637-640 (1997)). Affinity maturation technology provides a method to enhance the antigen neutralizing ability of antibodies (Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471 (2005); Wu et al. , J. Mol. Biol. 368:652 (2007)), and may increase the antigen-binding activity by introducing mutations into the amino acids of the CDR and/or framework regions of the antibody variable domain. Improving the antigen-binding properties of the antibody may improve the biological activity of the antibody in vitro or reduce the dosage, and may further improve the efficacy in vivo (Wu et al., J. Mol. Biol. 368:652-665 (2007)).

The amount of antigen that can be neutralized by one antibody molecule depends on the affinity of the antibody for this antigen; therefore, the antigen can be neutralized with a small amount of antibody by increasing the affinity. Antibody affinity for an antigen can be routinely increased using a variety of known methods (see, eg, Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471 (2005)). Moreover, if the antigen can be covalently bonded and the affinity can be infinite, one antibody molecule can theoretically neutralize one antigen molecule (when the antibody is bivalent, two antigens can be neutralized). However, one limitation of the development of therapeutic antibodies so far is that usually one antibody molecule binds and neutralizes only one antigen molecule (two antigen molecules when the antibody is divalent). Recently, it has been reported that antibodies bind to antigens in a pH-dependent manner. (hereinafter also referred to as "pH-dependent antibodies" or "pH-dependent binding antibodies"), which enable one antibody molecule to bind and neutralize multiple antigen molecules (see, e.g., WO2009/125825; Igawa et al., Nat. . Biotechnol. 28:1203-1207 (2010)). pH-dependent antibodies bind strongly to antigen under neutral conditions in plasma and dissociate from antigen under acidic conditions in the endosomes of cells. After dissociation from the antigen, FcRn is used to recirculate the antibody in the plasma and bind and neutralize another antigen molecule again. Therefore, one pH-dependent antibody can repeatedly bind and neutralize multiple antigen molecules.

Recent reports indicate that antibody recovery properties can be achieved by focusing on the difference in calcium (Ca) ion concentration in plasma and endosomes, and using antibodies that exhibit calcium-dependent antigen-antibody interactions (hereinafter also referred to as "Ca ion Concentration-dependent antibodies") (WO2012/073992). (Hereinafter, pH-dependent antibodies and "calcium ion concentration-dependent antibodies" are collectively referred to as "pH/Ca concentration-dependent antibodies.")

By binding to FcRn, IgG antibodies remain in the plasma for a long time. The binding between IgG antibodies and FcRn is strong under acidic pH conditions (eg, pH 5.8), but there is little binding under neutral pH conditions (eg, pH 7.4). IgG antibodies are brought into cells non-specifically and bind to FcRn under the acidic pH conditions of endosomes, thereby returning to the cell surface. This IgG then dissociates from FcRn under neutral pH conditions in plasma.

It has been reported that a pH-dependent antibody modified to increase binding to FcRn under neutral pH conditions has the ability to repeatedly bind and eliminate antigen molecules from plasma; therefore, administration of this antibody can eliminate antigen from plasma (WO2011/122011). According to this report, pH-dependent antibodies modified to increase binding to FcRn under neutral pH conditions (e.g., pH 7.4) can further accelerate antigen elimination compared to pH-dependent antibodies including the Fc region of native IgG antibodies ( WO2011/122011).

When mutations are introduced into the Fc region of an IgG antibody to eliminate its binding to FcRn under acidic pH conditions, it will no longer be recovered from the endosome into the plasma, which will significantly affect the retention of the antibody in the plasma. Relatedly, it has been reported that methods to increase FcRn binding under acidic pH conditions can be used as a method to improve plasma retention of IgG antibodies. Introducing amino acid modifications into the Fc region of IgG antibodies to enhance their FcRn binding under acidic pH conditions can enhance the efficiency of recycling from endosomes to plasma, thereby improving plasma retention. For example, modifications M252Y/S254T/T256E (YTE; Dall'Acqua et al., J. Biol. Chem. 281:23514-235249 (2006)), M428L/N434S (LS; Zalevsky et al., Nat. Biotechnol. 28: 157-159 (2010)) and N434H (Zheng et al., Clin. Pharm. & Ther. 89(2):283-290 (2011)) have been reported to have a longer antibody half-life than native IgG1.

However, in addition to concerns about the worsening of immunogenicity or the incidence of agglutination of such antibodies including Fc region variants with increased FcRn binding under neutral pH conditions or acidic pH conditions, there are also reports indicating that the administration of therapeutic antibodies In patients with preexisting conditions, there is increased binding to anti-drug antibodies (hereinafter also referred to as "pre-existing ADA") such as rheumatoid factor (WO2013/046722, WO2013/046704). WO2013/046704 reports that Fc region variants containing specific mutations (according to EU numbering, represented by two residue modifications of Q438R/S440E) have increased binding to FcRn under acidic pH conditions, and compared with unmodified natural Fc, Showed significantly reduced binding to rheumatoid factor. However, WO2013/046704 does not specifically prove that this Fc region variant has better plasma retention than antibodies with native Fc regions.

Safe and more advantageous Fc region variants with further improved plasma retention that do not show binding to pre-existing ADA are therefore needed.

Antibody-dependent cellular cytotoxicity (hereinafter referred to as "ADCC"), complement-dependent cytotoxicity (hereinafter referred to as "CDC"), and antibody-dependent cellular phagocytosis (ADCP) mediated by IgG antibodies Reporters function as effectors 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 the antibody receptor present on the surface of effector cells, such as killer cells, natural killer cells, or activated macrophages (here Within the scope of the disclosure, it is also called "Fcγ receptor", "FcgR", "Fc gamma receptor" or "FcγR"). In humans, FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb isoforms (isoforms) are reported as FcγR family proteins, and their respective allotypes have also been reported (Jefferis et al., Immunol. Lett. 82:57 -65 (Non-Patent Document 13)). Achieving a balance of antibody affinity for activating receptors, including FcγRIa, FcγRIIa, FcγRIIIa, or FcγRIIIb, respectively, and inhibitory receptors, including FcγRIIb, is important in optimizing antibody effector function.

To date, many techniques have been reported to increase or improve the activity of therapeutic antibodies against antigens. For example, the activity of an antibody binding to activating FcγR plays an important role in the cytotoxicity of the antibody, and thus antibodies have been developed that target membrane antigens and increase cytotoxicity due to enhanced binding of activating FcγR(s). See, for example, WO2000/042072; WO2006/019447; Lazar et al., Proc. Nat. Acad. Sci. USA. 103:4005-4010 (2006); Shinkawa et al., J. Biol. Chem. 278, 3466-3473 (2003); Clynes et al., Proc. Natl. Acad. Sci. U SA 95:652-656 (1998); Clynes et al., Nat. Med. 6:443-446 (2000)). Similarly, the binding activity to inhibitory FcγR (in humans, FcγRIIb) plays an important role in immunosuppressive activity and agonist activity. Therefore, antibodies targeting membrane antigens with increased inhibitory FcγR binding activity have been studied. . (Li et al., Proc. Nat. Acad. Sci. USA. 109 (27):10966-10971 (2012)). In addition, the impact of FcγR binding of antibodies that bind to water-soluble antigens is mainly examined from the perspective of side effects (Scappaticci et al., J. Natl. Cancer Inst. 99 (16): 1232-1239 (2007)). For example, when an antibody with increased FcγRIIb binding is used as a drug, the risk of developing anti-drug antibodies can be expected to be reduced (Desai et al., J. Immunol. 178(10):6217-6226 (2007)).

Recently, it has been reported that amino acid modifications are introduced into the Fc region of IgG antibodies to increase the activity of antibodies targeting soluble antigens and bind to activating and/or inhibitory FcγR(s), which can further accelerate the elimination of this antigen from serum. (WO2012/115241, WO2013/047752, WO2013/125667, WO2014/030728). Furthermore, an Fc region variant was identified that has little change in FcγRIIb-binding activity from the natural IgG antibody Fc region but has reduced activity against other activating FcγRs (WO2014/163101).

In contrast to antibodies with FcRn-mediated recycling mechanisms, plasma retention of soluble antigens is very short-lived, and soluble antigens can therefore be displayed by binding to antibodies with such recycling mechanisms (e.g., antibodies without pH/Ca concentration dependence) Increased plasma retention and plasma concentration. Therefore, for example, if a soluble antigen in plasma has multiple physiological functions, even if one physiological function is blocked due to antibody binding, the plasma concentration of the antigen may still worsen the pathogenic symptoms caused by other physiological functions. The plasma concentration of the antigen due to antibody binding may still worsen. Retention and/or increased plasma concentration. In this case, in addition to using the above-mentioned antibody modification methods to accelerate antigen elimination, such as using multiple pH/Ca concentration-dependent antibodies to form multivalent immune complexes with multiple antigens, there have also been reports indicating an increase in For binding methods of FcRn, FcγR(s), and complement receptors (WO2013/081143).

Even if the Fc region is unmodified, it has been reported that the isoelectric point (pI) of the antibody can be increased or decreased by modifying the amino acid residues to change the charge of the amino acid residues that may be exposed on the surface of the antibody variable region. Control the half-life of antibodies in the blood regardless of the type of target antigen or antibody without substantially reducing the antigen-binding activity of the antibody (WO2007/114319: Technology that mainly replaces amino acids in FR; WO2009/041643: mainly replaces amines in CDR base acid technology). These documents show that the plasma half-life of an antibody can be prolonged by reducing the isoelectric point of the antibody, and conversely, the plasma half-life of the antibody can be shortened by increasing the isoelectric point of the antibody.

Regarding modification of the charge of amino acid residues in the constant region of antibodies, it has been reported that modification of the charge of specific amino acid residues can promote the entry of antigen into cells, especially the charge of specific amino acid residues in the CH3 domain. To increase the isoelectric point value of the antibody, and it has also been documented that this modification should not interfere with the binding to FcRn (WO2014/145159). It has also been reported that modification of the charge of amino acid residues in the constant region of an antibody (mainly the CH1 domain) to reduce the isoelectric point value can extend the half-life of the antibody in plasma, and that combined amino acid residue mutations can increase FcRn. Binding can enhance binding to FcRn and extend the plasma half-life of the antibody (WO2012/016227).

However, it is unclear whether combining modification technologies designed to increase or decrease the isoelectric point value of the antibody with other modification technologies designed to increase or decrease binding to FcRn or FcγR(s) will result in increased plasma retention or increased plasma retention of the antibody. The effect of eliminating antigens from plasma.

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

The ECM is reported to be involved in the in vivo dynamics of proteins administered in vivo. Blood concentrations of the VEGF-Trap molecule administered subcutaneously as a fusion protein of VEGF receptor and Fc have been examined (Holash et al., Proc. Natl. Acad. Sci., 99(17):11393-11398 (2002) ). Subcutaneously administered VEGF-Trap molecules with high isoelectric points have low plasma concentrations and therefore low bioavailability. Modified VEGF-Trap molecules that use amino acid substitutions to lower isoelectric point values have higher plasma concentrations and their bioavailability can be improved. In addition, the change in bioavailability is related to the binding strength to ECM. It can be seen that the bioavailability of VEGF-Trap molecules administered subcutaneously depends on its binding strength to ECM in the subcutaneous site.

WO2012/093704 reports that there is an inverse relationship between antibody binding to ECM and plasma retention, so antibody molecules that are not bound to ECM have better plasma retention than antibodies that are bound to ECM.

As mentioned above, techniques for reducing extracellular matrix binding in order to improve protein bioavailability and plasma retention in vivo have been reported. Conversely, the advantages of increasing antibody binding to the ECM have not yet been elucidated.

Human IL-8 (interleukin 8) is a member of the chemotactic interleukin family and is 72 or 77 amino acid residues in length. The term "chemokine" is a general term that refers to a family of proteins with a molecular weight of 8-12 kDa and containing four cysteines that form intermolecular disulfide bonds. According to the arrangement of cysteine, chemokines are divided into CC chemokines, CXC chemokines, C chemokines, and CA3C chemokines. IL-8 is classified as a CXC chemokine, also known as CXCL8.

IL-8 exists in monomeric and homodimeric forms in solution. The IL-8 unit body consists of an antiparallel beta plate with a structure in which the C-terminal alpha helix spans and covers the beta sheet. If the IL-8 unit is the 72 amino acid form of IL-8, it includes between cysteine 7 and cysteine 34, and between cysteine 9 and cysteine 50 2 disulfide cross-links between. IL-8 homodyads are stabilized by non-covalent interactions between the beta sheets of the two units, because there is no covalent bond between the molecules of the homodyads.

IL-8 is expressed in various cells such as peripheral blood mononuclear cells, tissue macrophages, NK cells, fibroblasts and vascular epithelial cells in response to stimulation of inflammatory cytokines (Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014)).

In normal tissues, chemokines are generally undetectable or only weakly detectable, but they are strongly detected at inflamed sites and are involved in inducing inflammation by promoting the involvement of white blood cells in inflamed tissue sites. IL-8 is a pro-inflammatory chemokine that is known to activate neutrophils, promote the expression of cell adhesion molecules, and promote neutrophil adhesion to vascular endothelial cells. IL-8 also has neutrophil chemotactic ability. IL-8 produced in injured tissue will promote the chemotaxis of neutrophils that adhere to vascular endothelial cells and enter the tissue, and together with the infiltration of neutrophils, induce inflammation. . IL-8 is also known to be a potent angiogenic factor for endothelial cells and has been implicated in promoting tumor angiogenesis.

Inflammatory diseases associated with elevated (e.g., excessive) levels of IL-8, including inflammatory diseases of the skin, such as inflammatory keratosis (e.g., psoriasis), atopic dermatitis, contact dermatitis; chronic inflammatory conditions, autologous Immune diseases, such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and Behcet's disease; inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis; inflammatory liver diseases, such as hepatitis B , hepatitis C, alcoholic hepatitis, drug-induced allergic hepatitis; inflammatory kidney diseases, such as glomerulonephritis; inflammatory respiratory diseases, such as bronchitis and asthma; inflammatory chronic vascular diseases, such as atherosclerosis; multiple sclerosis disease, oral ulcers, vocal cord inflammation, and related inflammation caused by artificial organs and/or artificial blood vessels. Elevated (i.e., excessive) levels of IL-8 are also associated with malignancies such as ovarian, lung, prostate, gastric, breast, melanoma, head and neck, and kidney cancer; sepsis due to infection; cystic fibrosis; and pulmonary fibrosis. (See, e.g., Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014) (not incorporated by reference in its entirety).

High-affinity human anti-IL-8 antibodies have been developed as pharmaceutical compositions for several of these diseases (Desai et al., J. Immunol. 178(10):6217-6226 (2007)) , but not yet on the market. Currently, only one pharmaceutical composition including an IL-8 antibody is available, which is a mouse anti-IL-8 antibody, for topical treatment of psoriasis. New anti-IL-8 antibodies are anticipated for the treatment of disease.

In a non-exclusive aspect, it is an unqualified purpose of the embodiments of Disclosure A to provide molecules with improved pharmacokinetic properties for antibodies, such as ion concentration-dependent antigen binding properties, thereby improving antibody half-life and/or removal from plasma. Clear the antigen.

In a non-exclusive aspect, it is the unqualified purpose of the embodiments disclosed in B to provide safe and more advantageous Fc region variants that have longer half-lives and reduced binding to pre-existing anti-drug antibodies (ADAs) .

In a non-exclusive aspect, it is an unlimited purpose of embodiments disclosed in C to provide anti-IL-8 antibodies that have pH-dependent binding affinity for IL-8. Additional embodiments relate to anti-IL-8 antibodies that, when administered to an individual, have the effect of eliminating IL-8 faster than a reference antibody. In another embodiment, it is disclosed that C is an anti-IL-8 antibody that stably maintains its IL-8 neutralizing activity when administered to an individual. In some embodiments, anti-IL-8 antibodies exhibit lower immunogenicity. In other embodiments, Disclosure C relates to methods of making and using the anti-IL-8 antibodies described above. Another non-limiting purpose of disclosing C is to provide new anti-IL-8 that can be included in pharmaceutical compositions.

In a non-exclusive manner, within the scope of Disclosure A provided herein, the inventors unexpectedly discovered that ion concentration-dependent antibodies (antibodies including ion concentration-dependent antigen-binding domains ("antigen-binding activity depends on ion concentration conditions") The ability of the altered antigen-binding domain to eliminate antigen from plasma can be accelerated by modifying at least one amino acid residue exposed on the surface of the antibody to increase its isoelectric point (pI). In another non-exclusive aspect, the inventors discovered that an ion concentration-dependent antibody with an increased isoelectric point value can further increase the extracellular matrix binding of the antibody. Without wishing to be bound by a particular theory, the inventors have discovered that antigen elimination from plasma can be increased by increasing the binding of antibodies to the extracellular matrix.

In a non-exclusive aspect, within the scope of Disclosure B provided herein, the inventors conducted research on safe and advantageous Fc region variants that do not bind to anti-drug antibodies (pre-existing ADA) and may further improve plasma retention. conducted precise research. As a result, the inventor unexpectedly discovered that the amino acid residue mutation combination includes the substitution of amino acid at position 434 to Ala(A) according to the EU numbering method and 2 specific residue mutations (represented by Q438R/S440E according to the EU numbering method). Fc region variants are ideal for maintaining significantly reduced binding to rheumatoid factor while achieving plasma retention of the antibody.

In a non-exclusive manner, within the scope of Disclosure C provided herein, the inventors have produced some pH-dependent anti-IL-8 antibodies (anti-IL-8 that bind to IL-8 in a pH-dependent manner). antibody). From the results of various validations, the inventors identified that the pH-dependent anti-IL-8 antibody has the effect of eliminating IL-8 more rapidly than the reference antibody when administered to an individual. In some embodiments, the disclosure C relates to pH-dependent anti-IL-8 antibodies that stably maintain their IL-8 neutralizing activity. In additional non-limiting embodiments, the pH-dependent anti-IL-8 antibody has lower immunogenicity and superior performance levels.

Also within the scope of Disclosure C, the inventors successfully obtained an anti-IL-8 antibody including an Fc region, the FcRn binding affinity of the Fc region at acidic pH is increased compared to the FcRn binding affinity of the natural Fc region. In an alternative aspect, the inventors succeeded in obtaining an anti-IL-8 antibody that includes an Fc region whose binding affinity to pre-existing ADA is reduced compared to the binding affinity of the native Fc region to pre-existing ADA. . In an alternative approach, the inventors successfully obtained anti-IL-8 antibodies with an Fc region whose plasma half-life was increased compared to the plasma half-life of the native Fc region. In an alternative approach, the inventors succeeded in obtaining a pH-dependent anti-IL-8 antibody including an Fc region whose binding affinity to effector receptors is comparable to that of the native Fc region. Reduced affinity. In a different aspect, the inventors identified nucleic acids encoding the anti-IL-8 antibodies described above. In another aspect, the inventor obtained a host containing the above nucleic acid. In another aspect, the inventors developed a method for producing the above-mentioned anti-IL-8 antibody, including the step of cultivating the above-mentioned host. In another aspect, the inventors developed a method of promoting elimination of IL-8 in an individual relative to a reference antibody, comprising the step of 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 according to ion concentration conditions, wherein its isoelectric point (pI) is determined by modifying at least one amino acid residue that may be exposed on the surface of the antibody. Increase. [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 in which the antigen-binding activity is higher under high ion concentration conditions than under low ion concentration conditions. [4] The antibody according to any one of [1] to [3], wherein the ion concentration is hydrogen ion concentration (pH) or calcium ion concentration. [5] The antibody of [4], wherein, against the antigen, the ratio of the KD of the acidic pH range to the neutral pH range, that is, KD (acidic pH range) / KD (neutral pH range), is 2 or more high. [6] The antibody according to any one of [1] to [5], wherein in the antigen-binding domain, at least one amino acid residue is substituted with histidine acid or at least one histidine acid is inserted. [7] The antibody according to any one of [1] to [6], which can promote the elimination of the antigen from the plasma compared with before the antibody modification. [8] The antibody according to any one of [1] to [7], wherein the extracellular matrix binding activity of the antibody is increased compared to before modification of the antibody. [9] The antibody according to any one of [1] to [8], wherein the amino acid residue modification is 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) Substituting a negatively charged amino acid residue with an uncharged amino acid residue, (b) substituting a negatively charged amino acid residue with a positively charged amino acid residue, and (c) Substituting uncharged amino acid residues with positively charged amino acid residues. [11] The antibody according to any one of [1] to [10], wherein the antibody includes a variable region and/or a constant region, and the amino acid residue is modified into the variable region and/or the constant region. Modification of amino acid residues in the region. [12] The antibody of [11], wherein the variable region includes a complementarity determining region (CDR) and/or a framework region (FR). [13] The antibody of [12], wherein the variable region includes a heavy chain variable region and/or a light chain variable region, and the CDR or FR is selected from the group consisting of: Kabat numbering At least 1 amino acid residue at one position is modified: (a) 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44 among the FRs of the heavy chain variable region , 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 bits; (b) Positions 31, 61, 62, 63, 64, 65 and 97 of the CDR of the heavy chain variable region; (c) FR 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; and (d) Positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 of the CDR of the light chain variable region. [14] The antibody of [13], wherein at least 1 amino acid residue in one position selected from the group consisting of the following in the CDR and/or FR is modified: (a) FR 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105th; (b) Positions 31, 61, 62, 63, 64, 65 and 97 of the CDR of the heavy chain variable region; (c) Positions 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 of the FR of the light chain variable region; and (d) Positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 of the CDR of the light chain variable region. [15] The antibody according to any one of [11] to [14], wherein at least 1 amino acid residue in one position selected from the group consisting of the following in the constant region is modified: According to EU numbering law 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 1 amino acid residue in one position selected from the group consisting of the following in the constant region is modified: According to EU numbering law 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 1 amino acid residue in one position selected from the group consisting of the following in the constant region is modified: According to the EU numbering law, 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413. [18] The antibody according to any one of [1] to [17], wherein the constant region has Fc gamma receptor (FcγR) binding activity, and the FcγR binding activity under neutral pH conditions is higher than that of natural IgG. The constant region of the reference antibody is enhanced. [19] The antibody of [18], wherein the FcγR is FcγRIIb. [20] The antibody according to any one of [1] to [17], wherein the constant region has binding activity to one or more activating FcγRs selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa, and has binding activity to FcγRIIb, and compared to a reference antibody that is different from natural IgG only in the constant region, the FcγRIIb binding activity is maintained or improved and the binding activity to the activating FcγR is reduced. [21] The antibody according to any one of [1] to [20], wherein the constant region has FcRn-binding activity and is different from a natural IgG only in the constant region, at neutral pH Conditions (eg pH 7.4) enhance the FcRn binding activity of the constant region. [22] The antibody according to any one of [1] to [21], wherein the antibody is a multispecific antibody that binds to at least two antigens. [23] The antibody according to any one of [1] to [22], wherein the antibody is an IgG antibody. [24] A pharmaceutical composition including an antibody according to any one of [1] to [23]. [25] The pharmaceutical composition of [24], which is used to promote the elimination of antigens from plasma. [26] The pharmaceutical composition of [24] or [25], which is used to enhance the binding of antibodies to extracellular matrix. [27] A nucleic acid encoding an antibody according to any one of [1] to [23]. [28] A vector including the nucleic acid of [27]. [29] A host cell including the vector of [28]. [30] A method of producing an antibody, which includes an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, including the following steps: Host cells were cultured as in [29] and antibodies were collected from the cell culture. [30A] A method of producing an antibody, the antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, comprising the following steps: It is possible to modify at least 1 amino acid residue exposed on the surface of the antibody to increase the isoelectric point (pI). [30B] The method of producing an antibody as in [30A], wherein at least 1 amino acid residue is modified at one position in the CDR or FR selected from the group consisting of the following according to Kabat numbering: (a) Among the FRs of the heavy chain variable region, 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, Positions 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112; (b) The heavy chain variable region Positions 31, 61, 62, 63, 64, 65, and 97 in the CDR; (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, Positions 81, 85, 100, 103, 105, 106, 107, and 108; and (d) 24, 25, 26, 27, 52, 53, 54, 55, and 56 of the CDRs of the light chain variable region bit; or (II) The position in the constant region is selected from the group consisting of: According to EU numbering law 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 producing an antibody as in [30A] or [30B], wherein the amino acid residue modification is selected from the group consisting of: (a) Substituting negatively charged amino acid residues with uncharged amino acid residues; (b) Substituting negatively charged amino acid residues with positively charged amino acid residues; (c) Substituting uncharged amino acid residues with positively charged amino acid residues; (d) Substitution or insertion of histidine in CDR or FR. [32] The method of producing an antibody according to any one of [30], [30A] or [30B], more optionally including any one or more of the following steps: Compared to the reference antibody, (a) Select antibodies that promote elimination of the antigen from plasma; (b) Select antibodies with enhanced binding activity to extracellular matrix; (c) Select antibodies with enhanced FcγR-binding activity under neutral pH conditions (such as pH 7.4); (d) Select antibodies with enhanced FcγRIIb binding activity under neutral pH conditions (e.g. pH 7.4); (e) Select an antibody that has maintained or increased FcγRIIb binding activity and reduced binding activity to one or more activating FcγRs. The activating FcγRs should be selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc , FcγRIIIa, FcγRIIIb and FcγRIIa; (f) Select antibodies with enhanced FcRn-binding activity under neutral pH conditions (e.g., pH 7.4); (g) Select antibodies with increased isoelectric point (pI); (h) Confirm the isoelectric point (pI) of the collected antibodies and select antibodies with increased isoelectric point (pI); and (i) Select antibodies whose antigen-binding activity changes or increases according to ion concentration conditions.

In an alternative embodiment, disclosure A relates to, but is not limited to: [A1] An antibody having a constant region, wherein at least 1 amino acid residue selected from the same group of modification sites as the group of modification sites defined in [15] or [16] is modified in a constant region district. [A2] An antibody such as [A1], further comprising a heavy chain variable region and/or a light chain variable region, the variable region having CDRs and/or FRs, and being selected from the group consisting of [13] or [14] At least one amino acid residue in the defined group of modification sites with the same modification site is modified on the CDR and/or FR. [A3] An antibody having a constant region, at least one amino acid residue selected from the same group of modification sites as the group of modification sites defined in [15] or [16], modified in the constant region with Increase its isoelectric point value. [A4] An antibody such as [A3], which further has a heavy chain variable region and/or a light chain variable region, the variable region having CDRs and/or FRs, selected from the group consisting of [13] or [14] At least one amino acid residue in the defined group of modification sites with the same modification site is modified on the CDR and/or FR. [A5] An antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, the antibody having a constant region and being selected from the same modified sites as those defined in [15] or [16] At least one amino acid residue in the group is modified in the constant region. [A6] The antibody of [A5], further having a heavy chain variable region and/or a light chain variable region, the variable region having CDRs and/or FRs, selected from and defined in [13] or [14] At least one amino acid residue in the group of modification sites with the same modification site is modified on the CDR and/or FR. [A7] Use of an antibody according to any one of [1] to [23] and [A1] to [A6] for the manufacture of a medicine that promotes the elimination of antigens from plasma. [A8] Use of an antibody such as any one of [1] to [23] and [A1] to [A6], for the manufacture of a medicine that increases extracellular matrix binding. [A9] Use of an antibody according to any one of [1] to [23] and [A1] to [A6] for eliminating antigens from plasma. [A10] Use of an antibody according to any one of [1] to [23] and [A1] to [A6], for increasing extracellular matrix binding. [A11] An antibody obtained by any of the methods for producing antibodies such as [30], [30A], [30B], [31], [32].

According to various embodiments, disclosure A includes part or all of one or more of the items described in [1] to [30], [30A], [30B], [31], [32] and [A1] to [A11] A combination of elements, provided that such a combination does not technically conflict with common technical knowledge in the technical field. For example, in some embodiments, Disclosure A includes a method of making a modified antibody that includes an antigen-binding domain that promotes elimination of the antigen from plasma compared to the antibody before modification, including: (a) Modify at least 1 amino acid residue that may be exposed on the surface of the antibody, which is: (I) The position in the CDR or FR according to the Kabat numbering method is selected from the group consisting of: (a) 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, Positions 84, 85, 86, 105, 108, 110, and 112; (b) Positions 31, 61, 62, 63, 64, 65, and 97 of the CDR of the heavy chain variable region; (c) The light chain variable region 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; and (d) the light Positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 of the CDR of the chain variable region; or (II) Positions in the constant region selected from the group consisting of the following according to EU numbering: 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 positions; (b) Modify the antigen-binding domain so that the obtained antigen-binding activity changes according to ion concentration conditions, wherein (a) and (b) can be performed simultaneously or sequentially; (c) Culturing host cells to express nucleic acid encoding the modified antibody; and (d) collecting the modified antibody from the host cell culture. In another embodiment, the method may optionally further include one or more of the following steps: Compared with before modification of the antibody, (e) Select antibodies that promote elimination of the antigen from plasma; (f) Select antibodies with enhanced binding activity to extracellular matrix; (g) Select antibodies with enhanced FcγR-binding activity under neutral pH conditions (such as pH 7.4); (h) Select antibodies with enhanced FcγRIIb binding activity under neutral pH conditions (e.g. pH 7.4); (i) Select an antibody that has maintained or increased FcγRIIb binding activity and reduced binding activity to one or more activating FcγRs. The activating FcγRs should be selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc , FcγRIIIa, FcγRIIIb and FcγRIIa; (j) Select antibodies with enhanced FcRn-binding activity under neutral pH conditions (e.g., pH 7.4); (k) Select antibodies with increased isoelectric point (pI); (l) Confirm the isoelectric point (pI) of the collected antibodies and select antibodies with increased isoelectric point (pI); and (m) Select antibodies whose antigen-binding activity changes or increases according to ion concentration conditions.

Another embodiment of disclosure A relates to, for example, without limitation: [D1] A method of manufacturing a modified antibody that has a longer or shorter half-life in plasma compared to before modification, including the following steps: (a) Modify the nucleic acid encoding the antibody before modification to change the charge of at least 1 amino acid residue in one position selected from the group consisting of: According to EU numbering law 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) Cultivate host cells to express the nucleic acid; and (c) collecting the antibody from the host cell culture. [D2] A method of extending or shortening the half-life of antibodies in plasma, including the following steps: Modify at least 1 amino acid residue at a position selected from the group consisting of: According to EU numbering law 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359 , 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443.

In one embodiment, disclosure B relates to, but is not limited to: [33] An Fc region variant, including an FcRn binding domain, the FcRn binding domain includes: (a) According to EU numbering, Ala is at position 434; Glu, Arg, Ser, or Lys is at position 438; and Glu, Asp, or Gln is at position 440. [34] The Fc region variant of [33], wherein the FcRn binding domain includes: According to EU numbering, Ala is at position 434; Arg or Lys is at position 438; and Glu or Asp is at position 440. [35] The Fc region variant of [33] or [34], wherein the FcRn binding domain further includes: According to EU numbering, Ile or Leu is at position 428; and/or Ile, Leu, Val, Thr, or Phe is at position 436. [36] The Fc region variant of [35], wherein the FcRn binding domain includes: According to the EU numbering method, Leu is at position 428; and/or Val or Thr is at position 436. [37] The Fc region variant according to any one of [33] to [36], wherein the FcRn binding domain includes a combination of amino acid substitutions selected from the group consisting of: N434A/Q438R/S440E; N434A /Q438R/S440D; N434A/Q438K/S440E; N434A/Q438K/S440D; N434A/Y436T/Q438R/S440E; N434A/ Y436T/Q438R/S440D; 434A/Y436T/Q438K/S440D; N434A /Y436V/Q438R/S440E; N434A/Y436V/Q438R/S440D; N434A/Y436V/Q438K/ S440E; N434A/Y436V/Q438K/S440D; 434A/ R435H/F436T/Q438R/S440D ; N434A/R435H/F436T/Q438K/S440E; N434A/R435H/ F436T/Q438K/S440D; N434A/R435H/F436V/Q438R/S440E; 434A/R435H/F436V/Q438K/S440E ; N434A/R435H/F436V/Q438K/ S440D; M428L/N434A/Q438R/S440E; M428L/N434A/Q438R/S440D; 440D; M428L/N434A/Y436T/Q438R /S440E; M428L/N434A/Y436T/Q438R/S440D; M428L/N434A/Y436T/Q438K/S440E; M428L/ N434A/Y436T/Q438K/S440D; 440E; 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, according to EU numbering method. [38] The Fc region variant of [37], wherein the FcRn binding domain includes a combination of amino acid substitutions selected from the group consisting of: N434A/Q438R/S440E; N434A/Y436T/Q438R/S440E; N434A/Y436V/Q438R/S440E; M428L/N434A/Q438R/S440E; M428L/N434A/Y436T/Q438R/S440E; M4 28L/N434A/ Y436V/Q438R/S440E; L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E, according to the EU numbering method. [39] The Fc region variant according to any one of [33] to [38], wherein the FcRn binding activity under acidic pH conditions (for example, pH 5.8) is enhanced compared to the Fc region of natural IgG. [40] An Fc region variant as in any one of [33] to [39], wherein, Compared with the Fc region of natural IgG, the binding activity for anti-drug antibodies (ADA) under neutral pH conditions is not significantly improved. [41] The Fc region variant of [40], wherein the anti-drug antibody (ADA) is rheumatoid factor (RF). [42] The Fc region variant of any one of [33] to [41], wherein compared with the Fc region of native IgG, the plasma clearance rate (CL) is reduced, the plasma retention time is increased, or the plasma half-life (t1 /2) increase. [43] The Fc region variant of any one of [33] to [42], wherein plasma retention is increased compared to a reference Fc region variant including the following amino acid substitution combination: N434Y/Y436V/Q438R/S440E, according to EU numbering method. [44] An antibody comprising an Fc region variant according to any one of [33] to [43]. [45] The antibody of [44], wherein the antibody is an IgG antibody. [46] A pharmaceutical composition comprising an antibody such as [44] or [45]; [47] The pharmaceutical composition of [46] is used to increase the retention of antibodies in plasma. [48] A nucleic acid encoding an Fc region variant according to any one of [33] to [43] or an antibody according to [44] or [45]. [49] A vector comprising the nucleic acid of [48]. [50] A host cell including a vector such as [49]. [51] A method of manufacturing an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant, comprising the following steps: Host cells are cultured as in [50] and the Fc region variant or antibody containing the variant is collected from the cell culture. [52] The method of manufacturing an Fc region variant including an FcRn binding domain or an antibody including the variant according to [51], which more optionally includes any one or more of the following steps: (a) Select Fc region variants that have improved FcRn-binding activity under acidic pH conditions compared to the Fc region of native IgG; (b) Select Fc region variants that do not significantly improve anti-drug antibody (ADA) binding activity under neutral pH conditions compared to the Fc region of native IgG; (c) Selecting Fc region variants that increase plasma retention compared to the Fc region of native IgG; and (d) Select antibodies that include Fc region variants that promote elimination of the antigen from plasma compared to a reference antibody that includes the Fc region of native IgG. [53] A method of manufacturing an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant, comprising the following steps: Substitute amino acids such that the Fc region variant obtained or an antibody containing the variant includes Ala at position 434 according to EU numbering; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440 Bit.

In one embodiment, disclosure B relates to, for example, without limitation: [B1] Use of an Fc region variant according to any one of [33] to [43] or an antibody according to [44] or [45], for the manufacture of a medicine that increases plasma retention. [B2] Use of an Fc region variant according to any one of [33] to [43] or an antibody according to [44] or [45] to produce an Fc region compared to natural IgG at neutral pH Medicines whose conditions do not significantly enhance the binding activity of anti-drug antibodies (ADA). [B3] The use of an Fc region variant according to any one of [33] to [43] or an antibody according to [44] or [45], for the purpose of increasing retention in plasma. [B4] The use of an Fc region variant according to any one of [33] to [43] or an antibody according to [44] or [45], in order to react at neutral pH compared to the Fc region of native IgG. The conditions do not significantly increase the binding activity of anti-drug antibodies (ADA). [B5] An Fc region variant or an antibody containing the variant is obtained by using a method for producing an antibody according to any one of [51], [52] and [53].

According to various embodiments, it is disclosed that B includes a combination of part or all of one or more of the elements described in [33] to [53] and [B1] to [B5], provided that such combination does not technically conflict with Ordinary technical knowledge in this technical field is inconsistent. For example, in some embodiments, Disclosure B includes Fc region variants containing an FcRn binding domain, which may include: (a) Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering; (b) According to EU numbering, Ala is at position 434; Arg or Lys is at position 438; and Glu or Asp is at position 440; (c) According to the EU numbering system, Ile or Leu is at position 428; Ala is at position 434; Ile, Leu, Val, Thr, or Phe is at position 436; Glu, Arg, Ser, or Lys is at position 438; and Glu, Asp , or Gln at position 440; (d) According to the EU numbering system, Ile or Leu is at position 428; Ala is at position 434; Ile, Leu, Val, Thr, or Phe is at position 436; Arg or Lys is at position 438; and Glu or Asp is at position 440; (e) According to the EU numbering system, Leu is at position 428; Ala is at position 434; Val or Thr is at position 436; Glu, Arg, Ser, or Lys is at position 438; and Glu, Asp, or Gln is at position 440; or (f) According to EU numbering, Leu is at position 428; Ala is at position 434; Val or Thr is at position 436; Arg or Lys is at position 438; and Glu or Asp is at position 440.

In one embodiment, it is disclosed that C relates to, for example, but not limited to: [54] An isolated anti-IL-8 antibody that binds to human IL-8, including at least 1 amino acid substitution at any position in (a) to (f) below, and binds in a pH-dependent manner In IL-8: (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2, including the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3, including the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1, including the amino acid sequence of SEQ ID NO:70; (e) HVR-L2, including the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3, including the amino acid sequence of SEQ ID NO:72. [55] The anti-IL-8 antibody of [54] includes: tyrosine at position 9 of the amino acid sequence of SEQ ID NO:68, tyrosine at position 11 of the amino acid sequence of SEQ ID NO:68 Amino acid substitution of arginine at position 3 and tyrosine at position 3 of the amino acid sequence of SEQ ID NO:69. [56] The anti-IL-8 antibody of [54] or [55] further includes alanine at position 6 of the amino acid sequence of SEQ ID NO:68 and the amino acid sequence of SEQ ID NO:68 The amino acid substitution of glycine at position 8. [57] The anti-IL-8 antibody according to any one of [54] to [56], including asparagine at position 1 of the amino acid sequence of SEQ ID NO:71, :Amino acid substitution of leucine at position 5 of the amino acid sequence of SEQ ID NO:71 and glutamine at position 1 of the amino acid sequence of SEQ ID NO:72. [58] The anti-IL-8 antibody according to any one of [54] to [57], including (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 67, (b) HVR-H2, including the amino acid sequence of SEQ ID NO: 73, and (c) HVR-H3, including the amino acid sequence of SEQ ID NO: : Amino acid sequence of 74. [59] The anti-IL-8 antibody according to any one of [54] to [58], including (a) HVR-L1, including the amino acid sequence of SEQ ID NO:70, (b) HVR-L2, including the amino acid sequence of SEQ ID NO:75, and (c) HVR-L3, including the amino acid sequence of SEQ ID NO:76. [60] The anti-IL-8 antibody according to any one of [54] to [59], including the heavy chain variable region of SEQ ID NO:78 and the light chain variable region of SEQ ID NO:79. [61] The anti-IL-8 antibody according to any one of [54] to [60], comprising an Fc region having at least one property selected from the following (a) to (f): (a) The binding affinity for FcRn under acidic pH conditions is increased relative to the binding affinity for FcRn of the native Fc region; (b) the binding affinity for pre-existing ADA is reduced relative to the binding affinity of the native Fc region for pre-existing ADA; (c) The plasma half-life is increased relative to the plasma half-life of the native Fc region; (d) The plasma clearance rate of the Fc region is reduced relative to the plasma clearance rate of the native Fc region; (e) The binding affinity of the Fc region for the effector receptor is reduced relative to the binding affinity of the native Fc region for the effector receptor; (f) Increased binding to extracellular matrix. [62] The anti-IL-8 antibody of [61], wherein the Fc region includes amino acid substitutions at one or more positions selected from the group consisting of: 235, 236, 239 according to EU numbering, 327, 330, 331, 428, 434, 436, 438 and 440. [63] The anti-IL-8 antibody of [62], which includes an Fc region, and the Fc region includes one or more amino acid substitutions selected from the following constituent groups: L235R, G236R, S239K, A327G, A330S , P331S, M428L, N434A, Y436T, Q438R and S440E. [64] The anti-IL-8 antibody of [63], wherein the Fc region includes the following amino acid substitutions: L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E. [65] The anti-IL-8 antibody of [63], wherein the Fc region includes the following amino acid substitutions: L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E. [66] An anti-IL-8 antibody, comprising a heavy chain of the amino acid sequence of SEQ ID NO:81, and a light chain of the amino acid sequence of SEQ ID NO:82. [67] An anti-IL-8 antibody, comprising a heavy chain of the amino acid sequence of SEQ ID NO:80, and a light chain of the amino acid sequence of SEQ ID NO:82. [68] An isolated nucleic acid encoding an anti-IL-8 antibody according to any one of [54] to [67]. [69] A vector comprising the nucleic acid of [68]. [70] A host cell including the vector of [69]. [71] A method of producing an anti-IL-8 antibody, comprising the step of culturing a host as in [70]. [72] The method of producing an anti-IL-8 antibody as described in [71] includes the step of isolating the antibody from the culture supernatant. [73] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of [54] to [67], and a pharmaceutically acceptable carrier. [74] The anti-IL-8 antibody according to any one of [54] to [67] is used in a pharmaceutical composition. [75] The use of the anti-IL-8 antibody according to any one of [54] to [67] is for the treatment of conditions in which excess IL-8 exists. [76] The use of the anti-IL-8 antibody according to any one of [54] to [67] is used in the manufacture of pharmaceutical compositions for conditions in which excess IL-8 exists. [77] A method of treating a patient suffering from a condition in which excess IL-8 is present, comprising the step of administering to the individual an anti-IL-8 antibody according to any one of [54] to [67]. [78] A method of promoting elimination of IL-8 from an individual, comprising the following steps: The subject is administered an anti-IL-8 antibody as in any one of [54] to [67]. [79] A pharmaceutical composition comprising the anti-IL-8 antibody according to any one of [54] to [67], which binds to IL-8 and binds to the extracellular matrix. [80] A method of producing an anti-IL-8 antibody, the antibody comprising: a variable region with pH-dependent IL-8 binding activity, comprising the following steps: (a) Assessing the binding of anti-IL-8 antibodies to the extracellular matrix, (b) Select anti-IL-8 antibodies that bind strongly to the extracellular matrix, (c) culture a host containing a vector containing nucleic acid encoding the antibody, and (d) Separate the antibody from the culture solution.

In an alternative embodiment, C is disclosed to be about: [C1] The use of an anti-IL-8 antibody as any one of [54] to [67] and [C26] to [C31], to manufacture a pharmaceutical combination for inhibiting the accumulation of biologically active IL-8 things. [C2] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for the purpose of inhibiting the accumulation of biologically active IL-8. [C3] The use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is to produce a pharmaceutical composition for inhibiting angiogenesis. [C4] The use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for the purpose of inhibiting angiogenesis. [C5] Use of an anti-IL-8 antibody as any one of [54] to [67] and [C26] to [C31] to produce a pharmaceutical composition for inhibiting the promotion of neutrophil migration . [C6] Use of an anti-IL-8 antibody as any one of [54] to [67] and [C26] to [C31] for inhibiting the promotion of neutrophil migration. [C7] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for inhibiting the accumulation of biologically active IL-8. [C8] A method of inhibiting the accumulation of biologically active IL-8 by administering to an individual an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31]. [C9] A pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, including an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] . [C10] A use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for inhibiting angiogenesis. [C11] A method of inhibiting angiogenesis in an individual, comprising the following steps: An anti-IL-8 antibody such as any one of [54] to [67] and [C26] to [C31] is administered to the subject. [C12] A pharmaceutical composition for inhibiting angiogenesis, including an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31]. [C13] The anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] is used to inhibit the promotion of neutrophil migration. [C14] A method for inhibiting neutrophil migration promotion in an individual, comprising administering to the individual an anti-IL according to any one of [54] to [67] and [C26] to [C31] -8 Antibody steps. [C15] A pharmaceutical composition for inhibiting the promotion of neutrophil migration in an individual, including an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] . [C16] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is used to treat diseases in which IL-8 is excessive. [C17] The use of an anti-IL-8 antibody as any one of [54] to [67] and [C26] to [C31], for the manufacture of a medicine for treating a disease in which IL-8 is excessive composition. [C18] A use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for treating a condition in which IL-8 is excessive. [C19] A method of treating a condition in which IL-8 is excessive in an individual, comprising the steps of: administering to the individual anti-IL as any one of [54] to [67] and [C26] to [C31] -8 antibodies. [C20] A pharmaceutical composition for treating a condition in which IL-8 is excessive in an individual, including an anti-IL-8 according to any one of [54] to [67] and [C26] to [C31] antibody. [C21] The anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] is used to promote the elimination of IL-8. [C22] The use of an anti-IL-8 antibody as any one of [54] to [67] and [C26] to [C31], which is a pharmaceutical composition for treating IL-8 that promotes elimination. [C23] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for the purpose of promoting the elimination of IL-8. [C24] A method of promoting elimination of IL-8 in an individual, comprising the following steps: An anti-IL-8 antibody such as any one of [54] to [67] and [C26] to [C31] is administered to the individual. [C25] A pharmaceutical composition for promoting the elimination of IL-8, comprising the following steps: An anti-IL-8 antibody such as any one of [54] to [67] and [C26] to [C31] is administered to the individual. [C26] An anti-IL-8 antibody, comprising an Fc region, the Fc region comprising amino acid substitutions at one or more positions selected from the group consisting of: in accordance with EU numbering 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440. [C27] The anti-IL-8 antibody of [C26], which includes an Fc region, and the Fc region has at least one property selected from the following (a) to (f): (a) The binding affinity for FcRn under acidic pH conditions is increased relative to the binding affinity for FcRn of the native Fc region; (b) the binding affinity for pre-existing ADA is reduced relative to the binding affinity of the native Fc region for pre-existing ADA; (c) The plasma half-life is increased relative to the plasma half-life of the native Fc region; (d) The plasma clearance rate of the Fc region is reduced relative to the plasma clearance rate of the native Fc region; (e) The binding affinity of the Fc region for the effector receptor is reduced relative to the binding affinity of the native Fc region for the effector receptor; and (f) Increased binding to extracellular matrix. [C28] An anti-IL-8 antibody such as [C26] or [C27], comprising an Fc region, the 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 the EU numbering method. [C29] The anti-IL-8 antibody of [C28], comprising an Fc region, the Fc region comprising one or more amino acid substitutions selected from the group consisting of: (A) According to the EU number method L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E; or (B) L235R, G236R, A327G, A330S, P331S, M428L, N436T, Q438R, S44, S44, 0E. [C30] The anti-IL-8 antibody of [C26], which includes: a heavy chain containing the amino acid sequence of SEQ ID NO:81 and a light chain containing the amino acid sequence of SEQ ID NO:82. [C31] The anti-IL-8 antibody of [C26], which includes: a heavy chain containing the amino acid sequence of SEQ ID NO:80 and a light chain containing the amino acid sequence of SEQ ID NO:82. [C32] An isolated nucleic acid encoding an anti-IL-8 antibody as any one of [C26] to [C31]. [C33] A vector including a nucleic acid such as [C32]. [C34] A host cell including a vector such as [C33]. [C35] A method of producing anti-IL-8 antibodies, comprising culturing host cells such as [C34]. [C36] A method of producing an anti-IL-8 antibody according to any one of [C26] to [C31], further comprising the step of isolating the antibody from a host cell culture. [C37] A pharmaceutical composition, including: an anti-IL-8 antibody according to any one of [C26] to [C31], and a pharmaceutically acceptable carrier. [C38] A method of treating a patient suffering from a condition in which excess IL-8 is present, comprising the step of administering to the subject an anti-IL-8 antibody of any one of [C26] to [C31]. [C39] A method of promoting elimination of IL-8 from an individual, comprising the step of administering to the individual an anti-IL-8 antibody according to any one of [C26] to [C31]. [C40] A method of inhibiting IL-8, wherein the method includes contacting the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] with IL-8. [C41] The method of inhibiting IL-8 as in [C40], wherein the method inhibits the biological activity of IL-8.

According to various embodiments, disclosure C includes any of one or more elements described in the above [54] to [80], [C1] to [C39] as long as such combinations are not technically inconsistent with ordinary technical knowledge in the technical field. Part or whole of a combination.

Non-limiting embodiments disclosing A, B or C are described below. All implementations described in the following examples are intended to be understood correctly and are not limited to any patent practices, regulations, rules, or narrow interpretations of the contents described in the examples in the country where the patent rights for this application are sought.

reveal A or reveal B In some embodiments, disclosure A relates to an antibody, including an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, and whose isoelectric point (pI) is determined by at least 1 amino acid residue that may be exposed on the surface of the antibody. The increase (in the context of disclosure A, is also called "an ion concentration-dependent antibody with an increased isoelectric point value"; and the antigen-binding domain of this antibody is also called an "ion concentration-dependent antigen-binding domain with an increased isoelectric point value" "). The present invention is based in part on the inventors' unexpected discovery that antigen elimination from plasma can be facilitated by an ion concentration-dependent antibody whose isoelectric point (pI) is modified by at least one amino acid that may be exposed on the antibody surface. residues (e.g., when the antibody is administered in vivo); and the binding of the antibody to the extracellular matrix can be increased in an ion concentration-dependent manner by increasing (increasing) the isoelectric point value of the antibody. The present invention is also partly based on the inventor's unexpected discovery that this beneficial effect is brought about by combining two completely different concepts: ion concentration-dependent antigen-binding domain or ion concentration-dependent antibody; and isoelectric point (pI ) has been increased by modification of at least 1 amino acid residue that may be exposed on the surface of the antibody (also referred to as "an antibody with an increased isoelectric point value" within the context of Disclosure A; and the isoelectric point (pI) is Antibodies that are reduced (lowered) by modifying at least one amino acid residue that may be exposed on the surface of the antibody (also referred to as "antibodies with reduced isoelectric point values" within the scope of Disclosure A. Therefore, the invention falling within Disclosure A Classified as a pioneering invention that can lead to significant technological innovation in this technical field (such as the medical field).

Of course, for example, an antibody includes an antigen-binding domain and its isoelectric point is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody, and is further modified such that the antigen-binding activity of the antigen-binding domain depends on ion concentration conditions. The modification is also included in the scope of Disclosure A described here (herein, within the scope of Disclosure A, such an antibody is also called an "ion concentration-dependent antibody with an increased isoelectric point value").

Of course, for example, an antibody includes an ion concentration-dependent antigen-binding domain, and the charge of at least one amino acid residue that may be exposed on the surface of the antibody is different from that before the antibody is modified (natural antibodies (such as natural Ig antibodies, preferably It is at least 1 amino acid residue at the corresponding position of a natural IgG antibody), or a control or parent antibody (such as before antibody modification or before antibody library construction or during construction), and its net antibody pI increases, and Included are also included in Disclosure A herein (within the scope of Disclosure A described herein, such antibodies are also referred to as "ion concentration-dependent antibodies with increased isoelectric point values").

Of course, for example, an antibody includes an ion concentration-dependent antigen-binding domain, and its isoelectric point is higher than before modification of the antibody by modifying at least one amino acid residue that may be exposed on the surface of the antibody (natural antibodies (such as natural Ig) Antibodies, preferably natural IgG antibodies or control or parent antibodies (e.g., before antibody modification or before antibody library construction or during construction), are also included in Disclosure A (such antibodies are also referred to as "antibodies with increased isoelectric point values" Ion concentration-dependent antibodies" are within the scope of Disclosure A described herein).

Of course, for example, an antibody contains an ion concentration-dependent antigen-binding domain, in which at least one amino acid residue that may be exposed on the surface of the antibody is modified in order to increase the isoelectric point value of the antibody. This antibody is also included in Disclosure A (such as Antibodies are also called "ion concentration-dependent antibodies with increased isoelectric point values" within the scope of Disclosure A described herein).

Within the scope of disclosures A and B of this description, "amino acids" include not only natural but also non-natural amino acids. To the extent that A and B are disclosed herein, an amino acid or amino acid residue may be expressed by a single letter (eg, A) or a three-letter code (eg, Ala), or both (eg, Ala(A)).

When used in the context of revealing A and B, "modified amino acid", "modified amino acid residue" or equivalent terms can be understood as, but are not limited to, chemically modifying one molecule to one of the antibody amino acid sequences or Multiple (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) specific amino acids (residues), or addition, deletion, or substitution to the antibody amino acid sequence Or insert one or more (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) amino acids. Amino acid additions, deletions, substitutions or insertions can be performed on nucleic acids encoding amino acid sequences, for example using site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 ( 1985)) or overlap extension PCR; via affinity maturation of antibodies, or using chain drag of antibody heavy or light chains; or utilizing antigen panning-based selection using phage libraries (Smith et al., Method Enzymol. 217: 228-257 (1993)); and these alone or in appropriate combination. Such amino acid modification is not limited but is preferably by substituting one or more amino acid residues of the antibody amino acid sequence with different amino acids (individually). Amino acid addition, deletion, substitution or insertion and modification of amino acid sequences using humanization or chimerization can be implemented using methods known in the technical field. Transformation or modification of amino acids (residues), such as amino acid addition, deletion, substitution, or insertion, can also be performed on the antibody variable region or the antibody constant region of the antibody used to prepare the recombinant antibody used to disclose A or B.

In one embodiment, within the context of disclosures A and B described herein, a substituted amino acid (residue) refers to a substitution to a different amino acid (residue), which may be designed to modify, for example, (a) to (c) ) matters: (a) The structure of the polypeptide backbone within the patch or helical configuration; (b) The charge or hydrophobicity of the target site; or (c) The size of the side chain.

Amino acid residues are classified into the following groups according to the nature of the side chains in the structure: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu and Ile; (2) Neutral and hydrophilic: Cys , Ser, Thr, Asn and Gln; (3) Acidic: Asp and Glu; (4) Basic: His, Lys and Arg; (5) Residues that affect chain orientation: Gly and Pro; and (6) Aromatic Sex: Trp, Tyr and Phe.

The substitution of amino acid residues within the same group is called conservative substitution, and the substitution of amino acid residues between different groups is called non-conservative substitution. Amino acid residue substitutions may be conservative substitutions, non-conservative substitutions, or combinations thereof. There are several suitable methods for substituting amino acids with amino acids other than natural amino acids. (Wang et al., Annu. Rev. Biophys. Biomol. Struct. 35:225-249 (2006); Forster et al., Proc. Natl. Acad. Sci. USA 100(11):6353-6357 (2003) ). For example, a cell-free translation system may be used, including tRNA in which the unnatural amino acid is linked to an amber suppressor tRNA (Clover Direct (Protein Express)) complementary to the UAG codon (amber codon) which is the stop codon.

Within the context of disclosures A and B described herein, it should be understood that the "antigen" structure is not limited to a specific structure, as long as the antigen includes an epitope that will bind to the antibody. The antigen can be an inorganic or organic substance. The antigen can be any ligand, including various interleukins, such as interleukins, chemokines and cell growth factors. Alternatively, it is of course possible to use as antigen a receptor that is present in a soluble form or has been modified to be soluble in biological fluids such as plasma. Non-limiting examples of such soluble receptors include soluble IL-6 receptors, described in Mullberg et al., J. Immunol. 152(10):4958-4968 (1994). Antigens may be monovalent (eg, soluble IL-6 receptor) or polyvalent (eg, IgE).

In one embodiment, it is disclosed that the antigens that the antibodies A and B can bind to are preferably present in the biological fluid of the subject (for example, the biological fluid exemplified in WO2013/125667 is preferably plasma, interstitial fluid, lymph fluid, ascites, or pleural fluid) Soluble antigen (within the scope of disclosures A and B described here, the object to be administered (administered) to the antibody can be any animal such as humans, mice, etc.); however, the antigen can also be a membrane antigen.

Within the scope of disclosures A and B of this document, the term “half-life prolongation in plasma” or “half-life shortening in plasma” or equivalent terms of the target molecule (which may be an antigen or an antibody) may also be referred to as “half-life prolongation in plasma” or “half-life reduction in plasma”. In addition to the parameter expression of half-life (t1/2), it can be expressed by any other parameters, such as the mean residence time in plasma, clearance rate (CL) in plasma, and area under the concentration curve (AUC) (Pharmacokinetics: Enshuniyoru Rikai (Understanding through practice) Nanzando). These parameters can be specifically evaluated by performing non-compartmentalized analysis according to the experimental steps provided with the in vivo kinetic analysis software WinNonlin (Pharsight). It is known to those of ordinary skill in the art that these parameters are normally related to each other.

Within the context of Disclosures A and B described here, "antigenic determinant" refers to an antigenic determinant in an antigen, and refers to the site on the antigen to which the antigen-binding domain of an antibody binds. Thus epitopes can be defined, for example, structurally. Alternatively, an epitope may be defined by the antigen-binding activity of an antibody that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope may be designated by the amino acid residues that make up the epitope. Or when the epitope is a sugar chain, the epitope can be designated based on its specific sugar chain structure. It is revealed that the antigen-binding domains of A and B can bind to a single epitope or different epitopes on the antigen.

A linear epitope may be a primary amino acid sequence. Such linear epitopes generally include at least 3, and usually at least 5, such as 8 to 10 amino acids or 6 to 20 amino acids as unique sequences.

In the configuration of the epitope, generally the amino acids that make up the epitope do not exist continuously in primary sequence. Antibodies recognize the epitope configuration of the three-dimensional structure of a peptide or protein. Methods for determining epitope configuration include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance (Epitope Mapping Protocols in Methods in Molecular Biology (1996) ), Vol. 66, Morris (ed.)).

Within the scope of disclosures A and B described here, "antibody" is not particularly limited and is used in the broadest sense as long as it binds to the target antigen. Non-limiting examples of antibodies include widely known general antibodies (such as natural immunoglobulins ("Ig")), and molecules and variants derived therefrom, such as Fab, Fab', F(ab') ₂ , diabody, ScFv (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); EP404,097; WO93/11161; Peer et al., Nature Nanotechnology 2:751-760 (2007)), low molecular weight antibodies (minibodies) (Orita et al., Blood 105:562-566 (2005)), scaffold proteins, single-arm antibodies (including all embodiments of single-arm antibodies described in WO2005/063816), Multispecific antibodies (such as bispecific antibodies: antibodies that are specific for two different epitopes, including antibodies that recognize different antigens and antibodies that recognize different epitopes on the same antigen). Within the scope of disclosures A and B described here, the "bispecific antibody" is not limited, but it can be prepared as an antibody molecule having a common L chain as described in WO2005/035756, or using the method described in WO2008/119353 to combine 2 A general type of antibody with an IgG4-like constant region is mixed, resulting in an exchange between the two types of antibodies (known to those of ordinary skill in the art as a "Fab-arm exchange" method). In an alternative embodiment, the antibody may have the following structure: the heavy chain variable region and the light chain variable region are linked together to form a single chain (eg, sc(Fv) ₂ ). Alternatively it can be an antibody-like molecule (e.g. scFv-Fc), obtained by linking the Fc region (constant region lacking the CH1 domain) to scFv (or sc(Fv) ₂ ), with the heavy chain variable region (VH) linked In the light chain variable region (VL). A multispecific antibody composed of scFv-Fc having a (scFv) ₂ -Fc structure, and the first and second polypeptides are VH1-linker-VL1-Fc and VH2-linker-VL2-Fc respectively. Or it can be an antibody-like molecule, where a single domain antibody is linked to the Fc region (Marvin et al., Curr. Opin. Drug Discov. Devel. 9(2):184-193 (2006)), an Fc fusion protein (eg, immunoadhesin ) (US2013/0171138), its functional fragments, functionally equivalent substances, and sugar chain modified variants thereof. Here, natural IgG (for example, natural IgG1) refers to a polypeptide with the same amino acid sequence as a naturally occurring IgG (for example, natural IgG1), and belongs to the class of antibodies essentially encoded by immunoglobulin gamma genes. Natural IgG can be its spontaneous mutants, etc.

Generally, if the antibody structure is substantially the same or similar to natural IgG, its basic structure may be a Y-shaped structure with 4 chains (2 heavy chain polypeptides and 2 light chain polypeptides). Generally, heavy chains and light chains can be linked together through disulfide bonds (SS bonds) to form heterodimers. Such heterodyads can be linked together via disulfide bonds to form a Y-shaped heteroquaternary body. The two heavy or light chains may be the same or different from each other.

For example, an IgG antibody can be digested with papain and cleaved into a 2-unit Fab (region) and a 1-unit Fc (region). Papain will cleave the hinge region (also referred to as the hinge region in the context of disclosures A and B described here). "hinge"), the Fab region of the heavy chain is connected to the Fc region. Typically, the Fab region contains an antigen-binding domain. Phagocytic cells such as white blood cells and macrophages have receptors that bind to the Fc region (Fc receptor) and can recognize and phagocytose the antigen through Fc receptor antibodies that have bound to an antigen (opsonization) . At the same time, the Fc region is involved in mediating immune responses, such as ADCC or CDC, and has effector function, that is, inducing a response when the antibody binds to the antigen. This antibody effector function is known to vary depending on the immunoglobulin (structural isotype) type. The Fc region of the IgG class can refer to, for example, the interval from cysteine 226 or proline 230 (EU numbering method) to the C-terminus; but the Fc region is not limited thereto. The Fc region can be appropriately obtained by partially digesting the monoclonal antibody IgG1, IgG2, IgG3 or IgG4 antibody or others with a protease such as pepsin, and then extracting the adsorbed fraction from a protein A or protein G column.

Within the scope of disclosures A and B described here, the positions of amino acid residues in the variable region (CDR and/or FR) of the antibody are as shown in Kabat, and the positions of amino acid residues in the constant region or Fc region are as shown in Kabat. Amino acid positions are expressed according to EU numbering based on Kabat's (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)).

Within the context of disclosures A and B described herein, "library" refers to a sequence-variable molecule (population) such as multiple antibodies, the sequences of which may be the same as or different from each other; multiple fusion polypeptides containing such antibodies; or encoding Nucleic acids or polynucleotides for these amino acid sequences are described in detail in WO2013/125667 (eg paragraphs 0121-0125). This library may, for example, contain at least 10 ⁴ antibody molecules, preferably at least 10 ⁵ antibody molecules, more preferably at least 10 ⁶ antibody molecules, particularly preferably at least 10 ⁷ antibody molecules or more. The library may be a phage library. The term "consisting essentially of" means that the antibody may have different antigen-binding activities, corresponding to portions of a plurality of independent clones of different sequences in the library. In one embodiment, the antibody can be derived from animals that have been immunized with specific antigens, infected patients, people whose blood antibody levels have increased due to vaccination, or lymphocytes suffering from cancer or autoimmune diseases. The immune library constructed from antibody genes can be appropriately used as a random variable region library. In an alternative embodiment, an unaltered library containing unaltered sequences (unbiased library antibody sequences) constructed from antibody genes derived from healthy human lymphocytes may be appropriately used as a random variable region library (Gejima et al. ., Human antibody 11:121-129 (2002)); Cardoso et al., Scand. J. Immunol. 51:337-344 (2000)). An amino acid sequence containing an unchanged sequence may refer to one obtained from an unchanged library. In an alternative embodiment, a synthetic library of a set of synthetic oligonucleotides containing sequences encoding a codon of appropriate lengths may be substituted for the V gene from genomic DNA or the CDR sequences for reconstituting a functional V gene as appropriate. Random variable zone library. In this case, sequence variation is also observed in the CDR3 gene, and only the heavy chain CDR3 sequence may be replaced. A standard approach to generating amino acid diversity in the variable regions of such antibodies is to increase variation in the positions of amino acid residues that may be exposed on the antibody surface.

In one embodiment, if the antibody disclosed in A or B has a structure that is substantially identical or similar to a natural Ig antibody, it generally has a variable region ("V region") [heavy chain variable region ("VH region") and light chain variable region ("VL region")] and constant region ("C region") ["heavy chain constant region ("CH region") and light chain constant region ("CL region")]. The CH region is further divided into 3: CH1 to CH3. Generally, the Fab region of the heavy chain contains the VH region and CH1, and the Fc region of the heavy chain generally contains CH2 and CH3. Generally, the hinge region is between CH1 and CH2. The variable regions are generally complementary. Decision region ("CDR") and framework region ("FR"). Generally, the VH region and the VL region each have 3 CDRs (CDR1, CDR2, and CDR3) and 4 FRs (FR1, FR2, FR3, and FR4). Generally, the 6 CDRs in the variable regions of the heavy and light chains interact and form the antigen-binding domain of the antibody. On the other hand, if there is only a single CDR, the antigen-binding affinity is known to be lower than when there are 6 CDRs, but Still have the ability to recognize and bind to antigens.

Ig antibodies are divided into several classes (structural isotypes) based on structural differences in their constant regions. In many mammals, there are five types of immunoglobulins classified based on structural differences in their constant regions: IgG, IgA, IgM, IgD and IgE. Also, in the case of humans, there are 4 subclasses of IgG: IgG1, IgG2, IgG3, and IgG4; and 2 subclasses of IgA: IgA1 and IgA2. Heavy chains are divided into gamma chain, mu chain, alpha chain, delta chain and epsilon chain based on differences in constant regions. Based on these differences, there are 5 immunoglobulin classes (structural isotypes): IgG, IgM, IgA, IgD and IgE. . On the other hand, there are 2 types of light chains: lambda and kappa, and all immunoglobulins have one of these two.

In one embodiment, if it is disclosed that the antibody of A or B has a heavy chain, for example, the heavy chain can be any one of gamma chain, mu chain, alpha chain, delta chain and epsilon chain or can be derived from any one, if it is disclosed that The antibody of A or B has a light chain. For example, the light chain can be a kappa chain or a lambda chain, or derived from one of them. Also within the scope of disclosures A and B described herein, the antibody may be of any structural isotype (e.g., IgG, IgM, IgA, IgD, or IgE) and any subclass (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2; mouse IgG1, IgG2a, IgG2b and IgG3) or derived from any one, but is not limited thereto.

Within the scope of disclosures A and B described here, the "antigen-binding domain" can be any structure as long as it binds to the antigen of interest. Such domains may include, for example, the variable regions of antibody heavy and light chains (e.g., 1 to 6 CDRs); a module of about 35 amino acids called the A domain, which is included in Avimer, which is found in cell membranes in the body Protein (WO2004/044011 and WO2005/040229); Adnectin, which contains a 10Fn3 domain and binds to the protein in the glycoprotein fibronectin expressed on the cell membrane (WO2002/032925); Affibody, which has 58 amine groups as constituent protein A A scaffold for the IgG binding domain of the 3-helix bundle of acid (WO1995/001937); the designed Ankyrin Repeat Protein (DARPin), which is a region exposed on the surface of the Ankyrin repeat (AR) molecule, has the following Structure: primary unit with a turn including 33 amino acid residues, 2 antiparallel helices, and repeatedly stacked loops (WO2002/020565); Anticalin et al., which is a 4 loop region supporting 8 One side of the central torsion barrel structure of the antiparallel strands is highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003/029462); and by Ma A hollow region formed by a parallel plate structure within a shoe-like structure formed by the stacked repeats of the leucine-rich repeat (LRR) module of the variable lymphocyte receptor (VLR), which does not possess immunoglobulins The structure is used as an acquired immune system in jawless vertebrates such as lampreys and hagfishes (WO2008/016854). It is revealed that the A or B antigen binding domain should include those with IgG antibody heavy chain and light chain variable regions, more specifically ScFv, single chain antibody, Fv, scFv ₂ (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. The "metal ion" here can be any Group I element 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 except boron, IV elements except carbon and silicon. Elements, Group VIII elements, such as the iron and platinum group elements, elements belonging to subgroup A of groups V, VI and VII, and ions of metallic elements such as antimony, bismuth and polonium. Metal atoms have the property of releasing electrons and becoming cations. Called dissociative tendency. Metals with strong free hydrogenation are presumed to be chemically active.

In one embodiment of Disclosure A, the metal ion is preferably calcium ion, as detailed in WO2012/073992 and WO2013/125667.

In one embodiment of Disclosure A, the "ion concentration condition" may focus on the difference in biological behavior of the ion concentration-dependent antibody between low ion concentration and high ion concentration. Furthermore, "its antigen-binding activity changes according to ion concentration conditions" can refer to revealing that the ion concentration-dependent antigen-binding domain of A or B or the antigen-binding activity of the ion concentration-dependent antibody will change between low ion concentration and high ion concentration. Such situations include, for example, but are not limited to, higher (stronger) or lower (weaker) antigen-binding activity at high ion concentrations than at low ion concentrations.

In one embodiment of Disclosure A, the ion concentration may be hydrogen ion concentration (pH) or calcium ion concentration. When the ion concentration is hydrogen ion concentration (pH), the ion concentration-dependent antigen-binding domain may also be called "pH-dependent antigen-binding domain"; and when the ion concentration is calcium ion concentration, it may also be called "calcium ion concentration-dependent Antigen binding domain".

In one embodiment in the context of disclosure A, an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with an increased isoelectric point value, and an ion concentration-dependent antibody with an increased isoelectric point value , can be obtained from a library that mainly includes antibodies with different sequences (variability). The antigen-binding domain of the antibody contains at least one amino acid residue that causes the antigen-binding activity of the antigen-binding domain or the antibody to change according to ion concentration conditions. base. The antigen binding domain may suitably be located within the light chain variable region (which may be modified) and/or the heavy chain variable region (which may be modified). In order to construct a library, such a light chain or heavy chain variable region can be combined with a heavy chain or light chain variable region that has been constructed into a random variable region sequence library. If the ion concentration is a hydrogen or calcium ion concentration, non-limiting examples of the library include, for example: a heavy chain variable region that has been constructed as a random variable region sequence library, and a library that combines light chain variable region sequences, wherein the light chain variable region The amino acid residues of the germline sequence of the region sequence, such as SEQ ID NO:1 (Vk1), SEQ ID NO:2 (Vk2), SEQ ID NO:3 (Vk3) or SEQ ID NO:4 (Vk4), are replaced by At least 1 amino acid residue that changes antigen-binding activity depending on ion concentration. And if the ion concentration is calcium ion concentration, the library includes, for example, the heavy chain variable region sequence of SEQ ID NO:5 (6RL#9-IgG1) or SEQ ID NO:6 (6KC4-1#85-IgG1) and has been constructed as A light chain variable region library of random variable region sequences or a library of light chain variable region combinations with germline sequences.

In one embodiment, if the ion concentration is calcium ion concentration, the high calcium ion concentration is not particularly limited to a specific value; but the concentration can be selected from the group consisting of between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM, or between 400 μM and 1 mM. It is suitable to select a concentration between 500 μM and 2.5 mM, which is close to the plasma (blood) concentration of calcium ions in vivo. The low calcium ion concentration is not particularly limited to a specific value; but the concentration can be selected from between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, or between 1 μM and 5 μM, or Between 2 μM and 4 μM. It is suitable to select a concentration between 1 μM and 5 μM, which is close to the calcium ion concentration in early endosomes.

Whether the antigen-binding activity of the antigen-binding domain or the antibody containing this domain will change depending on the metal ion concentration (such as calcium ion concentration) can be easily determined by known methods, such as using the method described in Disclosure A, or the method described in WO2012/073992 method. For example, the antigen-binding activity of the antigen-binding domain or an antibody containing this domain can be determined and compared at low and high calcium ion concentrations. In this case, conditions other than calcium ion concentration should be the same. In addition, conditions other than the calcium ion concentration for determining the antigen-binding activity can be appropriately selected by those skilled in the art. This antigen-binding activity can be determined, for example, at 37°C in HEPES buffer conditions, or using BIACORE (GE Healthcare) or other equipment.

In one embodiment in the context of disclosure A, an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with an increased isoelectric point value, or an ion concentration-dependent antibody with an increased isoelectric point value. The antigen-binding activity is higher under high calcium ion concentration conditions than under low calcium ion concentration conditions. In this case, the ratio of the antigen-binding activity under low calcium ion concentration conditions to the antigen-binding activity under high calcium ion concentration conditions is not limited; but the KD (dissociation constant) of the antigen under low calcium ion concentration conditions is relative to the high calcium ion concentration The ratio of KD of the conditions, that is, KD (3 μM Ca)/KD (2 mM Ca), is preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. The upper limit of KD (3 μM Ca)/KD (2 mM Ca) is not limited and can be any value, such as 400, 1000 or 10000.

If the antigen is a soluble antigen, the dissociation constant (KD) can be used as the value of the antigen-binding activity. If the antigen is a membrane antigen, the apparent dissociation constant (KD) can be used. The dissociation constant (KD) and apparent dissociation constant (KD) can be determined by known methods, such as BIACORE (GE healthcare), Scatchard plot or flow cytometer.

Alternatively, for example, the dissociation rate constant (kd) can be used as another indicator to represent the binding activity ratio. If the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator of the antigen-binding activity ratio, the ratio of the dissociation rate constant (kd) under low calcium ion concentration conditions to the dissociation rate constant (kd) under high calcium ion concentration conditions , that is, kd (low calcium ion concentration conditions)/kd (high calcium ion concentration conditions) is preferably 2 or more, more preferably 5 or more, more preferably 10 or more, more preferably 30 or more . The upper limit of kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) is not limited and can be any value, such as 50, 100 or 200.

If the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as the value of the antigen-binding activity. If the antigen is a membrane antigen, the apparent dissociation rate constant (kd) can be used. The dissociation rate constant (kd) and apparent dissociation rate constant (kd) can be determined using known methods, such as BIACORE (GE healthcare) or a flow cytometer.

In one embodiment, the method for producing or screening calcium ion concentration-dependent antigen-binding domains or calcium ion concentration-dependent antibodies or libraries thereof whose antigen-binding activity is higher under high calcium ion concentration conditions than under low calcium ion concentration conditions is not limited. . Methods include, for example, those described in WO2012/073992 (eg, paragraphs 0200-0213).

This method may include, for example: (a) Determine the antigen-binding activity of the antigen-binding domain or antibody under low calcium ion concentration conditions; (b) Determine the antigen-binding activity of the antigen-binding domain or antibody under conditions of high calcium ion concentration; and (c) Select an antigen-binding domain or antibody whose antigen-binding activity under low calcium ion concentration conditions is lower than that under high calcium ion concentration conditions.

Or this method may include, for example: (a) Contact the antigen and the antigen-binding domain or antibody or library thereof under conditions of high calcium ion concentration; (b) incubate the antigen-binding domain or antibody bound to the antigen in step (a) under conditions of low calcium ion concentration; and (c) Isolating the antigen-binding domain or antibody dissociated in step (b).

Or this method may include, for example: (a) contacting an antigen with an antigen-binding domain or antibody or pool thereof under conditions of low calcium ion concentration; (b) Select an antigen-binding domain or antibody that does not bind to the antigen or has low antigen-binding ability in step (a); (c) Allow the antigen-binding domain or antibody selected in step (b) to bind to the 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).

Or this method may include, for example: (a) Contact the antigen-binding domain or antibody or its library with the column on which the antigen has been immobilized under conditions of high calcium ion concentration; (b) Elute the antigen-binding domain or antibody bound to the column in step (a) from the column under conditions of low calcium ion concentration; and (c) Isolate the antigen-binding domain or antibody eluted in step (b).

Or this method may include, for example: (a) Pass the antigen-binding domain or antibody or its library through a column with immobilized antigen under low calcium ion concentration conditions to collect the eluted antigen-binding domain or antibody that is not bound to the column; (b) causing the antigen-binding domain or antibody collected in step (a) to bind to this antigen under high calcium ion concentration conditions; and (c) Isolating the antigen-binding domain or antibody that bound the antigen in step (b).

Or this method may include, for example: (a) Contact the antigen with the antigen-binding domain or antibody or library thereof under conditions of high calcium ion concentration; (b) Obtaining the antigen-binding domain or antibody that binds to the antigen in step (a); (c) Cultivate the antigen-binding domain or antibody obtained in step (b) at a low calcium ion concentration; and (d) Isolated antigen-binding domains or antibodies whose antigen-binding activity in step (c) is weaker than the criteria selected in step (b).

Each step of these various screening methods can be repeated several times or the steps can be combined appropriately to obtain the optimal molecule. The above conditions can be appropriately selected for low and high calcium ion concentration conditions. Therefore, an ideal calcium ion concentration-dependent antigen-binding domain or calcium ion concentration-dependent antibody can be obtained.

In the context of Disclosure A, in one embodiment, the antigen-binding domain or antibody used as the starting material may be a modified antigen-binding domain or antibody by modifying at least 1 amino acid residue that may be exposed on its surface. The charge increases the isoelectric point value. In an alternative embodiment, if an amino acid that modifies the ion concentration-dependent binding activity of an antigen-binding domain is introduced into the sequence, at least one amino acid residue can and may be exposed on the surface of the antigen-binding domain or antibody. Charge modifications are imported together to increase the isoelectric point value.

Or in the context of this disclosure, for example, pre-existing antigen-binding domains or antibodies, pre-existing libraries (phage libraries, etc.); antibodies prepared from fusion tumors obtained by immunizing animals, or B cells derived from immunized animals; or Libraries thereof; or antigen-binding domains, antibodies, or libraries obtained by introducing mutations in natural or non-natural amino acids capable of chelating calcium (described below) (e.g., libraries with increased calcium-chelating amino acids, Or a library of calcium-chelable amino acids has been introduced into a specific site).

In one embodiment in the context of disclosure A, if the ion concentration is a calcium ion concentration, the type of amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain or the ion concentration-dependent antigen-binding domain that increases the isoelectric point value is not It is limited as long as it can form a calcium-binding motif. For example, calcium-binding motifs are known to those of ordinary skill in the art (eg Springer et al. (Cell 102:275-277 (2000)); Kawasaki et al. (Protein Prof. 2:305-490 (1995))) ; Moncrief et al. (J. Mol. Evol. 30:522-562 (1990)); Chauvaux et al. (Biochem. J. 265:261-265 (1990)); Bairoch et al. (FEBS Lett. 269 :454-456 (1990)); Davis (New Biol. 2:410-419 (1990)); Schaefer et al. (Genomics 25:638-643 (1995)); Economou et al. (EMBO J. 9: 349-354 (1990)); Wurzburg et al. (Structure. 14(6):1049-1058 (2006)). Therefore, if the antigen-binding domain is a random calcium-binding motif, such as a C-type lectin, such as ASGPR, The antigen-binding activity of CD23, MBR or DC-SIGN domains can be changed according to calcium ion concentration conditions. Such calcium-binding motifs can include, for example, in addition to the above, SEQ ID NO:7 (corresponding to "Vk5-2"). The calcium-binding motif contained in the antigen-binding domain is shown.

In one embodiment in the context of Disclosure A, if the ion concentration is a calcium ion concentration, an amino acid with metal chelating activity can be used as a modified ion concentration-dependent antigen-binding domain or ion concentration-dependent having an increased isoelectric point value Amino acids with binding activity in the sexual antigen-binding domain. For example, any amino acid can be appropriately used as the amino acid with metal chelating activity as long as it can form a calcium-binding motif. Specifically, such amino acids include those with electron donating properties. Amino acids preferably include, but are not limited to, Ser (S), Thr (T), Asn (N), Gln (Q), Asp (D) and Glu (E).

Amino acids with metal chelating activity are not limited to specific positions in the antigen-binding domain. In one embodiment, the amino acid can be located at any position in the heavy chain variable region and/or light chain variable region capable of forming an antigen-binding domain. It may include at least 1 amino acid residue based on the heavy chain and/or light chain that causes a change in the calcium ion concentration-dependent antigen-binding activity of the antibody, such as CDR (one or more of CDR1, CDR2, and CDR3) and/or FR (one or more of FR1, FR2, FR3 and FR4). This amino acid residue can be placed at one or more positions of positions 95, 96, 100a and 101 of the heavy chain CDR3 according to the Kabat numbering system; and at positions 30, 31 and 32 of the light chain CDR1 according to the Kabat numbering system. One or more positions; position 50 according to Kabat numbering of light chain CDR2; and/or position 92 according to Kabat numbering of light chain CDR3. Such amino acid residues may be placed individually or in combination.

Troponin C (Troponin C), calmodulin (calmodulin), parvalbumin (parvalbumin), myosin (myosin) light chain, etc. are known to have multiple calcium-binding sites. It is speculated that molecular evolution comes from a common origin. In one embodiment, one or more of the light chain CDR1, CDR2, and CDR3 can be designed to include its binding motif. For the above purpose, for example, cadherin domain; calmodulin-containing EF hand; protein kinase C-containing C2 domain; coagulation protein factor IX-containing Gla domain; asialoglycoprotein receptor or manna can be appropriately used Sugar-binding receptor C-type lectin; LDL receptor containing A domain; Annexin; thrombospondin type-3 domain; and EGF-like domain.

In one embodiment, if the ion concentration is hydrogen ion concentration (pH), protons, that is, the concentration condition of hydrogen nuclei, and hydrogen index (pH) are used synonymously. If the hydrogen ion activity of an aqueous solution is represented by aH ⁺ , the pH is defined as -log10aH ⁺ . If the ionic strength of the aqueous solution is low (for example, less than 10 ^-3 ), aH ⁺ is almost equal to the hydrogen ion strength. For example, at 25°C and 1 atmosphere, the ion product of water is Kw = aH ⁺ * aOH = 10 ^-14 ; therefore, for pure water, aH ⁺ = aOH = 10 ^-7 . In this case, pH = 7 is neutral, an aqueous solution with a pH less than 7 is acidic, and an aqueous solution with a pH greater than 7 is alkaline. Therefore, the hydrogen ion concentration condition can focus on the difference in biological behavior of the pH-dependent antibody at high hydrogen ion concentration (acidic pH range) and at low hydrogen ion concentration (neutral pH range) for hydrogen ion concentration conditions or pH conditions. For example, in the context of Disclosure A, "the antigen-binding activity under high hydrogen ion concentration (acidic pH range) conditions is lower than the antigen-binding activity under low hydrogen ion concentration conditions (neutral pH range)" means ion concentration-dependent antigen The antigen-binding activity of the binding domain, the ion concentration-dependent antibody, the ion concentration-dependent antigen-binding domain with an increased isoelectric point value, or the ion concentration-dependent antibody with an increased isoelectric point value is preferably 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 rather than a pH selected from pH 6.7 to pH 10.0, it is more preferably pH 5.5 to pH 6.5, more preferably pH 6.7 to pH 9.5, more preferably pH 7.0 to pH 9.0, and more preferably a weak pH of pH 7.0 to pH 8.0. Ideally the above expression means that the antigen-binding activity is weaker at a pH in early endosomes in vivo than at a plasma pH in vivo; and in particular may mean that the antigen-binding activity of an antibody, for example at pH 5.8, is weaker than at, for example, pH 5.8. pH 7.4.

Whether the antigen-binding activity of an antigen-binding domain or an antibody containing this domain changes depending on hydrogen ion concentration conditions can be easily assessed by known methods, such as using the context of Disclosure A or the analytical method described in WO2009/125825. For example, the antigen-binding activity of an antigen-binding domain or an antibody containing this domain toward the antigen of interest can be determined at low and high hydrogen ion concentrations and compared. In this case, conditions other than hydrogen ion concentration are preferably the same. In addition, conditions other than the hydrogen ion concentration for determining the antigen-binding activity can be appropriately selected by those skilled in the art. This antigen-binding activity can be determined, for example, at 37°C in HEPES buffer conditions, or using BIACORE (GE Healthcare) or other equipment.

Within the scope described in Disclosure A, unless otherwise specified by the context, the "neutral pH range" (also known as "low hydrogen ion concentration", "high pH", "neutral pH conditions" or "neutral pH") is not particularly limited is a specific value; but it is preferably selected from pH 6.7 to pH 10.0, from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0. The neutral pH range is preferably pH 7.4, which is close to the pH in plasma (blood) in vivo, but for convenience of measurement, for example, pH 7.0 can be used.

Within the scope described in Disclosure A, the "acidic pH range" (also referred to as "high hydrogen ion concentration", "low pH", "acidic pH conditions" or "acidic pH") is not specifically limited to a specific value unless the context specifically indicates otherwise. ; But it should be selected from pH 4.0 to pH 6.5, from pH 4.5 to pH 6.5, from pH 5.0 to pH 6.5 or from pH 5.5 to pH 6.5. The acidic pH range is preferably pH 5.8, which is connected to the living hydrogen ion concentration in the early endosome, but for convenience of measurement, for example, pH 6.0 can be used.

In one embodiment in the context of disclosure A, if the ion concentration is a hydrogen ion concentration, an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with an increased isoelectric point value, or an isoelectric point value. Increased ion concentration-dependent antigen-binding activity of the antibody is higher at neutral pH than at acidic pH. In this case, the ratio of the antigen-binding activity under neutral pH conditions relative to the antigen-binding activity under acidic pH conditions is not limited; however, the dissociation constant (KD) of the antigen under acidic pH conditions relative to the KD under neutral pH conditions is The ratio, KD (acidic pH range)/KD (neutral pH range), (eg KD (pH 5.8)/KD (pH 7.4)) can be 2 or more; 10 or more; or 40 or more. The upper limit of KD (acidic pH range)/KD (neutral pH range) is not limited and can be any value, such as 400, 1000 or 10000.

In an alternative embodiment, the dissociation rate constant (kd) can also be used as an indicator to represent the above-mentioned binding activity ratio. If the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an index representing the binding activity ratio, the ratio of the dissociation rate constant (kd) under high hydrogen ion concentration conditions to the dissociation rate constant (kd) under low hydrogen ion concentration conditions, 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) is not limited and can be any value, such as 50, 100 or 200.

If the antigen is a soluble antigen, the value of the antigen-binding activity can be represented by the dissociation rate constant (kd). If the antigen is a membrane antigen, such a value can be represented by the apparent dissociation rate constant (apparent kd). The dissociation rate constant (kd) and the apparent dissociation rate constant (apparent kd) can be determined by known methods, for example using BIACORE (GE healthcare) or a flow cytometer.

In one embodiment, methods for producing or screening pH-dependent antigen-binding domains or pH-dependent antibodies or libraries thereof whose antigen-binding activity is higher under neutral pH conditions than under acidic pH conditions are not limited. Such methods include, for example, those described in WO2009/125825 (eg, paragraphs 0158-0190).

Such methods may include, for example: (a) Determine the antigen-binding activity of the antigen-binding domain or antibody under acidic pH conditions; (b) Determine the antigen-binding activity of the antigen-binding domain or antibody under neutral pH conditions; and (c) Select an antigen-binding domain or antibody whose antigen-binding activity is lower under acidic pH conditions than under neutral pH conditions.

Or this method may include, for example: (a) contacting the antigen with the antigen-binding domain or antibody or library thereof under neutral pH conditions; (b) incubate the antigen-binding domain or antibody that binds to the antigen in step (a) under acidic pH conditions; and (c) Isolating the antigen-binding domain or antibody dissociated in step (b).

Or this method may include, for example: (a) contacting the antigen with the antigen-binding domain or antibody or library thereof under acidic pH conditions; (b) Select the antigen-binding domain or antibody that does not bind to the antigen or has low antigen-binding ability in step (a); (c) Allow the antigen to bind to the antigen-binding domain or antibody selected in step (b) under neutral pH conditions; and (d) Isolating the antigen-binding domain or antibody that binds to the antigen in step (c).

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

Or this method may include, for example: (a) Allowing the antigen-binding domain or antibody or its library to pass through the column with immobilized antigen under acidic pH conditions to collect the eluted antigen-binding domain or antibody that is not bound to the column; (b) allow the antigen-binding domain or antibody collected in step (a) to bind to the antigen under neutral pH conditions; and (c) Isolating the antigen-binding domain or antibody that binds to the antigen in step (b).

Or this method may include, for example: (a) contacting the antigen with the antigen-binding domain or antibody or library thereof under neutral pH conditions; (b) The antigen-binding domain or antibody obtained in step (a) that binds to the antigen; (c) incubate the antigen-binding domain or antibody obtained in step (b) under acidic pH conditions; and (d) Isolated antigen-binding domains or antibodies whose antigen-binding activity in step (c) is weaker than the criteria selected in step (b).

Each step of these various screening methods can be repeated several times or the steps can be combined appropriately to obtain the optimal molecule. The above conditions can be appropriately selected for acidic and neutral pH conditions. Therefore, ideal pH-dependent antigen-binding domains or pH-dependent antibodies can be obtained.

In the context of Disclosure A, in one embodiment, the antigen-binding domain or antibody used as the starting material may be a modified antigen-binding domain or antibody by modifying at least 1 amino acid residue that may be exposed on its surface. The charge increases the isoelectric point value. In an alternative embodiment, if an amino acid that modifies the ion concentration-dependent binding activity of an antigen-binding domain is introduced into the sequence, at least one amino acid residue can and may be exposed on the surface of the antigen-binding domain or antibody. Charge modifications are imported together to increase the isoelectric point value.

Or in the context of this disclosure, for example, pre-existing antigen-binding domains or antibodies, pre-existing libraries (phage libraries, etc.); antibodies prepared from fusion tumors obtained by immunizing animals, or B cells derived from immunized animals; or Its library; or an antigen-binding domain, antibody or library obtained by introducing natural or non-natural amino acid mutations with a side chain having a pKa 4.0-8.0 (described below) (for example, with a side chain having a pKa 4.0-8.0 A library of natural or non-natural amino acid mutations, or a library of natural or non-natural amino acid mutations with side chains with pKa 4.0-8.0 introduced at specific locations). Such a preferred antigen-binding domain may have, for example, an amino acid sequence in which at least one amino acid residue is substituted with an amino acid with a side chain pKa of 4.0-8.0 and/or is inserted with a side chain pKa of 4.0-8.0. 4.0-8.0 amino acids, as described in WO2009/125825.

In an embodiment disclosed in the context of A, the location where the mutation of an amino acid with a side chain pKa of 4.0-8.0 is introduced is not limited. This mutation can be introduced at any location, as long as the antigen-binding activity is compared to before the substitution or insertion. It suffices to become weaker in the acidic pH range than in the neutral pH range (the KD (acidic pH range)/KD (neutral pH range) value increases or the kd (acidic pH range)/kd (neutral pH range) value increases) . If the antibody has a variable region or CDR, this site may be within the variable region or CDR. The number of substituted or inserted amino acids can be appropriately determined by those with ordinary knowledge in the technical field; and the number can be one or more. In addition, in addition to the above substitution or insertion, other amino acids may also be deleted, added, inserted and/or substituted or modified. Substitution or insertion of amino acids with side chain pKa 4.0-8.0 can be carried out in a random manner using scanning methods, such as histidine scanning, in which the propylamine in the alanine scanning known to those skilled in the art is Acid is replaced with histidine, and/or is selected from the antigen-binding domain or antibody. Because these amino acids are randomly substituted or inserted into mutations, the KD (acidic pH range)/KD (neutral pH range) value is higher than before the mutation or Antibodies or libraries thereof with increased kd (acidic pH range)/kd (neutral pH range) values.

In addition, the antigen-binding domain or antibody is preferably such that the antigen-binding activity in the neutral pH range before and after such mutations does not significantly decrease, does not substantially decrease, is substantially equal or increases; and in other words, the activity can be maintained at at least 10% or higher, preferably It is 50% or higher, more preferably 80% or higher, more preferably 90% or higher or even higher. If the binding activity of the antigen-binding domain or antibody is reduced due to substitution or insertion of amino acids with pKa 4.0-8.0, the binding activity can be reduced by substitution, deletion, addition, or insertion of one or more amino acids outside the above-mentioned substitution or insertion site. The parts of the body are restored or increased.

In an alternative embodiment, amino acids with side chain pKa 4.0-8.0 can be placed anywhere within the heavy and/or light chain variable regions forming the antigen binding domain. At least 1 amino acid residue with a side chain pKa 4.0-8.0 may be located, for example, in the CDR (one or more of CDR1, CDR2, and CDR3) and/or FR (FR1, one or more of FR2, FR3 and FR4). Such amino acid residues include, but are not limited to, amino acid residues at one or more positions 24, 27, 28, 31, 32 and 34 of the light chain variable region CDR1 according to Kabat numbering; light chain variable Amino acid residues at one or more positions 50, 51, 52, 53, 54, 55 and 56 of the CDR2 region according to Kabat numbering; and/or 89 according to Kabat numbering of the light chain variable region CDR3 , 90, 91, 92, 93, 94 and 95A amino acid residues at one or more positions. These amino acid residues can be placed individually or in combination as long as the antigen-binding activity of the antibody changes according to the hydrogen ion concentration conditions.

In one embodiment, within the scope of disclosure A, any amino acid residue can be appropriately used as an amino acid residue that changes the antigen-binding activity of the antigen-binding domain or antibody under hydrogen ion concentration conditions. Specifically such amino acid residues may include side chain pKa 4.0-8.0. Amino acids with electron donating properties 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)). Amino acid residues preferably include, for example, amino acids with side chain pKa 6.0-7.0, especially His (H).

Within the context of disclosure A, unless otherwise indicated and unless inconsistent with the context, it is understood that the isoelectric point (pI) may be a theoretically or experimentally determined isoelectric point, also referred to as "pI".

The pI value can be determined experimentally, for example using isoelectric focusing electrophoresis. The theoretical pI value can be calculated using gene and amino acid sequence analysis software (Genetyx, etc.).

In one embodiment, whether compared with the antibody before modification (natural antibody (such as a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody (such as an antibody before modification or library construction or under construction)) The antibody with increased isoelectric point value or the increased isoelectric point value of the antibody revealing A can be determined by the above method. In addition, or instead of the above method, the method can be used, for example, mice, rats, rabbits, dogs. , Antibody pharmacokinetic test in monkey or human plasma, determined by a combination such as BIACORE, cell proliferation assay, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA) or fluorescent immunoassay.

Within the scope described in Disclosure A, "amino acid residues that may be exposed on the surface" generally refers to amino acid residues located on the surface of the polypeptide constituting the antibody. "Amino acid residues located on the surface of a polypeptide" may refer to amino acid residues whose side chains contact solvent molecules (usually mostly water molecules). However, the side chains do not necessarily have to completely contact the solvent molecules. If even a part of the side chains contacts the solvent molecules, the amino acid residue is still defined as an "amino acid located on the surface." Amino acid residues located on the surface of the polypeptide may include amino acid residues located close to the surface of the antibody and thus may have mutual charge interactions with amino acid residues even if only a portion of the side chains are in contact with the solvent molecule. Those skilled in the art can use commercial software to create homology models of polypeptides or antibodies, such as homology simulation methods. Alternatively methods such as X-ray crystallization may be used. The amino acid residues that may be exposed on the surface can be determined, for example, using the axes of a three-dimensional model of the antibody obtained with a computer program such as the InsightII program (Accelrys). Surface exposure can be determined using algorithms known in the art (eg Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558() 1983)). The surface-exposed sites can be determined using software suitable for protein modeling and three-dimensional structural information obtained from the antibody. Software available for this purpose includes, for example, SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires user input For the size parameter, the "size" of the probe used in the calculation can be set to a radius of about 1.4 angstroms or less. Pacios also records software for determining the surface exposure area and area for personal computers (Pacios, Comput. Chem18(4):377- 386 (1994); J. Mol. Modelling. 1:46-53 (1995)). Based on the above information, as described above, the appropriate amino acid residues constituting the polypeptide surface of the antibody can be selected.

Methods to increase the isoelectric point of a protein, such as reducing the number of amino acids in side chains that are negatively charged at neutral pH (such as aspartic acid and glutamic acid) and/or increasing the number of amine groups in positively charged side chains Acid number (such as arginine, lysine, and histidine). For an amino acid residue with a negatively charged side chain, the negative charge under pH conditions is -1, which is sufficiently larger than the pKa of its side chain. This is a theory well known to those with ordinary knowledge in the technical field. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the negative charge of the side chain under neutral pH conditions (for example, in a solution with pH 7.0) is expressed as -1. On the contrary, for amino acid residues with positively charged side chains, the positive expression under pH conditions is +1, which is 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 positive charge of the side chain under neutral pH conditions (for example, in a solution with pH 7.0) is expressed as +1. Amino acid residues whose side chains are uncharged under neutral pH conditions (for example, in a solution of pH 7.0) are known to have 15 types of natural amino acids, namely alanine, cysteine, phenylalanine, glycine, Isoleucine, leucine, methionine, aspartic acid, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. It is of course understood that the amino acid used to change the isoelectric point value may be a non-natural amino acid.

From the above, as a method to increase the isoelectric point of a protein under neutral pH conditions (for example, in a solution at pH 7.0), a charge modification of +1 can be given to the protein of interest, for example, by using the amino acid sequence of the protein, aspartic acid (residue) or glutamic acid (residue) (whose side chain has a negative charge of -1) is replaced by an amino acid (residue) with an uncharged side chain. The protein of interest can also be given a charge modification of +1, for example by replacing an amino acid (residue) with an uncharged side chain with arginine or lysine (the side chain of which has a positive charge +1) for amine group Acid (residue). Alternatively, the protein of interest can be given a charge modification of +2, for example by substituting aspartic acid or glutamic acid (whose side chain has a negative charge of -1) with arginine or lysine (whose side chain has a positive charge). +1). Or in order to increase the isoelectric point of the protein, amino acids with uncharged side chains and/or amino acids with positively charged side chains can be added to or inserted into the amino acid sequence of the protein, or amino acids with positively charged side chains can be deleted. The amino acid sequence of the protein contains amino acids with uncharged side chains and/or amino acids with negatively charged side chains. It should be understood that in addition to the charges from the side chains, for example, the N-terminal and C-terminal amino acid residues of the protein have charges from the main chain (NH ³⁺ of the amine group at the N-terminus and COO ^- of the carbonyl group at the C-terminus) . Therefore, the isoelectric point of a protein can also be increased by adding, deleting, substituting or inserting functional groups from the main chain.

One of ordinary skill in the art will appreciate the change in net charge obtained by modifying one or more amino acids (residues) of an amino acid sequence, focusing on the presence or intensity of the charge of the amino acid (residues). Or the effect of the isoelectric point of the protein does not only (or substantially) depend on the amino acid sequence constituting the antibody or the type of the target antigen, but more on the addition, deletion, substitution or insertion of the amino acid residues type and number.

Antibodies whose isoelectric point is increased by modifying at least 1 amino acid residue that may be exposed on the surface of the antibody ("antibody with increased isoelectric point value" or "antibody with increased isoelectric point") can enter more quickly Cells may promote elimination of antigens from plasma, as described or taught, for example, in WO2007/114319, WO2009/041643, WO2014/145159 or WO2012/016227.

Among several antibody structural isotypes, IgG antibodies, for example, have significantly larger molecular weights, and their main metabolic pathway is not excretion through the kidneys. IgG antibodies, molecules containing part of the Fc region, are known to be recycled via the salvage pathway via FcRn, and therefore have a long in vivo half-life. It is speculated that IgG antibodies are mainly metabolized in endothelial cells through metabolic pathways (He et al., J. Immunol. 160(2):1029-1035 (1998)). Specifically, it is believed that if it does not specifically enter endothelial cells, IgG antibodies are recovered by binding to FcRn, and the IgG antibodies that cannot bind are metabolized. If the Fc region is modified to reduce FcRn binding activity, the plasma half-life of IgG antibodies will be shortened. On the other hand, the plasma half-life of antibodies with increased isoelectric point has been shown to depend on the isoelectric point in a highly relevant manner, as described for example in WO2007/114319 and WO2009/041643. Specifically, the above-mentioned literature records that the plasma half-life of an antibody with an increased isoelectric point will still decrease if the amino acid sequence constituting Fc is not modified, which may lead to immunogenicity. This result suggests that isoelectric point increasing technology can be used even for major metabolism. Any type of antibody molecule excreted by the kidney, such as scFv, Fab or Fc fusion proteins, is also broadly applicable.

The pH concentration of biological fluids, such as plasma, is in the neutral pH range. Without being bound by a particular theory, it is believed that in biological fluids, as the isoelectric point value increases, the net positive charge of the antibody with an increased isoelectric point increases. Therefore, the antibody will be more strongly charged by the net positive charge than the antibody without an increased isoelectric point. Negative physiochemical Coulomb interactions at the endothelial cell surface; bind the antibody through non-specific binding and enter the cell, resulting in shortened half-life of the antibody in the plasma or increased elimination of the antigen from the plasma. It also increases the isoelectric point of the antibody and enhances the uptake of the antibody (or antigen/antibody complex) into cells and/or intracellular permeability. It is believed to reduce the concentration of the antibody in the plasma, reduce the bioavailability of the antibody, and/or Shortening the half-life of this antibody in plasma; and this phenomenon is expected to commonly occur in vivo, regardless of cell type, tissue type, organ type, etc. And if the antibody and antigen form a complex and enter the cell, not only the isoelectric point of the antibody but also the isoelectric point of the antigen will also affect the decrease or increase in the uptake into the cell.

In one embodiment, methods of producing or screening antibodies with increased isoelectric points are described, for example, in WO2007/114319 (eg, paragraphs 0060-0087), WO2009/041643 (eg, paragraphs 0115-), WO2014/145159, and WO2012/016227. Such methods may include, for example: (a) Modify the nucleic acid encoding an antibody including at least one amino acid residue that may be exposed on the surface of the antibody to modify the charge of the amino acid residue to increase the isoelectric point value of the antibody; (b) Cultivate host cells to express the nucleic acid; and (c) Collect antibodies from host cell cultures.

Or this method may include, for example: (a') modifying a nucleic acid encoding an antibody that includes at least 1 amino acid residue that may be exposed on the surface of the antibody to modify the charge of the amino acid residue; (b') culturing host cells to express the nucleic acid; (c') collecting antibodies from host cell cultures; and (d') (Confirm or measure as necessary) Select an antibody with an increased isoelectric point compared to the antibody before modification. Here, the antibody as the starting material, the antibody before modification, or the reference antibody may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying amino acid residues, amino acids that change the binding activity of the ion concentration-dependent antigen-binding domain can be included in the sequence.

Alternatively the method may simply comprise culturing the host cells obtained in step (b) or (b') and collecting the antibodies from the cell culture.

In an alternative embodiment, the method may be, for example, a method of producing a multispecific antibody including a first polypeptide and a second polypeptide and optionally a third polypeptide and a fourth polypeptide, including: (A) Modify the nucleic acid encoding the first polypeptide and/or the second polypeptide and optionally the third polypeptide and/or the fourth polypeptide, any one or more of which include at least 1 amine group that may be exposed on the surface of the polypeptide Acid residue to modify the charge of the amino acid residue to increase the isoelectric point value of the antibody; (B) Culturing host cells to express the nucleic acid; and (C) Collection of multispecific antibodies from host cell cultures.

Or this method may include, for example: (A') Modify the nucleic acid encoding the first polypeptide and/or the second polypeptide and optionally the third polypeptide and/or the fourth polypeptide, any one or more of which include at least 1 amine that may be exposed on the surface of the polypeptide amino acid residue to modify the charge of the amino acid residue so as to increase the charge change of the amino acid residue; (B') Culturing host cells to express the nucleic acid; (C') collecting multispecific antibodies from host cell cultures; and (D') (Confirm if necessary and) Select an antibody with an increased isoelectric point compared to the antibody before modification.

The antibody or the antibody before modification or the reference antibody used here as starting material can be, for example, an ion concentration-dependent antibody. Alternatively, when modifying amino acid residues, amino acids that change the binding activity of the ion concentration-dependent antigen-binding domain can be included in the sequence.

Alternatively the method may simply comprise culturing the host cells obtained in step (B) or (B') and collecting the antibodies from the cell culture. In this case, the polypeptide to be modified of the nucleic acid is preferably a homomultimer of the first polypeptide, a homomultimer of the second polypeptide, or a heteromultimer of the first and second polypeptides (the third polypeptide if necessary). the same multimer, the same multimer of the fourth polypeptide, or the heteropolymer of the third and fourth polypeptides).

In an alternative embodiment, the method may be, for example, a method of producing a humanized or human antibody with a reduced half-life in plasma, comprising: a method selected from the group consisting of CDRs of human origin, CDRs of animal origin other than human, and synthetic CDRs. An antibody of the group CDR; human-derived FR; and human constant region, (1) modifying at least 1 amino acid residue that may be exposed on the surface of at least one region selected from the group consisting of CDR, FR and constant region The amino acid residues at the corresponding positions before modification become amino acid residues with different charges, so that the isoelectric point of the antibody is increased.

Alternatively, the method may include, for example, CDRs selected from the group consisting of CDRs of human origin, CDRs of animal origin other than humans, and synthetic CDRs; human-derived FRs; and antibodies with human constant regions, (I') modifications selected from the group consisting of CDRs , at least one amino acid residue in at least one region in the group consisting of FR and constant region that may be exposed on the surface becomes an amino acid residue with a different charge than the amino acid residue at the corresponding position before modification; and (II') (Confirm if necessary and) Select an antibody with an increased isoelectric point compared to the antibody before modification.

The antibody or the antibody before modification or the reference antibody used here as starting material can be, for example, an ion concentration-dependent antibody. Alternatively, when modifying amino acid residues, amino acids that change the binding activity of the ion concentration-dependent antigen-binding domain can be included in the sequence.

Or, for example, pre-existing antigen-binding domains or antibodies, pre-existing libraries (phage libraries, etc.) can be used; antibodies prepared from fusion tumors obtained by immunizing animals, or B cells or libraries thereof derived from immunized animals; or in accordance with the above In any embodiment, the above-mentioned antigen-binding domain antibody or library thereof is modified to increase the isoelectric point of the above-mentioned antigen-binding domain antibody or library thereof for at least one amino acid residue that may be exposed on the surface.

In one embodiment of the antibody disclosed in A, the isoelectric point value may be suitably increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more or by at least 0.6, 0.7, 0.8, 0.9 or more , and significantly shorten the half-life of this antibody in plasma, the isoelectric point value can be increased, for example, by at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more or by at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 or more or an increase of 3.0 or more, compared to the antibody before modification or transformation (natural antibody (such as natural Ig antibody, preferably natural IgG antibody) or control or parent antibody ( For example, the antibody before modification or library construction or during construction)). A person with ordinary knowledge in this technical field can appropriately and routinely determine the optimal isoelectricity of the antibody that reveals A according to the purpose, considering the balance between pharmacological effects and toxicity, such as the number of antibody or antigen-binding domains or the isoelectric point of the antigen. pip value. Without being bound by a particular theory, it is believed that an antibody disclosed in A would be beneficial in one embodiment that does not simply shuttle between plasma and cellular endosomes and bind repeatedly as a single antibody molecule due to the presence of an ion concentration-dependent antigen-binding domain. For polyantigens, the net positive charge of the antibody increases due to the increase in isoelectric point, allowing the antibody to be rapidly taken up into cells. These properties may shorten the half-life of the antibody in plasma, increase the extracellular matrix binding activity of the antibody, or enhance elimination of the antigen from plasma. The optimal isoelectric point value can be determined to benefit from these properties.

In an embodiment disclosed in the context of A, compared with the antibody before modification or before at least 1 amino acid residue is modified to increase the isoelectric point value (natural antibody (such as a natural Ig antibody, preferably a natural IgG Antibody) or a control or parent antibody (such as an antibody before modification of the antibody or before or during library construction), which can be an ion concentration-dependent antibody), and the increase in the isoelectric point value in a ion concentration-dependent manner reveals that the antibody of A should increase Eliminate antigens from plasma, for example, at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times , 4.75 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times or 10 times or more (when the antibody is administered into a living body) or Its extracellular matrix binding activity can be suitably increased, for example, by at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25x, 4.5x, 4.75x or 5x or more.

In an embodiment disclosed in the context of A, compared to the introduction of an ion concentration-dependent antigen-binding domain (natural antibody (such as a natural Ig antibody, preferably a natural IgG antibody) into this antibody) or a control or parent antibody (for example, before antibody modification or before or during library construction), which may be an antibody with an increased isoelectric point), the ion concentration-dependent increase in isoelectric point reveals that the antibody of A preferably enhances the elimination of the antigen from plasma, for example, by at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times, 4.75 times, 5 times, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold or 10-fold or more (when the antibody is administered in vivo) or its extracellular matrix binding activity can It is advisable to increase, for example, at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times, 4.75 times or 5 times or more.

In one embodiment, the evaluation reveals whether the extracellular matrix binding activity of the antibody A is better than that of the antibody before modification or transformation (natural antibody (such as a natural Ig antibody, which can be a natural IgG antibody)) or a control or parent antibody. (For example, the antibody before antibody modification or before or during library construction), it can be an ion concentration-dependent antibody or antibody with an increased isoelectric point) and the analysis method is not limited. For example, the analysis method can be performed using an ELISA system, which detects the binding between an antibody and an extracellular matrix by adding the antibody to an extracellular matrix immobilized plate and adding a labeled antibody against this antibody. Alternatively, as described in Examples 1 to 4 here and WO2012/093704, electrochemiluminescence (ECL) can also be used, which can detect extracellular matrix binding ability with high sensitivity. This method can be performed, for example, using an ECL system, in which a mixture of antibodies and ruthenium antibodies is added to an extracellular matrix-immobilized plate, and the binding between this antibody and the extracellular matrix is measured based on the electrochemiluminescence of ruthenium. The concentration of the antibody to be added can be set appropriately; the concentration added can be high to increase the sensitivity of detecting extracellular matrix binding. Such extracellular matrix may be derived from animals or plants as long as it contains glycoproteins such as collagen, proteoglycans, fibronectin, laminin, entactin, fibrin, and perlecan ; Preferably extracellular matrix from animals. For example, extracellular matrices from animals such as humans, mice, rats, monkeys, rabbits or dogs can be used. For example, natural extracellular matrix derived from humans can be used as an indicator of antibody pharmacodynamics in human plasma. The conditions for evaluating extracellular matrix binding of antibodies are preferably in the neutral pH range, about pH 7.4, which are physiological conditions; however, the conditions do not necessarily have to be in the neutral range, and binding can also be in the acidic pH range (e.g., about pH 6.0) evaluate. Alternatively, when assessing extracellular matrix binding of an antibody, the assay can be performed in the presence of an antigen molecule to which the antibody binds and assess the binding activity of the antigen-antibody complex to the extracellular matrix.

In one embodiment, it is disclosed that the antibody of A is (substantially) compared to a natural antibody (such as a natural Ig antibody, preferably a natural IgG antibodies) or reference antibodies (e.g., antibodies before modification of the antibody or before or during library construction) may retain their antigen-binding activity. In this case, "(substantially) retaining the antigen-binding activity" may refer to at least 50% or more, preferably 60% or more, and more preferably 70% compared to the binding activity of the antibody before modification or transformation. % or 75% or more, preferably 80%, 85%, 90% or 95% or more activity. Or it is revealed that the antibody of A only needs to maintain a level of binding activity that allows it to function when binding to the antigen; therefore, the affinity determined at 37°C under physiological conditions can be, for example, 100 nM or less, preferably 50 nM or less. , more preferably 10 nM or less, still more preferably 1 nM or less.

In one embodiment of Disclosure A, the expression "modification of at least 1 amino acid residue that may be exposed on the surface of the antibody" or equivalent expressions may refer to at least 1 amino acid residue of the antibody that may be exposed on the surface. Implement one or more additions, deletions, substitutions and insertions. Such modifications preferably include substitution of at least 1 amino acid residue.

This substituted amino acid residue may include, for example, substituting an amino acid residue with a negatively charged side chain in the amino acid sequence of the antibody of interest with an amino acid residue with an uncharged side chain, or an amino acid residue with an uncharged side chain. Amino acid residues with side chains are substituted with amino acid residues with positively charged side chains, and amino acid residues with negatively charged side chains are substituted with amino acid residues with positively charged side chains. , can be implemented individually or in appropriate combination. Insertion or addition of amino acid residues may include, for example, insertion or addition of amino acids with uncharged charges and/or insertion or addition of amino acids with positively charged side chains to the amino acid sequence of the antibody of interest. , which can be implemented individually or in appropriate combination. Deletion of amino acid residues may include, for example, deletion of amino acids with uncharged charges and/or deletion of amino acids with negatively charged side chains in the amino acid sequence of the antibody of interest, which may be performed individually or in appropriate combinations.

One of ordinary skill in the art can appropriately combine one or more of these additions, deletions, substitutions and insertions into the amino acid sequence of the antibody of interest. Modifications that result in a reduction in the local charge of an amino acid residue are also acceptable as long as the net isoelectric point of the antibody revealing A is increased. For example, if desired, an antibody whose isoelectric point value has been increased (too much) can be modified to decrease the isoelectric point (slightly). It is also acceptable to modify at least one amino acid residue to reduce the local charge of the amino acid residue for other purposes (such as increasing antibody stability or reducing immunogenicity) at the same time or at different times. Such antibodies include antibodies from libraries constructed for a specific purpose.

In one embodiment, among the amino acids (residues) used to modify at least 1 amino acid residue that can be exposed on the surface of the antibody, the natural amino acids are as follows: The amino acids with negatively charged side chains can be Glu (E) or Asp (D); the uncharged amino acid in the side chain can be Ala (A), Asn (N), Cys (C), Gln (Q), Gly (G), His (H), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) or Val (V); Amino acids with positively charged side chains can be His (H), Lys (K) or Arg (R).

As detailed in Examples 1 to 4, in a solution with neutral pH (for example, pH 7.0), lysine and arginine are present as residues in the antibody and are almost 100% positively charged, while histidine is Existing in the form of residues in antibodies, only about 9% are positively charged, and most of the rest are presumed to have no charge. Therefore, it is better to choose Lys(K) or Arg(R) as the amino acid with positively charged side chain.

In one embodiment, the antibody of disclosure A preferably has a variable region and/or a constant region. In addition, the variable region should have a heavy chain variable region and/or a light chain variable region, and/or should have CDRs (such as one or more of CDR1, CDR2, and CDR3) and/or FR (such as FR1, FR2, One or more of FR3 and FR4). The constant region should preferably include a heavy chain constant region and/or a light chain constant region, in terms of sequence and type, for example, an IgG type constant region (preferably human IgG1, human IgG2, human IgG3 or human IgG4 type constant region, human kappa chain constant region region, and human lambda chain constant region). Modified variants of these constant regions may also be used.

In one embodiment, modification of at least 1 amino acid residue that may be exposed on the surface of the antibody may modify a single amino acid or a combination of multiple amino acids. An ideal approach would be to introduce a combination of multiple amino acid substitutions at sites exposed to amino acids on the antibody surface. There is no restriction, but it is preferable that such a plurality of amino acid substitutions be introduced into positions that are sterically close to each other. When an amino acid that may be exposed on the antibody surface (preferably but not limited to an amino acid with a negatively charged side chain (such as Glu (E) or Asp (D)) is replaced with a positively charged side chain (such as Lys ( K) or Arg (R)); or if a positively charged pre-existing amino acid is used (e.g. Lys (K) or Arg (R)), for example, an amino acid stereoscopically close to the amino acid can be or multiple amino acids (which, depending on the state, may include amino acids embedded within the antibody molecule) are substituted with positively charged amino acids to create a dense state of local positive charge at the steric proximity site. Here "stereoproximity" "Site" is not particularly limited; it may mean that one or more amino acid substitutions are introduced within, for example, 20 angstroms, preferably within 15 angstroms, and more preferably within 10 angstroms. Whether the amino acid substitution site of concern is exposed to the antibody molecule The surface or whether the amino acid substituted site is in close proximity to other amino acid substituted sites or pre-existing amino acids as described above can be evaluated using known methods such as X-ray crystallography.

In addition to the above methods, methods of providing multiple positive charges at sites sterically close to each other may include the use of amino acids that are inherently positively charged in the natural IgG constant region. Such amino acids include, for example: arginine at positions 255, 292, 301, 344, 355 and 416 according to EU numbering; and arginine at positions 121, 133, 147, 205, 210, 213, 214, according to EU numbering. Lysine at positions 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360, 370, 392, 409, 414, and 439. Multiple positive charges can be obtained by substituting sites sterically close to these positively charged amino acids with positively charged amino acids.

If it is revealed that the antibody of A has a variable region (which can be modified), the amino acid residues that are not obscured by antigen binding (i.e., those that may still be exposed on the surface) can be modified, and/or no amino acid modifications can be introduced At sites blocked by antigen binding, amino acid modifications that do not (substantially) inhibit antigen binding may be implemented. If the amino acid residues present in the ion concentration-dependent binding domain that may be exposed on the surface of the antibody molecule have been modified, the amino acids in the antigen-binding domain can be modified so that the modification does not (substantially) reduce the ability to change according to ion concentration conditions. The binding activity of the amino acid residues responsible for the antigen-binding activity of the antibody (for example, in calcium-binding motifs or histidine insertion sites and/or histidine substitution sites) or the amino acid residues can be modified in such a way that they can be modified according to ion concentration conditions. Parts other than amino acid residues that change the antigen-binding activity of the antibody. On the other hand, if the amino acid residues present in the ion concentration-dependent binding domain that can be exposed on the surface of the antibody molecule have been modified, the amino acid residues that can change the antigen-binding activity of the antibody according to the ion concentration conditions can be selected. be of such type or location that the isoelectric point of the antibody does not fall below an acceptable level. If the isoelectric point of the antibody is below an acceptable level, the overall isoelectric point of the antibody can be increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody molecule.

There is no limitation. The FR sequence with a high isoelectric point is preferably selected from the human germline FR sequence or equivalent region sequences. These amino acids may sometimes be modified.

If the antibody disclosed in A has a constant region (which may be modified) with an FcγR binding domain (which may be a binding domain for any of the FcγR isotypes and subtypes described below) and/or an FcRn binding domain, at least 1 of the constant region The site for modification of the amino acid residues that may be exposed on the surface may optionally be amino acid residues other than amino acid residues in the FcγR binding domain and/or FcRn binding domain. Alternatively, if the modification site is selected from amino acid residues within the FcγR-binding domain and/or FcRn-binding domain, it should be selected that does not (substantially) affect the binding activity or binding affinity to FcγR and/or FcRn, or if it does Effect is a biologically or pharmacologically acceptable site.

In one embodiment, at least 1 amino acid residue of the variable region (which may be modified) is modified to produce an A-disclosing antibody with an increased isoelectric point by modifying at least 1 amino acid residue that may be exposed on the surface. The position of the residue is not limited; but such position may be selected from the group consisting of the following according to Kabat numbering: (a) FR 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, Positions 84, 85, 86, 105, 108, 110 and 112; (b) Positions 31, 61, 62, 63, 64, 65 and 97 of the CDR of the heavy chain variable region; (c) Positions of the light chain variable region FR of 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; and (d) CDR 24 of the light chain variable region , 25, 26, 27, 52, 53, 54, 55 and 56, wherein the amino acid modified at each position can be selected from any of the above amino acids, in terms of side chain charge, such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S) or Asn (N) but not limited thereto. 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 can be used in combination with other positions that have a sufficient effect of increasing the isoelectric point value of the antibody to assist in increasing the isoelectric point value of the antibody revealing A. Such a position that assists in increasing the isoelectric point value may be, for example, for the light chain variable region, selected from the group consisting of: 27, 52, 56, 65 and 69 according to Kabat numbering.

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

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

Non-limiting examples of combinations of amino acid position modifications in the heavy chain variable region are, for example, any 2 or more positions selected from the group consisting of the following positions according to Kabat numbering: positions 16, 43, 64 and 105; Any 2 or more positions from the group consisting of: positions 77, 82a, and 82b; positions 77 and 85; positions 41 and 44; positions 82a and 82b; positions 82 and 82b; positions 82b and 83; or 63 And position 64, wherein the modified amino acid at each position can be selected from any of the above-mentioned amino acids, in terms of side chain charge, such as Lys (K), Arg (R), Gln (Q), Gly (G) , Ser (S) or Asn (N) but not limited to this.

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

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

Non-limiting examples of combinations of amino acid position modifications in the light chain variable region are, for example, positions 24 and 27 according to Kabat numbering; positions 25 and 26; positions 41 and 42; positions 42 and 76; positions 52 and 56; 65 and 79 positions; 74 and 77 positions; 76 and 79 positions; selected from any 2 or more positions from the group of the following positions: 16, 24, and 27 positions; selected from the group of the following positions Any 2 or more positions: 24, 27, and 37 positions; any 2 or more positions: 25, 26, and 37 positions; selected from the group of the following positions; selected from the group of the following positions Any 2 or more positions: 27, 76, and 79; any 2 or more positions: 41, 74, and 77; selected from the group of the following positions; selected from the group of the following positions Any 2 or more positions: Position 41, 76, and 79; Select any 2 or more positions from the group consisting of: Positions 24, 27, 41, and 42; Select from the group consisting of the positions Any 2 or more positions of: 24, 27, 52, and 56 positions; any 2 or more positions of: 24, 27, 65, and 69 positions; selected from the group consisting of Any 2 or more positions from the group of positions: positions 24, 27, 74, and 77; any 2 or more positions from the group consisting of positions: 24, 27, 76, and 79; Select any 2 or more positions from the group of the following positions: 25, 26, 52, and 56; Select any 2 or more positions from the group of the positions: 25, 26, 65, and 69 positions; selected from any 2 or more positions from the group of the following positions: 25, 26, 76, and 79 positions; selected from any 2 or more positions from the group of the following positions: 27, Positions 41, 74, and 77; any 2 or more positions selected from the group consisting of the following positions: Positions 27, 41, 76, and 79; any 2 or more positions selected from the group consisting of the following positions Position: Positions 52, 56, 74, and 77; Select any 2 or more from the group that constitutes the position: Positions 52, 56, 76, and 79; Select any from the group that constitutes the position below 2 or more positions: 65, 69, 76, and 79 positions; any 2 or more positions: 65, 69, 74, and 77 positions; selected from the group of the following positions; selected from the group of the following positions Any 2 or more positions in the group: 18, 24, 45, 79, and 107 positions; any 2 or more positions in the group consisting of the following positions: 27, 52, 56, 74, and 77 positions ;Select any 2 or more positions from the group consisting of: 27, 52, 56, 76, and 79; Select any 2 or more positions from the group consisting of: 27, 65 , 69, 74, and 77 positions; choose any 2 or more positions from the group of the following positions: 27, 65, 69, 76, and 79 positions; choose any 2 from the group of the following positions or more positions: positions 41, 52, 56, 74, and 77; selected from any 2 or more positions from the group consisting of: positions 41, 52, 56, 76, and 79; selected from the group consisting of Any 2 or more positions from the group of positions: 41, 65, 69, 74, and 77; Select any 2 or more positions from the following group of positions: 41, 65, 69, 76, and 79 positions; any 2 or more positions selected from the group of the following positions: 24, 27, 41, 42, 65, and 69 positions; any 2 or more positions selected from the group of the following positions Positions: 24, 27, 52, 56, 65, and 69; selected from any 2 or more of the group consisting of positions: 24, 27, 65, 69, 74, and 77; selected from the following Any 2 or more positions from the group of positions: 24, 27, 65, 69, 76, and 79; any 2 or more positions from the group of positions: 24, 27, 41 , 42, 74, and 77 positions; selected from any 2 or more positions from the group of the following positions: 24, 27, 52, 56, 74, and 77 positions; selected from the group of the following positions Any 2 or more positions: 24, 27, 41, 42, 76, and 79; any 2 or more positions selected from the group consisting of: 24, 27, 52, 56, 76, and 79 Position; Select any 2 or more positions from the group of the following positions: 24, 27, 74, 76, 77, and 79 Position; Choose any 2 or more positions from the group of the positions below: Positions 52, 56, 65, 69, 74, and 77; or any 2 or more positions selected from the group consisting of: positions 52, 56, 65, 69, 76, and 79, where each position The modified amino acid can be selected from any of the above-mentioned amino acids, such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S) or Asn (N) in terms of side chain charge. ) but is not limited to this.

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; /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; /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, the effect of increasing the isoelectric point by modifying some amino acid residues in the variable region has been explained or proven based on theoretical evidence, homology models or experimental techniques and is not exclusive (or substantial). (above) depends on the amino acid sequence that constitutes the antibody itself or the type of target antigen, and more on the type and number of substituted amino acid residues. It has been demonstrated that even after some amino acid modifications, the antigen-binding activity against several types of antigens is (substantially) maintained or at least maintained with a high probability as one of ordinary skill in the art would expect.

For example, WO2009/041643 specifically points out that in the heavy chain FR of the humanized glypican 3 antibody shown in SEQ ID NO:8, the ideal modification sites of the amino acid residues that may be exposed on the surface are 1 and 3 according to the Kabat numbering system. ,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 bits. It has also been reported that the amino acid residue at position 97 according to Kabat numbering is more ideal because it is exposed on almost all antibody surfaces. WO2009/041643 also discloses that the amino acid residues at positions 52, 54, 62, 63, 65, and 66 of the heavy chain CDR of the antibody are ideal. Also disclosed are amino acid residues 1, 3, 7, 8, 9, 11, 12, 16, 17, and 18 of the light chain FR of the humanized glypican 3 antibody shown in SEQ ID NO: 9 according to Kabat numbering. ,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 are ideal. It is also revealed that the amino acid residues at positions 24, 27, 33, 55 and 59 of the light chain CDR of this antibody are ideal. Furthermore, WO2009/041643 specifically indicates that amino acid residues 31, 64 and 65 of the heavy chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 10 according to Kabat numbering are ideal sites because they can Allows modification of amino acid residues that may be exposed on the surface while maintaining antigen-binding activity. It is also revealed that the amino acid residues 24, 27, 53, and 55 of the light chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 11 according to Kabat numbering are more ideal. It is also specifically pointed out that position 31 of the amino acid residue of the heavy chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 12 according to the Kabat numbering method is an ideal site because it allows modifications to be exposed on the surface. amino acid residues to maintain antigen-binding activity. It is also revealed that the amino acid residues 24, 53, 54, and 55 of the light chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 13 according to Kabat numbering are more ideal. WO2009/041643 also reveals that the amino acid residues 61, 62, 64 and 65 of the heavy chain CDR of the anti-human glypican 3 antibody shown in SEQ ID NO: 14 according to the Kabat numbering method are ideal positions because they can allow Modification of amino acid residues that may be exposed on the surface maintains antigen-binding activity. It is also revealed that the amino acid residues 24 and 27 of the light chain CDR of the anti-human glypican 3 antibody shown in SEQ ID NO: 15 according to Kabat numbering are ideal positions. It is also revealed that the amino acid residues 61, 62, 64 and 65 of the heavy chain CDR of the anti-human IL-31 receptor antibody shown in SEQ ID NO: 16 according to Kabat numbering are ideal positions because they can allow Modification of amino acid residues that may be exposed on the surface maintains antigen-binding activity. WO2009/041643 also reveals that the amino acid residues 24 and 54 of the light chain CDR of the anti-human IL-31 receptor antibody shown in SEQ ID NO: 17 according to Kabat numbering are ideal positions. Likewise, WO2007/114319 reports antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and hB26-PF shows a change in isoelectric point during isoelectric focusing, and has the same binding activity to its antigen, Factor IXa or Factor X, compared to the antibody before modification or transformation. It has also been reported that if these antibodies are administered to mice, the isoelectric point of each antibody is highly correlated with its clearance rate (CL) in plasma, residence time in plasma, and half-life (T1/2) in plasma. WO2007/114319 also proves that the amino acid residues at positions 10, 12, 23, 39, 43, 97, and 105 of the variable region are ideal as amino acid residue modification sites that may be exposed on the surface.

In an alternative or further embodiment, it is possible, for example, to use known methods such as The homology simulation method of the homology model constructed by the region (but not limited to this) is used to identify the amino acid residues that may be exposed on the surface of the antibody constant region to determine the method for producing A-revealing antibodies with increased isoelectric points. The modification site of at least one amino acid residue. 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, this site should be selected from the group consisting of the following: in accordance with EU numbering 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, and should be selected from the group consisting of the following Group: 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 are also preferably selected from the group consisting of: 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, the amino acid at each position can be selected from the above-mentioned amino acids after modification. In terms of side chain charge, such as Lys (K), Arg (R), Gln (Q) or Asn (N) but not limited to Here it is. When selecting a modification site for at least one amino acid residue, for example, from the above group, for example, the modified amino acid type of each site can be as follows: At position 254: 254K, 254R, 254Q or 254N; At position 258, 258K, 258R, 258Q or 258N; At position 281: 281K, 281R, 281Q or 281N; At position 282: 282K, 282R, 282Q or 282N; At position 285: 285K, 285R, 285Q or 285N; At position 309: 309K, 309R, 309Q or 309N; At position 311: 311K, 311R, 311Q or 311N; At position 315: 315K, 315R, 315Q or 315N; At position 327: 327K, 327R, 327Q or 327N; At position 330: 330K, 330R, 330Q or 330N; At position 342: 342K, 342R, 342Q or 342N; At position 311: 343K, 343R, 343Q or 343N; At position 345: 345K, 345R, 345Q or 345N; at position 356: 356K, 356R, 356Q or 356N; At position 358: 358K, 358R, 358Q or 358N; At position 359: 359K, 359R, 359Q or 359N; At position 361: 361K, 361R, 361Q or 361N; At position 362: 362K, 362R, 362Q or 362N; At position 384: 384K, 384R, 384Q or 384N; At position 385: 385K, 385R, 385Q or 385N; At position 386: 386K, 386R, 386Q or 386N; At position 387: 387K, 387R, 387Q or 387N; At position 389: 389K, 389R, 389Q or 389N; At position 399: 399K, 399R, 399Q or 399N; At position 400: 400K, 400R, 400Q or 400N; At position 401: 401K, 401R, 401Q or 401N; At position 402: 402K, 402R, 402Q or 402N; At position 413: 413K, 413R, 413Q or 413N; At position 418: 418K, 418R, 418Q or 418N; At position 419: 419K, 419R, 419Q or 419N; At position 421: 421K, 421R, 421Q or 421N; At position 433: 433K, 433R, 433Q or 433N; At position 434: 434K, 434R, 434Q or 434N; and at position 443: 443K, 443R, 443Q or 443N.

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

In one embodiment of the antibody disclosed in A, the net isoelectric point of the antibody can be modified by at least one amine that may be exposed on the surface of the variable region (which may be modified) and the constant region (which may be modified) as described above. Increased by amino acid residues.

Within the scope of disclosures A and B described herein, when the antibody disclosed in A or B is an IgG type antibody or a molecule derived therefrom, the heavy chain constant region of the antibody may include IgG1 type, IgG2 type, IgG3 type or IgG4 type. constant region. In disclosure A or B, the heavy chain constant region may be a human heavy chain constant region but is not limited thereto. Several subtypes of human IgG are known to exist. Specifically, it is reported that the amino acid sequence of the human IgG constant region has some differences among individuals (Methods Mol. Biol. 882:635-80 (2012); Sequences of proteins of immunological interest, NIH Publication No. 91-3242) . Examples include human IgG1 constant region (SEQ ID NO:18), human IgG2 constant region (SEQ ID NO:19), human IgG3 constant region (SEQ ID NO:20) and human IgG4 constant region (SEQ ID NO:21).

Among them, for example, G1m1, 17 and G1m3 are known allotypes against human IgG1. The amino acid sequences of this subtype are different: G1m1 and 17 have aspartic acid at position 356 and leucine at position 358 according to the EU numbering method, while G1m3 has glutamic acid at position 356 and 358 according to the EU numbering method. Methionine. However, there are no reports suggesting that these reported subtypes have significant differences in primary antibody functions and properties. Therefore, those with ordinary knowledge in this technical field can easily predict various evaluations using specific subtypes. The results are not limited to the used subtypes but examples can be obtained and the same effect can be predicted in any subtype. Within the scope of disclosures A and B described herein, when expressed as "human IgG1", "human IgG2", "human IgG3" or "human IgG4", this subtype is not limited to a specific subtype and may include all reported subtypes .

In an alternative or further embodiment of disclosure A or B, the light chain constant region of the antibody may comprise a 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. There are reports, such as Sequences of proteins of immunological interest, NIH Publication No. 91-3242, that some paratype sequences are derived from genetic polymorphisms in the human kappa chain constant region and the human lambda chain constant region. Such subtypes include, for example, human kappa chain constant region (SEQ ID NO:22) and human lambda chain constant region (SEQ ID NO:23). However, there are no reports suggesting that these reported subtypes have significant differences in primary antibody functions and properties. Therefore, those with ordinary knowledge in this technical field can easily understand that when referring to the specific subtypes within the scope of disclosure A and B described herein, any subtype can be expected to have the same effect (hereinafter also collectively referred to as natural (human) IgG (type) constant region).

In addition, the Fc region of a natural IgG antibody constitutes part of the constant region of a natural IgG antibody. Therefore, if the antibody disclosed in A or B is, for example, an IgG-type antibody or a molecule derived therefrom, the antibody may include natural IgG (IgG1, IgG2 , IgG3 or IgG4 type) (hereinafter also collectively referred to as the natural (human) IgG (type) Fc region), the Fc region contained in the constant region. The Fc region of natural IgG may refer to an Fc region that has the same amino acid sequence as the Fc region of naturally occurring IgG. Specific examples of the Fc region of natural human IgG may include the above-mentioned human IgG1 constant region (SEQ ID NO: 18), human IgG2 constant region (SEQ ID NO: 19), human IgG3 constant region (SEQ ID NO: 20) or human The Fc region (Fc region of IgG class) contained in the IgG4 constant region (SEQ ID NO: 21) may refer, for example, from cysteine 226 to the C terminus according to EU numbering or from proline 230 to proline according to EU numbering. C end).

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

Within the scope described in Disclosure A, WO2013/081143 reports that, for example, an ion concentration-dependent antibody (multivalent antigen-antibody complex) can form a multivalent immune complex with a multimeric antigen and reacts by recognizing 2 or more of the monomeric antigens. Multiple epitopes can form multivalent immune complexes, multispecific ion concentration-dependent antibodies or multi-antibody binding site ion concentration-dependent antibodies (multivalent antigen-antibody complexes), which can bind more strongly to FcγR, FcRn, Complement receptor, due to binding avidity (between multiple epitopes and multiple antibodies) via at least 2 or more multivalent constant regions (which may be modified) or Fc regions (which may be modified) included in this antibody molecule The sum of the binding strengths of the binding sites), so the antibody can be taken up into cells faster. Therefore, when at least one amino acid residue that can be exposed on the surface of the antibody is modified to increase the isoelectric point, the above-mentioned ion concentration-dependent antibody can form a multivalent immune complex with the multimeric antigen or the monomeric antigen, and can As an antibody that reveals A (an ion concentration-dependent antibody with increased isoelectric point value). Those of ordinary skill in the art should understand that antibodies that are capable of forming multivalent immune complexes with multimeric antigens or monomeric antigens have an increased isoelectric point value in a concentration-dependent manner compared to antibodies that are unable to form multivalent immune complexes. Antibodies with increased ion concentration-dependent values can be taken up into cells faster. Those with ordinary skill in this technical field will also understand that in one embodiment, the activity of the antibody revealing A in binding to FcRn and/or FcγR can be increased under neutral pH conditions. In this case, it can be combined with multimeric antigens or monomers. The antigen forms a multivalent immune complex and the isoelectric point value increases in a concentration-dependent manner and the antibody is absorbed into the cell more quickly.

In one embodiment, the antibody disclosed in A may be a single-arm antibody (including all embodiments of the single-arm antibody described in WO2005/063816). Generally, single-arm antibodies are antibodies that lack the two Fab regions that are common in IgG antibodies, and can be produced by the method described in WO2005/063816 without limitation. There is no limit to the structure of an IgG-type antibody with a heavy chain, such as VH-CH1-hinge-CH2-CH3. If the Fab region is cut closer to the N-terminus than the hinge (such as VH or CH1), the antibody will In the form of inclusion of additional sequences, when the Fab region is cut closer to the C-terminus than the hinge (for example, CH2), the Fc region will have an incomplete form. Therefore, there is no limitation. From the viewpoint of the stability of the antibody molecule, a single-arm antibody is preferably produced by cutting the hinge region (hinge) of one of the two Fab regions of an IgG antibody. More preferably, the cleaved heavy chain is connected to the uncleaved heavy chain using intramolecular disulfide bonds. WO2005/063816 has reported that such single-arm antibodies have better stability than Fab molecules. Antibodies with increased or decreased isoelectric point values can also be produced by preparing such single-arm antibodies. And if the antibody with an increased isoelectric point value introduced into the ion concentration-dependent antigen-binding domain is a single-arm antibody, compared with the antibody without an ion concentration-dependent antigen-binding domain with an increased isoelectric point value, the half-life of this antibody in plasma is It can be further shortened, the cellular uptake of the antibody can be further enhanced, the elimination of the antigen from the plasma can be further enhanced, or the affinity of the antibody for the extracellular matrix can be further increased.

Without being bound by a particular theory, if the cellular uptake acceleration effect of a single-arm antibody is expected in one embodiment, it is conceivable that, but not limited to, the isoelectric point value of the soluble antigen is lower than the isoelectric point value of the antibody. The net isoelectric point of a complex consisting of an antibody and an antigen can be calculated by considering the complex as a single molecule using known methods. In this case, the lower the isoelectric point of the soluble antigen, the lower the net isoelectric point of the complex; and the greater the isoelectric point value of the soluble antigen, the greater the net isoelectric point of the complex. When a normal IgG antibody molecule (with 2 Fabs) binds to a single low isoelectric point soluble antigen compared to two low isoelectric point soluble antigens, the net isoelectric point of the latter complex is lower. When such a normal antibody is converted into a single-arm antibody, only one antigen can bind to a single molecule of this antibody; the reduction in the isoelectric point of the complex caused by binding to the second antigen can be inhibited. In other words, it is believed that when the isoelectric point value of the soluble antigen is based on the isoelectric point value of the antibody, it is converted into a single-arm antibody compared to a normal antibody, allowing the isoelectric point of the complex to increase, thereby accelerating uptake into cells.

There is no limit. When the isoelectric point value of the Fab of a normal IgG type antibody molecule (with 2 Fabs) is lower than the isoelectric point value of the Fc, the transformation into a single-arm antibody will increase the complex of the single-arm antibody and the antigen. Net isoelectric point. Furthermore, when converting to a single-arm antibody in this way, from the viewpoint of the stability of the single-arm antibody, one of the Fabs should be cut at the hinge region located at the interface between Fab and Fc. In this case, by selecting a site that will increase the isoelectric point value of the single-arm antibody to a desired degree, an effective increase in the isoelectric point value can be expected.

Therefore, a person with ordinary knowledge in this technical field will understand that it does not exclude (or substantially) depend on the amino acid sequence of the antibody itself and the type of soluble antigen, by calculating the theoretical isoelectric point value (theoretical Fc, etc.) of the antibody. The relationship between the theoretical isoelectric point value and the Fab theoretical isoelectric point value) and the theoretical isoelectric point value of the soluble antigen, and predicting the difference in the theoretical isoelectric point value, in order to transform this antibody into a single-arm antibody, the isoelectric point value of the antibody can be Increased, and subsequently cellular uptake of this antigen may be accelerated.

In one embodiment, the antibody disclosed in A or B may be a multispecific antibody, and the multispecific antibody may be, but is not limited to, a bispecific antibody. The multispecific antibody may be a multispecific antibody containing a first polypeptide and a second polypeptide. Here, "multispecific antibodies containing the first polypeptide and the second polypeptide" refer to antibodies that bind to at least 2 or more types of different antigens or at least 2 or more types of epitopes of the same antigen. The first polypeptide and the second polypeptide preferably include a heavy chain variable region, more preferably a variable region containing CDRs and/or FRs. In another embodiment, the first polypeptide and the second polypeptide preferably each contain a heavy chain constant region. In another embodiment, the multispecific antibody may include a third polypeptide and a fourth polypeptide, each containing a light chain variable region and preferably a light chain constant region. In this case, the first to fourth polypeptides can be assembled together to form a multispecific antibody.

In one embodiment, if the antibody disclosed in A is a multispecific antibody and the multispecific antibody contains a heavy chain constant region to lower its isoelectric point value, for example, the following sequence can be used: IgG2 or IgG4 sequence at position 137; IgG1, IgG2 or IgG4 sequence at position 196; IgG2 or IgG4 sequence at position 203; IgG2 sequence at position 214; IgG1, IgG3 or IgG4 sequence at position 217; IgG1, IgG3 or IgG4 sequence at position 233; The IgG4 sequence at position 274; the IgG2, IgG3 or IgG4 sequence at position 274; the IgG1, IgG2 or IgG4 sequence at position 276; the IgG4 sequence at position 355; the IgG3 sequence at position 392; the IgG4 sequence at position 419; or The IgG1, IgG2 or IgG4 sequence at position 435. At the same time, the isoelectric point value can be increased, for example, the following sequences can be used: IgG1 or IgG3 sequence at position 137; IgG3 sequence at position 196; IgG1 or IgG3 sequence at position 203; IgG1, IgG3 or IgG4 sequence at position 214; The IgG2 sequence at position 217; the IgG2 sequence at position 233; the IgG1, IgG2 or IgG3 sequence at position 268; the IgG1 sequence at position 274; the IgG3 sequence at position 276; the IgG1, IgG2 or IgG3 sequence at position 355; The IgG1, IgG2 or IgG4 sequence at position 392; the IgG1, IgG2 or IgG3 sequence at position 419; or the IgG3 sequence at position 435.

In one embodiment, if the antibody disclosed in A has two heavy chain constant regions, the isoelectric point values of the two heavy chain constant regions can be the same or different from each other. Such heavy chain constant regions may be IgG1, IgG2, IgG3 and IgG4 heavy chain constant regions that originally have different isoelectric points. Alternatively, an isoelectric point difference can be introduced between the two heavy chain constant regions. In order to introduce such an isoelectric point difference into the constant region, the modification site of at least one amino acid residue can be as the above position or according to the EU numbering method. The selection of the heavy chain constant region described in WO2009/041643 is composed of the following The positions of the groups are: 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419 and 435. The amino acid residue at position 297 of the glycated site may be modified to remove the sugar chain because removing the sugar chain from the heavy chain constant region will cause an isoelectric point difference.

In one embodiment, the antibody disclosed in A or B can be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody of mammalian origin. Monoclonal antibodies include those produced by fusion tumors or by host cells transformed by genetic engineering techniques with expression vectors carrying antibody genes. The antibody revealing A or B can be, for example, an antibody such as a chimeric antibody, a humanized antibody, or an antibody produced using affinity maturation or a molecule derived therefrom.

In one embodiment, the antibody disclosed in A or B can be from any animal species without limitation (such as humans; or non-human animals, such as mice, rats, hamsters, rabbits, monkeys, Malay monkeys, rhesus monkeys, Arabian baboons , chimpanzee, goat, sheep, dog, cow or camel) or any bird; and the antibody is preferably from a human, monkey or mouse.

In one embodiment, the antibody disclosed in A or B may be an Ig type antibody, preferably an IgG type antibody.

Within the context of Disclosures A and B described herein, Fc receptor (also referred to as "FcR") refers to a receptor protein or molecule derived therefrom that can bind to the Fc region of an immunoglobulin (antibody) or an Fc Zone variant. Within the scope of disclosure A described here, for example, Fc receptors for IgG, IgA, IgE, and IgM are known to be FcγR, FcαR, FcεR, and FcμR, respectively. Within the scope of disclosures A and B described here, Fc The receptor may also be, for example, FcRn (also known as "nascent Fc receptor").

Within the scope described in Disclosure A, "FcγR" may refer to a receptor protein that can bind to the Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody, or a molecule derived therefrom or an Fc region variant, and may include a receptor protein that is substantially composed of One or more or all members of the protein family encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) includes isoforms FcγRIIa (including subtypes H131 (H type) and R131 (R type)) , FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including subtypes V158 and F158) and FcγRIIIb (including subtypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and All unidentified human FcγRs and FcγR isotypes and paratypes. Furthermore, FcγRIIb1 and FcγRIIb2 have been reported to be cleavage variants of human FcγRIIb (hFcγRIIb). There has also been a report of a cleavage variant called FcγRIIb3 (Brooks et al., J. Exp. Med, 170: 1369-1385 (1989)). In addition to the above methods, hFcγRIIb includes, for example, all cleavage variants registered at NCBI under the numbers NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992.3. hFcγRIIb also includes all reported gene polymorphisms, such as FcγRIIb (Li et al., Arthritis Rheum. 48:3242-3252 (2003), Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005) ; Kyogoku et al., Arthritis Rheum. 46(5):1242-1254 (2002)), and all genetic polymorphisms to be reported in the future.

The FcγR can be from any organism, including but not limited to humans, mice, rats, rabbits or monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as all unidentified mouse FcγRs, and FcγR isotypes and subtypes. Such ideal FcγRs include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), or FcγRIIIB (CD16). FcγR is in the membrane form in vivo, so it can be artificially converted into the appropriate soluble form and used in experimental systems.

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

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

In FcγRI (CD64) including FcγRIa, FcγRIb, FcγRIc, and FcγRIII (CD16) including FcγRIIIa (including subtypes V158 and F158), the α chain that binds to the Fc region of IgG is linked to the common γ chain with ITAM , this ITAM transmits activation signals within cells. FcγRIIIb (including subtypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPI anchor protein. The cytoplasmic domain of FcγRII (CD32), which includes FcγRIIa (including subtypes H131 and R131) and FcγRIIc isoforms, contains ITAM. These receptors are expressed on many immune cells such as macrophages, fat cells and antigen-presenting cells. These receptors bind to the Fc region of IgG and transmit activation signals that promote the phagocytosis of macrophages, produce inflammatory cytokines, degranulate fat cells, and increase the function of antigen-presenting cells. Within the scope of disclosures A and B here, FcγRs that have the ability to transmit activation signals as described above are also called activated FcγRs.

The cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM, which transmits inhibitory signals. In B cells, the cross-linking between FcγRIIb and the B cell receptor (BCR) inhibits the activation signal from the BCR, resulting in the inhibition of BCR antibody production. In macrophages, cross-linking of FcγRIII and FcγRIIb inhibits phagocytosis and the ability to produce inflammatory cytokines. Within the context of disclosures A and B, FcγRs capable of transmitting inhibitory signals as described above are also called inhibitory Fcγ receptors.

Within the scope described in disclosure A, whether the binding activity of the antibody or Fc region (variant) to various FcγRs is increased, (substantially) maintained, or reduced compared to before the modification of the antibody or Fc region (variant), can be based on the There are methods for evaluation known to those of ordinary skill in the technical field. Such a method is not particularly limited, and methods described in the examples can be used, such as BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010) which mainly uses surface plasmon resonance (SPR) phenomena. ). Alternatively, ELISA and fluorescence-activated cell selection (FACS) and ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) can be used. In these assays, the extracellular domain of human FcγR can be used (eg WO2013/047752).

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

Within the scope of Disclosure A, in one embodiment, if the antibody of Disclosure A or B has a constant region (which may be modified), the constant region may have an Fc region or an Fc region variant (preferably a human Fc region or a human Fc region variant), and within the scope of disclosures A and B described herein, there should be an FcγR binding domain and an FcRn binding domain.

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

In one embodiment, when it is revealed that the antibody of A has FcγR-binding activity, it is preferred that the FcγR-binding activity of the antibody under neutral pH conditions is increased compared to a reference antibody containing a native IgG constant region. Considering the point of comparing the FcγR binding activity between the two, it is not limited but it is preferable to reveal that the antibody of A and the reference antibody containing the natural IgG constant region have the same amino acid sequence in this region (such as the variable region), rather than this The constant region of the antibody disclosed in A is modified at one or more amino acid residues.

In one embodiment, when it is revealed that the antibody of A has FcγR binding activity or increased FcγR binding activity under neutral pH conditions (e.g., pH 7.4), without being bound by theory, it is believed that this antibody combination possesses the following properties: The property of shuttling between cellular endosomes and the ability of a single antibody molecule to repeatedly bind to multiple antigens through the presence of an ion concentration-dependent antigen-binding domain; through the increase in the isoelectric point and positive charge of the entire antibody, it is rapidly The property of uptake into cells; and the property of rapid uptake into cells by increasing FcγR binding activity under neutral pH conditions. As a result, the half-life of the antibody in plasma can be further shortened or the binding activity of the antibody to the extracellular matrix can be further increased or the elimination of the antigen from the plasma can be further accelerated; therefore, it is beneficial to disclose the antibody of A. One of ordinary skill in the art can routinely determine the optimal isoelectric point value of the antibody to benefit from these properties.

In one embodiment, the FcγR binding activity of an FcγR binding domain is higher than the FcγR binding activity of the Fc region or constant region of natural human IgG, wherein the sugar chain linked to position 297 according to EU numbering is a fucose-containing sugar. Chains can be prepared by modifying the amino acid residues of the Fc region or constant region of natural human IgG (see WO2013/047752). Also, a domain that binds to any structure of FcγR can be used as the FcγR binding domain. In this case, the FcyR-binding domain can be prepared without introducing amino acid modifications, or its affinity for FcyR can be increased by introducing additional modifications. Such FcγR binding domains may include Fab fragment antibodies that bind to FcγRIIIa, single domain antibodies from camelids, and single chain Fv antibodies, such as Schlapschly et al. (Protein Eng. Des. Sel. 22 (3): 175-188 (2009) ), Behar et al. (Protein Eng. Des. Sel. 21(1):1-10 (2008)) and Kipriyanov et al., J Immunol. 169(1):137-144 (2002), and The FcγRI-binding cyclic peptide described in Bonetto et al., FASEB J. 23(2):575-585 (2009). Is the FcγR-binding activity of an FcγR-binding domain higher than that of the FcγR-binding domain linked to position 297 according to EU numbering? The FcγR-binding activity of the Fc region or constant region of a natural human IgG in which the sugar chain is a fucose-containing sugar chain can be appropriately evaluated using the method described above.

In one embodiment of Disclosure A, the starting FcyR binding domain preferably includes, for example, a (human) IgG Fc region or a (human) IgG constant region. Any Fc region or constant region can be used as the starting Fc region or starting constant region as long as a variant of the starting Fc region or starting constant region can bind to human FcγR in the neutral pH range. The Fc region or constant region obtained by further modifying an Fc region or a starting constant region in which amino acid residues have been modified from the Fc region or constant region can also be used as the Fc of Disclosure A. area or constant area. The starting Fc region or starting constant region may refer to the polypeptide itself, a composition containing the starting Fc region or starting constant region, or the amino acid sequence encoding the starting Fc region or starting constant region. The starting Fc region or starting constant region may include a known Fc region or a known constant region produced using recombinant techniques. The source of the starting Fc region or the starting constant region is not limited and can be obtained from non-human animals or human organisms. In addition, FcrR binding domains can be obtained from Malay monkeys, marmosets, rhesus monkeys, chimpanzees or humans. The starting Fc region or starting constant region is preferably obtained from human IgG1; but 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 a suitable starting FcγR binding domain, and also means that within the scope of disclosure A described herein, IgG classes or subtypes derived from any organism The Fc region or constant region of a subclass can be used as the initial Fc region or the initial constant region. Examples of natural IgG variants or modified types documented in publicly known literature include Strohl, Curr. Opin. Biotechnol. 20(6):685-691 (2009); Presta, Curr. Opin. Immunol. 20(4): 460-470 (2008); Davis et al., Protein Eng. Des. Sel. 23(4):195-202 (2010); WO2009/086320, WO2008/092117; WO2007/041635; and WO2006/105338, but not Limited to this.

In one embodiment, the amino acid residues of the starting FcγR binding domain, the starting Fc region, or the starting constant region may include, for example, one or more mutations: e.g., substitutions to be different from those in the starting Fc region or starting constant region. Amino acid residues; insertion of one or more amino acid residues into the amino acid residues of the starting Fc region or the starting constant region; or deletion of one or more amines from the starting Fc region or the starting constant region acid residues. The amino acid sequence of the modified Fc region or constant region is preferably a part of the amino acid sequence of the Fc region or constant region that does not occur naturally. Such variants must have less than 100% sequence identity or similarity with the original Fc region or the original constant region. For example, the variant has about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100% relative to the amino acid sequence of the starting Fc region or starting constant region. %, more preferably about 90% to less than 100%, more preferably about 95% to less than 100% amino acid sequence identity or similarity. In a non-limiting example, it is disclosed that the modified Fc region or constant region of A is different from the starting Fc region or starting constant region by at least one amino acid.

In one embodiment, the Fc region or constant region having FcγR binding activity in the acidic pH range and/or the neutral pH range may be included in the antibody disclosed in Disclosure A and may be obtained by any method. Specifically, variants of the Fc region or constant region that have FcγR-binding activity in the neutral pH range can be obtained by using amino acids of human IgG antibodies that can serve as the starting Fc region or starting constant region. Suitably modified IgG antibody Fc regions or IgG antibody constant regions may include, for example, the Fc region or constant region of human IgG (IgG1, IgG2, IgG3 or IgG4, or variants thereof), and spontaneous mutants generated therefrom. For the Fc region or constant region of human IgG1, human IgG2, human IgG3 or human IgG4 antibodies, some paratype sequences due to genetic polymorphism are recorded in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, available Use either of them to reveal A. In particular, for the human IgG1 sequence, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM.

Within the scope of disclosure A, in another embodiment, the modification to other amino acids is not limited, as long as the variant has FcγR binding activity in the neutral pH range. Such modified amino acid positions are reported in, for example: WO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072, WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635, WO200 8/092117、WO2005/ 070963, WO2006/020114, WO2006/116260, WO2006/023403, WO2013/047752, WO2006/019447, WO2012/115241, WO2013/125667, WO2014/030728, WO2014/1631 01. WO2013/118858, and WO2014/030750.

The position of amino acid modification that increases the FcγR binding activity of the constant region or Fc region in the neutral pH range may include, for example, one or more positions selected from the group consisting of: Fc region or constant region of a human IgG antibody The position of EU numbering method, 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, Positions 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 are recorded in WO2013/047752L. Modification of such amino acid residues may increase FcγR binding in the Fc region or constant region of IgG antibodies under neutral pH conditions. WO2013/047752 records that the ideal modification of the IgG type constant region or Fc region is, for example, the modification of one or more amino acid residues selected from the group consisting of: According to the EU numbering method, the amino acid at position 221 becomes: Lys or Tyr; the amino acid at position 222 becomes any one of Phe, Trp, Glu, and Tyr; the amino acid at position 223 becomes any one of Phe, Trp, Glu, and Lys; the amino group at position 224 The acid becomes any one of Phe, Trp, Glu and Tyr; the amino acid at position 225 becomes any one of toGlu, Lys, and Trp; the amino acid at position 227 becomes toGlu, Gly, Lys, and Tyr Any one of them; the amino acid at position 228 becomes any one of Glu, Gly, Lys, and Tyr; the amino acid at position 230 becomes any one of Ala, Glu, Gly, and Tyr; the amino acid at position 231 becomes any one of Ala, Glu, Gly, and Tyr; The amino acid becomes any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 232 becomes any one of Glu, Gly, Lys, and Tyr; the amino acid at position 233 becomes Ala , Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 becomes Ala, Asp, Any of Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 becomes Ala, Asp, Glu , Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 becomes Ala, Asp, Glu, Any of Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 becomes Asp, Glu, Phe, His , Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 238 becomes Asp, Glu, Phe, Gly, His, Any of Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 239 becomes Asp, Glu, Phe, Gly, His, Ile, Lys , Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 240 becomes any one of toAla, Ile, Met, and Thr; the amino acid at position 241 The amino acid becomes any one of Asp, Glu, Leu, Arg, Trp, and Tyr; the amino acid at position 243 becomes any one of toGlu, Leu, Gln, Arg, Trp, and Tyr; the amino acid at position 244 becomes The amino acid at position 245 becomes His; the amino acid at position 245 becomes Ala; the amino acid at position 246 becomes any one of Asp, Glu, His, and Tyr; the amino acid at position 247 becomes Ala, Phe, Any one of Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; the amino acid at position 249 becomes any one of Glu, His, Gln, and Tyr; the amino acid at position 250 becomes Glu or Gln; the amino acid at position 251 becomes Phe; the amino acid at position 254 becomes any one of Phe, Met, and Tyr; the amino acid at position 255 becomes any one of Glu, Leu, and Tyr The amino acid at position 256 becomes any one of Ala, Met, and Pro; the amino acid at position 258 becomes any one of Asp, Glu, His, Ser, and Tyr; the amino acid at position 260 becomes It becomes any one of Asp, Glu, His, and Tyr; the amino acid at position 262 becomes any one of Ala, Glu, Phe, Ile, and Thr; the amino acid at position 263 becomes Ala, Ile, Any of Met, and Thr; the amino acid at position 264 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Any of Tyr; the amino acid at position 265 becomes Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr Any one of them; the amino acid at position 266 becomes any one of Ala, Ile, Met, and Thr; the amino acid at position 267 becomes Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Any of Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 268 becomes Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg , Thr, Val, and Trp; the amino acid at position 269 becomes Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr Any one of them; the amino acid at position 270 becomes any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amine at position 271 The amino acid becomes any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amine group at position 272 The acid changes to any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 273 changes to Phe or Ile; the amino acid at position 274 becomes any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 275 The amino acid becomes Leu or Trp; the amino acid at position 276 becomes Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr. Any one; the amino acid at position 278 becomes any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; 279 The amino acid at position 280 becomes Ala; the amino acid at position 280 becomes any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid at position 281 becomes Asp, Any one of Lys, Pro, and Tyr; the amino acid at position 282 becomes any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 283 becomes Ala, Gly, His, Ile, Any one of Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid at position 284 becomes any one of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid at position 285 becomes Any one of Asp, Glu, Lys, Gln, Trp, and Tyr; the amino acid at position 286 becomes any one of Glu, Gly, Pro, and Tyr; the amino acid at position 288 becomes Asn, Asp, Glu, and Tyr; the amino acid at position 290 becomes Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid at position 291 becomes Asp, Any one of Glu, Gly, His, Ile, Gln, and Thr; the amino acid at position 292 becomes any one of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid at position 293 becomes to any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 294 becomes Phe, Gly, His, Ile, or Lys , Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Any of Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 296 becomes Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln , Arg, Ser, Thr, and Val; the amino acid at position 297 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 298 becomes any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; The amino acid at position 299 becomes any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; 300 The amino acid at position 301 becomes Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; the amino acid at position 301 becomes Any one of Asp, Glu, His, and Tyr; the amino acid at position 302 becomes Ile; the amino acid at position 303 becomes any one of Asp, Gly, and Tyr; the amino acid at position 304 becomes Any one of Asp, His, Leu, Asn, and Thr; the amino acid at position 305 becomes any one of Glu, Ile, Thr, and Tyr; the amino acid at position 311 becomes Ala, Asp, Asn, Any of Thr, Val, and Tyr; the amino acid at position 313 becomes Phe; the amino acid at position 315 becomes Leu; the amino acid at position 317 becomes Glu or Gln; the amino acid at position 318 becomes The acid becomes any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 becomes Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Any one of Ser, Thr, Val, Trp, and Tyr; the amino acid at position 322 becomes any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr The amino acid at position 323 becomes Ile; the amino acid at position 324 becomes any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr ;The amino acid at position 325 becomes any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 326 becomes any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 327 becomes It 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 at position 328 becomes Ala, Any of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 329 becomes Asp, Glu , Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 330 becomes Cys, Glu, Phe, Any of Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 becomes Asp, Phe, His, Ile, Leu , Met, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 332 becomes Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Any of Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 333 becomes Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr , Val, and Tyr; the amino acid at position 334 becomes any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; the amino acid at position 335 becomes Asp, Phe, or Gly , His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 becomes any one of Glu, Lys, and Tyr; the amino acid at position 337 The amino acid becomes any one of Glu, His, and Asn; the amino acid at position 339 becomes any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr; The amino acid at position 376 becomes Ala or Val; the amino acid at position 377 becomes Gly or Lys; the amino acid at position 378 becomes Asp; the amino acid at position 379 becomes Asn; the amino acid at position 380 becomes Asn; The amino acid at position 382 becomes Ala, Asn, and Ser; the amino acid at position 382 becomes Ala or Ile; the amino acid at position 385 becomes Glu; the amino acid at position 392 becomes Thr; The amino acid at position 421 becomes Lys; the amino acid at position 427 becomes Asn; the amino acid at position 428 becomes Phe or Leu; the amino acid at position 429 becomes Met ; The amino acid at position 434 becomes Trp; the amino acid at position 436 becomes Ile; and the amino acid at position 440 becomes any one of Gly, His, Ile, Leu, and Tyr. The number of amino acids to be modified is not particularly limited. Amino acid modifications can be made at only 1 position or at 2 or more positions. Combinations of amino acid modifications at 2 or more positions are shown in Table 5 of WO2013/047752. Modifications of these amino acid residues can also be appropriately introduced into A-disclosing antibodies.

In one embodiment, the binding activity of the (FcγR-binding domain) of the antibody of A to (human) FcγR, such as to any one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, can be higher than that of the natural FcγR. The binding activity of the IgG (the Fc region or constant region) or the reference antibody containing the initial Fc region or the initial constant region. For example, the FcγR-binding activity of the antibody revealing A (FcγR-binding domain) may be 55% or more, 60% or more, 65% or more, 70% or more, compared to the FcγR-binding activity of the reference antibody. 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, especially 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 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times greater than the FcγR binding activity of the reference antibody times or more, 7.5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more More, 80 times or more, 90 times or more or 100 times or more.

In yet another embodiment, the level of increase in binding activity (in the neutral pH range) for inhibitory FcγRs (FcγRIIb-1 and/or FcγRIIb-2) can be greater than for activating FcγRs (FcγRIa: FcγRIb; FcγRIc; FcγRIIIa, including Subtype V158; FcγRIIIa, including subtype F158; FcγRIIIb, including subtype FcγRIIIb-NA1; FcγRIIIb, including subtype FcγRIIIb-NA2; FcγRIIa, including subtype H131; or FcγRIIa, including subtype R131) is an increase in binding activity level.

In one embodiment, the antibody disclosed in A may have binding activity to FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

In one embodiment, it is disclosed that the ideal FcγR binding domain of A also includes an FcγR binding domain that has greater binding activity to a specific FcγR than to other FcγRs (FcγR binding domain, selective FcγR binding activity). If an antibody (or its Fc region is used as the FcγR binding domain) is used, a single antibody molecule can only bind to a single FcγR molecule. Therefore, a single antibody molecule that is bound to an inhibitory FcγR cannot bind to other activating FcγRs, and a single antibody molecule that is bound to an activating FcγR cannot bind to other activating FcγRs or inhibitory FcγRs.

As mentioned above, activating FcγRs preferably include, for example, FcγRI (CD64), such as FcγRIa, FcγRIb or FcγRIc; and FcγRIII (CD16), such as FcγRIIIa (such as subtype V158 or F158) or FcγRIIIb (such as subtype FcγRIIIb-NA1 or FcγRIIIb-NA2). . Inhibitory FcγRs suitably include, for example, FcγRIIb (such as FcγRIIb-1 or FcγRIIb-2).

In one embodiment, an FcγR binding domain with greater binding activity for inhibitory FcγR than for activating FcγR can be used as the selective FcγR binding domain contained in the antibody of Disclosure A. Such selective FcγR binding domains may include, for example, an FcγR that has higher binding activity to FcγRIIb (such as FcγRIIb-1 and/or FcγRIIb-2) than an activating FcγR selected from one or more of the group consisting of: Binding domains: FcγRI (CD64) such as FcγRIa, FcγRIb or FcγRIc; FcγRIII (CD16) such as FcγRIIIa (such as paratype V158 or F158) or FcγRIIIb (such as FcγRIIIb-NA1 or FcγRIIIb-NA2); FcγRII (CD32) such as FcγRIIa (including paratypes) type H131 or R131); and FcγRIIc.

In addition, whether the FcγR binding domain has selective binding activity can be evaluated by comparing the binding activities for FcγR determined by the above method, for example, by comparing the KD value for activating FcγR divided by the KD value for inhibitory FcγR. The value (ratio), more specifically, compares the FcγR selectivity index shown in the following formula: [Formula 1] FcγR selectivity index = KD value for activating FcγR/KD value for inhibitory FcγR

In Formula 1, the KD value for an activating FcγR refers to the KD value for one or more of the following: FcγRIa; FcγRIb; FcγRIc; FcγRIIIa, including subtype V158 and/or F158; FcγRIIIb including FcγRIIIb-NA1 and/or FcγRIIIb -NA2; FcγRIIa, including subtype H131 and/or R131; and FcγRIIc; and the KD value against inhibitory FcγR refers to the KD value against FcγRIIb-1 and/or FcγRIIb-2. The activating FcγR and inhibitory FcγR used to determine the KD value can be selected in any combination. For example, a value (ratio) obtained by dividing the KD value for FcγRIIa including subtype H131 by the KD value for FcγRIIb-1 and/or FcγRIIb-2 can be used, but is not limited to this.

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

In one embodiment, it is suitable to use the Fc region-containing variant or the constant region variant of human IgG (IgG1, IgG2, IgG3 or IgG4) in which the amino acid at position 238 or 328 according to the EU numbering method is Asp or Glu. Antibody) in the antibody of Disclosure A as an Fc region variant or a constant region variant, because as specifically disclosed in WO2013/125667, WO2012/115241, and WO2013/047752, it is specific for FcγRIIb-1 and/or FcγRIIb-2 Compared to FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, including subtype V158, FcγRIIIa, including subtype F158; FcγRIIIb, including subtype FcγRIIIb-NA1; FcγRIIIb, including subtype FcγRIIIb-NA2; FcγRIIa, including subtype H131; FcγRIIa, Including subtype R131, and/or FcγRIIc has higher binding activity. In such embodiments, it is disclosed that the antibody of A has binding activity to all activating FcγRs (herein, selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, FcγRIIa) and FcγRIIb, and its FcγRIIb binding activity , compared to a reference antibody containing a native IgG constant region or a native IgG Fc region, is maintained or increased, and/or its binding activity for all activating FcγRs is reduced.

In one embodiment of the antibody of Disclosure A containing an Fc region variant or a constant region variant, the binding activity for FcγRIIb may be maintained or increased compared to a reference antibody having a native IgG constant region or Fc region. , its binding activity to FcγRIIa (H type) and FcγRIIa (R type) may be reduced. Such antibodies may have increased binding selectivity for FcγRIIb compared to FcγRIIa.

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

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

The above modifications may be in a single position alone or in combination at 2 or more positions. Alternatively, such desirable modifications may include, for example, variants of the human constant region or human Fc region of human IgG (IgG1, IgG2, IgG3 or IgG4) as shown in Tables 14 to 15, 17 to 24, and 26 to 28 of WO2013/047752. , the amino acid at position 238 according to the EU numbering method is Asp, and the amino acid at position 271 according to the EU numbering method is Gly. In addition, it can replace 233, 234, 237, 264, 265, 266 according to the EU numbering method. , 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and one or more positions of 396. In this case, such variants may include, but are not limited to, variants of the human constant region or human Fc region that contain amino acids according to one or more of the EU numberings: Bit 233 is Asp, bit 234 is Tyr, bit 237 is Asp, bit 264 is Ile, bit 265 is Glu, bit 266 is any one of Phe, Met, and Leu; bit 267 is one of Ala, Glu, Gly, and Gln. Any one; 268 bits are Asp or Glu; 269 bits are Asp; 272 bits are any one of Asp, Phe, Ile, Met, Asn and Gln; 296 bits are Asp; 326 bits are Ala or Asp; 327 bits is Gly; 330 bits are Lys or Arg; 331 bits are Ser; 332 bits are Thr; 333 bits are any one of Thr, Lys, and Arg; 396 bits are Asp, Glu, Phe, Ile, Lys, Leu, Met Any one of , Gln, Arg and Tyr.

In an alternative embodiment, an antibody of Disclosure A containing an Fc region variant or a constant region variant may maintain or increase binding activity for FcγRIIb and FcγRIIa compared to a reference antibody containing a native IgG constant or Fc region. (H type) has reduced binding activity to FcγRIIa (R type). The ideal amino acid substitution position of such a variant may be as described in WO2014/030728, for example, the amino acid at position 238 according to EU numbering, and at least 1 amino acid selected from the group consisting of: According to EU Numbering method: 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 bits.

More preferably, this variant has Asp at position 238 according to the EU numbering method and has at least one amino acid selected from the following amino acid groups: Asp at position 233, Tyr at position 234, and Tyr at position 235 according to the EU numbering method. Bit 237 is Phe, bit 237 is Asp, bit 264 is Ile, bit 265 is Glu; bit 266 is Phe, Leu or Met; bit 267 is Ala, Glu, Gly or Gln; bit 268 is Asp, Gln or Glu; bit 269 is Asp; 271 is Gly; 272 is Asp, Phe, Ile, Met, Asn, Pro or Gln; 274 is Gln; 296 is Asp or Phe; 326 is Ala or Asp; 327 is Gly; 330 Bit 331 is Lys, Arg or Ser; bit 331 is Ser; bit 332 is Lys, Arg, Ser or Thr; bit 333 is Lys, Arg, Ser or Thr; bit 334 is Arg, Ser or Thr; bit 355 is Ala or Gln ; Bit 356 is Glu; Bit 358 is Met; Bit 396 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Bit 409 is Arg; bit 419 is Glu.

In an alternative embodiment, an antibody of Disclosure A containing an Fc region variant or a constant region variant maintains binding activity for FcγRIIb compared to a reference antibody containing a native IgG constant region or Fc region and for all The binding activity of activating FcγRs, especially FcγRIIa (R-type), is reduced. The ideal amino acid substitution position of such a variant can be as reported in WO2014/163101. For example, in addition to the amino acid at position 238 according to EU numbering, there is at least 1 amino acid position selected from the following: According to EU numbering 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330 of the law. More preferably, this variant is Asp at position 238 according to the EU numbering method, and has at least 1 amino acid selected from the amino acid group: According to the EU numbering method, position 235 is Phe; position 237 is Gln or Asp; Bit 241 is Met or Leu; bit 268 is Pro; bit 295 is Met or Val; bit 296 is Glu, His, Asn, or Asp; bit 298 is Ala or Met; bit 323 is Ile; bit 324 is Asn or His; and Bit 330 is His or Tyr.

Within the scope described in Disclosure A, the level of "maintenance of binding activity to FcγRIIb" may be, but is not limited to, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater. Large, 80% or larger, 81% or larger, 82% or larger, 83% or larger, 84% or larger, 85% or larger, 86% or larger, 87% or larger, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101% or greater, 102% or greater, 103% or greater Large, 104% or greater, 105% or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 120% or greater, 130% or greater, 140% or greater, 150% or greater, 175% or greater or 2 times or greater.

Within the scope described in Disclosure A, the above-mentioned "binding activity reduction for all activating FcγRs, especially for FcγRIIa (R type)" level may 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, antibodies disclosing A or B may comprise such variants.

Within the context of disclosures A and B of this document, unlike FcγR, which belongs to the immunoglobulin superfamily, "FcRn", specifically human FcRn, is structurally similar to major histocompatibility complex (MHC) class I polypeptides. , and show 22% to 29% sequence identity with MHC class I molecules (Ghetie et al., Immunol. Today 18(12), 592-598 (1997)). The FcRn system is expressed as a heterobinary body, consisting of a soluble Beta or light chain (Beta2 microglobulin) complexed with a membrane-penetrating α or heavy chain. Like MHC, the α chain of FcRn contains three extracellular domains (α1, α2, and α3), and its short cytoplasmic domain tethers the protein to the cell surface. The α1 and α2 domains interact with the FcRn binding domain of the Fc region of this 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 the transmission of IgG from the mother to the fetus. Furthermore, FcRn is expressed in the small intestine of neonatal rodents, and FcRn is involved in transporting maternal IgG from digested colostrum or milk across the bristle margin of the epithelium. FcRn is also expressed in various tissues and endothelial cell lines in various species. FcRn is also expressed in adult human vascular endothelium, muscle vasculature, and hepatic sinusoids. It is believed that FcRn plays a role in maintaining plasma IgG concentrations by binding to IgG and recycling IgG to serum. Generally, the binding of FcRn to IgG molecules is strictly pH dependent. Optimal binding is observed in the acidic pH range below pH 7.0.

The polynucleotide and amino acid sequences of human FcRn can be derived, for example, from the precursors of NM_004107.4 and NP_004098.1 (including the signal sequence), respectively (RefSeq access numbers are shown in parentheses).

This precursor forms a complex with human Beta2-microglobulin in vivo. Therefore, through known recombinant expression technology, soluble human FcRn that forms a complex with human Beta2-microglobulin can be produced for appropriate use in various experimental systems. Such soluble human FcRn can be used to assess the FcRn binding activity of antibodies or Fc region variants. In disclosure A or B, FcRn is not particularly limited as long as it is in a form that can bind to the FcRn binding domain; however, ideally FcRn can be human FcRn.

Within the scope of disclosures A and B of this document, when an antibody or Fc region variant has FcRn-binding activity, it may have an "FcRn-binding domain," preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited, as long as the antibody has binding activity or affinity for FcRn at acidic pH and/or neutral pH; or it may be a domain that has the activity of directly or indirectly binding to FcRn. Such domains include, but are not limited to, Fc region type immunoglobulins of IgG, albumin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides and anti-FcRn scaffold molecules, which have the activity of directly binding to FcRn; and binding to A molecule of IgG or albumin that has the activity of indirectly binding to FcRn. In revealing A or B, it is also possible to use domains with FcRn-binding activity in the acidic pH range and/or in the neutral pH range. If this domain originally has FcRn binding activity in the acidic pH range and/or in the neutral pH range, it can be used without further modification. If this domain has only weak or no FcRn-binding activity in the acidic pH range and/or in the neutral pH range, the amino acid residues in the FcRn-binding domain of the antibody or Fc region variant can be modified to have a binding activity in the acidic pH range and /or FcRn binding activity in the neutral pH range. Alternatively, amino acids that originally have FcRn-binding activity in the acidic pH range and/or neutral pH range can be modified to further increase their FcRn-binding activity. The FcRn binding activity in the acidic pH range and/or in the neutral pH range before and after amino acid modification can be compared to identify amino acid modifications of interest to the FcRn binding domain.

The FcRn binding domain is preferably a region that directly binds to FcRn. Such ideal FcRn binding domains include, for example, the constant and Fc regions of antibodies. However, it can bind to the region of polypeptides with FcRn-binding activity, such as albumin and IgG, and can bind to FcRn indirectly via albumin and IgG. Therefore, the FcRn-binding region may be a region that binds to a polypeptide with binding activity to albumin or IgG. There is no limitation. In order to promote the elimination of antigens from plasma, an FcRn-binding domain with a large FcRn-binding activity at neutral pH is preferable. In order to promote antibody retention in plasma, an FcRn-binding domain with a large FcRn-binding activity at acidic pH is preferable. area. For example, an FcRn-binding domain that originally has greater FcRn-binding activity at neutral pH or acidic pH can be selected. Alternatively, the amino acids of the antibody or Fc region variant can be modified to have FcRn-binding activity at neutral pH or acidic pH. Alternatively, pre-existing FcRn binding activity at neutral pH or acidic pH can be increased.

Within the scope of disclosures A and B described herein, whether the FcRn-binding activity of the antibody or Fc region (variant) is increased, (substantially) maintained, or reduced compared to before modification of the antibody or Fc region (variant), Known methods can be used, such as those described in the examples here and eg BIACORE, Scatchard plot and flow cytometry (see WO2013/046722). The extracellular domain of human FcRn can be used as the soluble antigen in these assays. Conditions other than pH when measuring the FcRn-binding activity of an antibody or Fc region (variant) can be appropriately selected by those skilled in the art. The analysis method can be performed under conditions such as MES buffer and 37°C, as described in WO2009/125825. The FcRn-binding activity of an antibody or Fc region (variant) can be assessed, for example, by loading FcRn onto an antibody-immobilized chip as an analyte.

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

Regarding the pH conditions for measuring the binding activity of FcRn to the FcRn-binding domain contained in the antibody or Fc region (variant), acidic pH conditions or neutral pH conditions can be appropriately used. For the temperature conditions for measuring the binding activity (binding affinity) between FcRn and the FcRn binding domain, any temperature between 10°C and 50°C can be used. In order to determine the binding activity (binding affinity) between FcRn and the human FcRn binding domain, it is advisable to Use a temperature of 15℃ to 40℃. It is more suitable to use any temperature from 20℃ to 35℃. For example, you can use 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, Any one of 33, 34, and 35°C. A non-limiting example of such a temperature may be 25°C.

In one embodiment, when it is revealed that the antibody of A or B has FcRn-binding activity, it has an FcRn-binding domain, preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited, as long as the antibody has binding activity or affinity for FcRn at acidic pH and/or neutral pH, and it can be a domain that has the activity of directly or indirectly binding to FcRn. In a specific embodiment, the antibody disclosed in A or B preferably has increased FcRn binding activity under neutral pH conditions compared to a reference antibody containing the constant region of a native IgG (see WO2013/046722). From the perspective of comparing the FcRn-binding activity between the two, it is suitable but not limited to: the antibody revealing A or B and the reference antibody containing the constant region of natural IgG, except that one or more amino acid residues have been modified. The regions other than the constant region of the base (such as the variable region) have the same amino acid sequence.

In one embodiment, within the scope of Disclosure A described herein, when the antibody of Disclosure A has increased FcRn-binding activity under neutral pH conditions, without being bound by a particular theory, the antibodies of Disclosure A may possess 2 or more in combination. Multiple following properties: shuttling between plasma and cellular endosomes, repeatedly binding to multiple antigens with a single antibody molecule with an ion concentration-dependent antigen-binding domain; increasing the isoelectric point value and increasing the positive charge of the entire antibody, and Rapidly taken up into cells; and rapidly taken up into cells by increasing FcRn binding activity under neutral pH conditions. As a result, the half-life of the antibody in the plasma can be further shortened or the binding activity of the antibody to the extracellular matrix can be further increased, or the elimination of the antigen from the plasma can be further accelerated. One of ordinary skill in the art can determine the optimal isoelectric point value of the antibody disclosed in A to benefit from such properties.

Within the scope of what is revealed in this record is A and B. According to Yeung et al. (J. Immunol. 182:7663-7671 (2009)), in the acidic pH range (pH 6.0), the activity of natural human IgG1 binding to human FcRn is KD 1.7 μM, while in the neutral pH range This activity was barely detectable. Therefore, in order to increase the FcRn-binding activity in the neutral pH range, it is appropriate to use the following as an antibody revealing A or B: an antibody or constant region variant or Fc region variant whose human FcRn-binding activity in the acidic pH range is KD 20 μM or higher, and its human FcRn-binding activity in the neutral pH range is comparable to or stronger than that of natural human IgG; preferably it is an antibody or constant region variant or Fc region variant, and its human FcRn-binding activity is within An acidic pH range of KD 2.0 μM or greater, and its human FcRn binding activity in a neutral pH range of KD 40 μM or greater; and more preferably an antibody or constant region variant or Fc region variant whose human FcRn The binding activity is KD 0.5 μM or better in the acidic pH range, and its human FcRn binding activity is KD 15 μM or better in the neutral pH range. The KD value was determined according to the method described by Yeung et al. (J. Immunol. 182:7663-7671 (2009) (by immobilizing the antibody on the chip and loading human FcRn as the analyte)).

Within the scope of disclosures A and B described here, a domain having any structure that binds to FcRn can be used as an FcRn-binding domain. In this case, it is not necessary to introduce amino acid modifications to create the FcRn-binding domain, or additional modifications may be introduced to increase the affinity for FcRn.

Within the context of disclosures A and B described herein, a starting FcRn binding domain may include, for example, the Fc region or constant region of a (human) IgG. Any Fc region or constant region may be used as the starting Fc region or starting constant region as long as a variant of the starting Fc region or starting constant region binds to FcRn in the acidic pH range and/or in the neutral pH range. . Alternatively, an Fc region or a constant region obtained by modifying a starting Fc region or a starting constant region (the Fc region or constant region of which has been modified) can also be appropriately used as the Fc region or constant region. The starting Fc region or starting constant region may include a known Fc region produced recombinantly. Depending on the context, the starting Fc region or starting constant region may refer to the polypeptide itself, a composition containing the starting Fc region or starting constant region, or the amino acid sequence encoding the starting Fc region or starting constant region. The origin of the initial Fc region or the initial constant region is not limited and can be obtained from any non-human animal or human organism. It is also known that the FcRn binding domain can be obtained from Malay monkeys, marmosets, rhesus monkeys, chimpanzees and humans. The starting Fc region or starting constant region can 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 a suitable starting FcRn binding domain, and that an Fc region or constant region of any biologically derived IgG class or subclass can be used as a starting Fc region or as the starting constant region. Examples of native IgG variants or modified forms are described, for example, in Strohl, Curr. Opin. Biotechnol. 20(6):685-691 (2009); Presta, Curr. Opin. Immunol. 20(4):460 -470 (2008); Davis et al., Protein Eng. Des. Sel. 23(4):195-202 (2010), WO2009/086320, WO2008/092117; WO2007/041635; and WO2006/105338).

Within the scope of disclosures A and B described herein, the amino acid residues of the starting FcRn binding domain, the starting Fc region, or the starting constant region may include, for example, one or more mutations: for example, substitutions to and starting Fc region or a mutation in which the amino acid residues of the starting constant region are different amino acid residues; inserting one or more amino acid residues into the starting Fc region or the amino acid residues in the starting 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 modified amino acid sequence of the Fc region or constant region preferably contains at least a portion of the amino acid sequence of the Fc region or constant region that does not occur naturally. Such variants must have less than 100% sequence identity or similarity with the original Fc region or the original constant region. For example, the amino acid sequence of the variant and the amino acid sequence of the initial Fc region or the initial constant region are about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% Identity or similarity of at least less than 100%, more preferably about 90% to less than 100%, still more preferably about 95% to less than 100%. In a non-limiting example, the modified Fc region or constant region between disclosure A or B differs from the starting Fc region or starting constant region by at least one amino acid.

Within the scope of disclosures A and B described here, the Fc region or constant region having FcRn-binding activity in the acidic pH range and/or the neutral pH range can be obtained by any method. Specifically, variants of the Fc region or constant region that have FcRn-binding activity in the acidic pH range and/or in the neutral pH range can be modified by modifying the human IgG-type antibody that can serve as the starting Fc region or starting constant region. Amino acids are obtained. Suitably modified Fc or constant regions of IgG type antibodies include, for example, those of human IgG (IgG1, IgG2, IgG3 and IgG4 and variants thereof), and mutants spontaneously generated therefrom are also included in this IgG Fc region or constant region. For the Fc region or constant region of human IgG1, human IgG2, human IgG3 and human IgG4 antibodies, some subtype sequences due to gene polymorphism are recorded in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, where Either term can be used in revealing A or B. In particular, for the human IgG1 sequence, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM.

Revealing an embodiment of A or B, the modification to other amino acids is not particularly limited, as long as the obtained variant has FcRn-binding activity in the acidic pH range and/or in the neutral pH range, preferably in the neutral pH range. , the amino acid modification site that increases the FcRn-binding activity under neutral pH conditions is described in, for example, WO2013/046722. Such modification sites include, for example, one or more positions selected from the group consisting of: the Fc region or the constant region of a human IgG antibody, according to EU numbering 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 positions 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442, as recorded in WO2013/046722. WO2013/046722 also records that part of the ideal modification of the Fc region or constant region is, for example, modification of one or more amino acids selected from the group consisting of: According to the EU numbering system, the amino acid at position 256 becomes Pro, The amino acid at position 280 becomes Lys, the amino acid at position 339 becomes Thr, the amino acid at position 385 becomes His, the amino acid at position 428 becomes Leu, and the amino acid at position 434 becomes Trp, Tyr, Phe, Ala or His. The number of amino acids to be modified is not particularly limited, and the modification can be performed on a single position alone or at 2 or more positions. Modification of these amino acid residues can enhance FcRn binding of the Fc region or constant region of the IgG type antibody under neutral pH conditions. Modifications of these amino acid residues can also be appropriately introduced into antibodies revealing A or B.

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

In one embodiment, it is disclosed that the FcRn-binding activity of the FcRn-binding domain of antibody A or B is compared to a reference antibody containing the Fc region or constant region of a native IgG, or contains an increased starting Fc region or starting constant region The reference antibody is increased. That is, it is revealed that the FcRn-binding activity of the Fc region variant or constant region variant of A or B or the FcRn-binding activity of an antibody containing such a variant is greater than the FcRn-binding activity of the reference antibody). It can also mean that if compared to the FcRn-binding activity of the reference antibody, the antibody revealing A or B can be, for example: 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 100% or greater, 105% or greater, preferably 110% or greater, 115% or greater, 120% or greater, 125% or greater, more preferably 130% or greater, 135% or greater, 140% or greater, 145% or greater, 150% or greater , 155% or greater, 160% or greater, 165% or greater, 170% or greater, 175% or greater, 180% or greater, 185% or greater, 190% or greater, 195 % or greater, 2 times or greater, 2.5 times or greater, 3 times or greater, 3.5 times or greater, 4 times or greater, 4.5 times or greater or 5 times or greater.

In one embodiment, the amino acid sequence of the antibody to be modified to reveal A or B is preferably a human sequence (found in natural human-derived antibodies), so that when the antibody is administered into the living body (preferably into the human body), the amino acid sequence does not increase. Immunogenicity of antibodies. Alternatively, after modification, mutations can be introduced at positions other than the amino acid modification site, so that one or more of the FRs (FR1, FR2, FR3, and FR4) replace the adult sequence. Methods for substituting FR adult sequences are known in the art, including but not limited to the reporters Ono et al., Mol. Immunol. 36(6):387-395 (1999). Humanized methods are known in the art, including but not limited to those reported in Methods 36(1):43-60 (2005).

In one embodiment, the framework region sequences (also referred to as "FR sequences") of the heavy chain and/or light chain variable regions of the antibodies disclosing A or B may include human germline framework sequences. When the framework sequences are entirely human germline sequences, it is expected that the antibody will induce little or no immunogenic response when administered to humans (eg, for the treatment or prevention of a disease).

The FR sequence may suitably include, for example, a fully human FR sequence such as that shown in V-Base (vbase.mrc-cpe.cam.ac.uk/). These FR sequences can be used to reveal A or B as appropriate. The germline sequences can be grouped according to their similarities (Tomlinson et al. (J. Mol. Biol. 227:776-798 (1992); Williams et al. (Eur. J. Immunol. 23:1456-1461 (1993) ); and Cox et al. (Nat. Genetics 7:162-168 (1994)). The ideal germline sequence can be appropriately selected from: Vκ, which is classified into 7 subgroups; Vλ, which is classified into 10 subgroups; and VH, which are classified into 7 subgroups.

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

Complete human Vκ sequences 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 is classified as subgroup Vk1; A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, which is classified as subgroup Vk2; A11, A27, L2, L6, L10, L16 , L20, and L25, which are classified as subgroup Vk3; B3, which are classified as subgroup Vk4; B2 (also known as "Vk5-2"), which are classified as subgroup Vk5; or A10, A14, and A26 , which is classified as subgroup Vk6 (Kawasaki et al. (Eur. J. Immunol. 31:1017-1028 (2001)); (Hoppe Seyler Biol. Chem. 374:1001-1022 (1993)); Brensing-Kuppers et al. al. (Gene 191:173-181 (1997)).

Fully human Vλ sequences 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 are classified into subgroup VL1; V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19 are classified as subgroup VL2; V3-2, V3-3, and V3-4 are classified as subgroup VL3; V4-1, V4-2, V4-3, V4-4, and V4-6, classified as subgroup VL4; or V5-1, V5-2, V5-4, and V5-6, classified as subgroup VL5 (Kawasaki et al. Genome Res. 7:250- 261 (1997)).

Generally, such FR sequences may differ from each other in one or more amino acid residues. These FR sequences can be used to modify antibody amino acid residues. Modified fully human FR sequences that may be used also include, for example, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (see, e.g., Kabat et al. (1991) supra; Wu et al. (J. Exp. Med) . 132:211-250 (1970)).

Within the context of disclosures A and B described herein, "flexible residues" may refer to amino acid residue variations that exist at positions that exhibit high amino acid diversity, where the light or heavy chain is variable Regions contain amino acids that are somewhat different when compared to known amino acid sequences and/or natural antibody or antigen-binding domains. Positions showing high diversity are generally located in CDRs. Information provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) is useful for determining where there is high diversity in known and/or natural antibodies. Some online databases (vbase.mrc-cpe.cam.ac.uk/, bioinf.org.uk/abs/index.html) provide many collections of human light and heavy chain sequences and their positions. This sequence and position information is useful in determining the location of flexible residues. There is no limit. For example, when the amino acid residue at a specific position has variability, it is 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, this position can be judged to show (high) diversity.

In one embodiment, it will be understood that when the antibody disclosed for A or B contains all or a portion of the light chain variable region and/or the heavy chain variable region, the antibody may optionally include one or more appropriate flexible residues. For example, the heavy chain and/or light chain variable region sequence selected as having an FR sequence, which originally contains amino acid residues that will change the antigen-binding activity of the antibody according to ion concentration (hydrogen ion concentration or calcium ion concentration) conditions, can be It is designed to contain other amino acid residues in addition to these amino acid residues. In this case, the number and position of flexible residues, for example, may not be limited to specific embodiments, as long as the antigen-binding activity of the antibody disclosed A or B changes according to ion concentration conditions. Specifically, the CDR sequence and/or FR sequence of the heavy chain and/or light chain may include at least 1 flexible residue. For example, if the ion concentration is calcium ion concentration, the flexible residues that can be introduced into the light chain variable region sequence (the aforementioned Vk5-2) include, but are not limited to, one or more amino acid residue positions shown in Table 1 or Table 2. . Likewise suitable flexible residues may be introduced into, for example, ion concentration dependent antibodies or ion concentration independent antibodies containing all or part of the light chain variable region and/or the heavy chain variable region, to which the antibody may be exposed. At least one amino acid residue on the surface is modified to increase the isoelectric point value.

[Table 1] (Positions are shown according to Kabat numbering.)

[Table 2] (Positions are shown according to Kabat numbering.)

In one embodiment, when a chimeric antibody is humanized, one or more amino acid residues that may be exposed on the surface of the antibody are modified to increase the isoelectric point value of the chimeric antibody to produce a Chimeric antibodies without such modifications have shorter plasma half-lives than humanized antibodies revealing A or B. Modification of potentially surface-exposed amino acid residues of a humanized antibody can be performed before or simultaneously with humanization of the antibody. Alternatively, by using a humanized antibody as a starting material, the amino acid residues that may be exposed on the surface can be modified to further modify the isoelectric point value of the humanized antibody.

Adams et al. (Cancer Immunol. Immunother. 55(6):717-727 (2006)) reported humanized antibodies, trastuzumab (antigen: HER2), bevacizumab (antigen: VEGF) and pertuzumab (antigen: HER2), using the same human Antibody FR sequences are humanized and have pharmacokinetics that are generally comparable to those in plasma. Specifically, when humanized using the same FR sequence, the understanding of plasma pharmacokinetics is roughly comparable. According to one embodiment of Disclosure A, in addition to the humanization step, by modifying the amino acid residues exposed on the antibody surface to increase the isoelectric point value of the antibody, the concentration of the antigen in the plasma is reduced. In embodiments disclosing an alternative to either A or B, human antibodies may be used. By modifying the amino acid residues that may be exposed on the surface of human antibodies produced from human antibody libraries, the number of mice, recombinant cells, etc. that produce human antibodies can be increased, and the isoelectric point value and original production of human antibodies can be increased. The ability of human antibodies to eliminate antigens from plasma.

In one embodiment, the antibody of Disclosure A may include modified sugar chains. Antibodies with modified sugar chains include, for example, modified glycosylated antibodies (WO99/54342), fucose-deficient antibodies (WO00/61739; WO02/31140, WO2006/067847; WO2006/067913), and antibodies with centrates Antibodies against GlcNAc sugar chains (WO02/79255).

In one embodiment, antibodies revealing A or B can be used, for example, in techniques that demonstrate anti-tumor activity against cancer cells, or techniques that promote the elimination of bioharmful antigens from plasma.

In an alternative embodiment, disclosure A or B is directed to a library of ion concentration-dependent antigen-binding domains with increased isoelectric points or ion concentration-dependent antibodies with increased isoelectric points, as described above.

In an alternative embodiment, it is disclosed that A or B is related to a nucleic acid (polynucleotide) encoding an ion concentration-dependent antigen-binding domain with an increased isoelectric point or an ion concentration-dependent antibody with an increased isoelectric point. ). In a specific embodiment, the nucleic acid can be obtained using appropriate known methods. 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, WO2013/047 752、WO2013/125667、WO2014/ 030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, and WO2012/093704, each of which is incorporated by reference in its entirety.

In one embodiment, the nucleic acid disclosed in A or B may be an isolated or purified nucleic acid. The nucleic acid encoding an antibody to this disclosure A or B can be any gene and can be DNA or RNA or other nucleic acid analogs.

In disclosures A and B described here, if the amino acid of the antibody is modified, the amino acid sequence of the antibody before modification may be a known sequence or the amino acid sequence of a newly obtained antibody. It can be obtained, for example, from an antibody library or by selection of nucleic acids encoding antibodies derived from monoclonal antibody-producing B cells derived from fusion tumors. The method for obtaining nucleic acids encoding antibodies from fusion tumors can use the following technology: using the antigen of interest or cells expressing the antigen of interest as the sensitizing antigen (sensitizing antigen), immunization is carried out with conventional immunization methods; the obtained immune cells and Known parental cells are fused using known cell fusion methods; cells that produce monoclonal antibodies (fusion tumors) are screened using known screening methods; and reverse transcriptase is used to synthesize the variable variable of this antibody from the obtained fusion tumor mRNA. The cDNA of the region (V region); and linking this cDNA to the DNA encoding the constant region (C region) of the antibody of interest.

Inducible antigens used to obtain nucleic acids encoding the above-mentioned heavy chain and light chain, including but not limited to: complete antigens with immunogenicity; and incomplete antigens, including haptens without immunogenicity. For example, the entire protein of interest or partial peptides of this protein can be used. Also included are substances composed of polysaccharides, nucleic acids, lipids and other compositions known to have antigenic potential. Therefore, in some embodiments, the antigen of the antibody disclosed in A or B is not particularly limited. This antigen can be produced using, for example, baculovirus-based methods (see, for example, WO98/46777). Fusion tumors can be generated according to the method of G. Kohler and C. Milstein, Methods Enzymol. 73:3-46 (1981)). When the immunogenicity of the antigen is low, immunization can be achieved by linking the antigen with an immunogenic macromolecule such as albumin. Alternatively, if desired, a soluble antigen can be prepared by linking the antigen to other molecules. When using a transmembrane molecule such as a membrane antigen (eg, a receptor) as an antigen, a portion of the extracellular region of the membrane antigen can be used as a fragment, or cells expressing the transmembrane molecule on their surface can be used as an immunogen.

In some embodiments, antibody-producing cells can be obtained by immunizing an animal with an appropriate induction antigen as described above. Alternatively, antibody-producing cells can be prepared by immunizing lymphocytes capable of producing antibodies in a test tube. A variety of mammals are available for immunization, and other routine antibody production procedures are available. Commonly used animals include rodents, lagomorphs and primates. Animals may include, for example, rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates including monkeys such as Malayan monkeys, rhesus monkeys, baboons, and chimpanzees. In addition, transgenic animals carrying human antibody gene stocks are also known, and these animals can be used to obtain human antibodies (see, for example, WO96/34096; Mendez et al., Nat. Genet. 15:146-156 (1997); WO93/12227, WO92/03918, WO94/02602, WO96/34096, and WO96/33735). Instead of using such genetically modified animals, human lymphocytes can also be induced in a test tube with the desired antigen or cells expressing the antigen, and the infected lymphocytes can be fused with human myeloma cells such as U266. The desired human antibody having antigen-binding activity was obtained (JP Pat. Publ. No. H01-59878).

Animal immunization can be performed, for example, by appropriately diluting and suspending the antigen in phosphate buffered saline (PBS), physiological saline or others, and mixing it with an adjuvant to emulsify if necessary; and then injecting the antigen into the animal intraperitoneally or subcutaneously. It is then advisable to administer the infectious antigen mixed with Freud's incomplete adjuvant several times every 4 to 21 days. The production of antibodies can be known, 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 prepared using conventional fusion agents (such as polyethylene glycol) (Goding, Antibodies: Principles and Practice, Academic Press, 1986, 59-103) and myeloma cells. Fusion to create fusionomas. If necessary, the fusion tumors can be cultured and expanded, and the binding specificity of the antibodies produced by the fusion tumors can be assessed by, for example, immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA). If desired, the antibody-producing fusion tumors with determined specificity, affinity, or activity of interest can then be subpopulated using, for example, limiting dilution.

Nucleic acids encoding the selected antibodies can be obtained from fusion tumors or antibody-producing cells (infectious lymphocytes, etc.) using probes that specifically bind to the antibodies (for example, oligonucleotides complementary to sequences encoding the constant regions of the antibodies). Selective breeding. Alternatively, the nucleic acid can be cloned from mRNA using RT-PCR. The heavy and light chains used to generate antibodies revealing A or B can be derived, for example, from antibodies belonging to any Ig antibody class and subclass, preferably IgG.

In one embodiment, the nucleic acid encoding the amino acid sequence constituting the heavy chain (all or part thereof) and/or the light chain (all or part thereof) of the antibody revealing A or B is modified, for example, using genetic engineering techniques. Recombinant antibodies with artificial sequences modified, for example, to reduce heterogeneous antigenicity against humans, such as chimeric antibodies or humanized antibodies, may be suitably prepared by, for example, encoding and antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies The nucleotide residues of the amino acid sequences associated with the antibody, sheep antibody or camel antibody components are prepared. Chimeric antibodies can be obtained by, for example, connecting DNA encoding the variable region of a mouse-derived antibody and DNA encoding the constant region of a human antibody, encapsulating the connected DNA coding sequence into an expression vector, and then obtaining The recombinant vector is introduced into the host to express this gene. Humanized antibodies, also known as reshaped human antibodies, are antibodies in which a human antibody FR is linked in-frame to an antibody CDR isolated from a non-human mammal, such as a mouse, to form a coding sequence. DNA sequences encoding such humanized antibodies can be synthesized by overlap extension PCR using a number of oligonucleotides as templates. The materials and experimental methods of overlap extension PCR are described in WO98/13388, etc. For example, DNA encoding the amino acid sequence of an antibody variable region revealing A or B can be obtained by overlap extension PCR using a number of oligonucleotides designed to have overlapping nucleotide sequences. The overlapping DNA is then ligated in-frame to the DNA encoding the constant region to form the coding sequence. The DNA linked in the above manner can be inserted into an expression vector to allow the DNA to be expressed, and the obtained vector can be introduced into a host or host cell. Antibodies encoded by this DNA can be expressed using feeder hosts or cultured host cells. The expressed antibodies can be appropriately purified from the culture medium of the host etc. (EP239400; WO96/02576). The FRs of humanized antibodies linked via CDRs can be selected, for example, to allow the CDRs to form an antigen-binding site suitable for the antigen. If necessary, the amino acid residues constituting the FR of the variable region of the selected antibody can be modified with appropriate substitutions.

In one embodiment, to express an antibody or fragment thereof that reveals A or B, the nucleic acid cassette can be cloned into an appropriate vector. For this purpose, several types of vectors can be used, such as phagemid vectors. In general, phagemid vectors may contain various elements, including regulatory sequences such as promoters or signal sequences, phenotypic selection genes, origins of replication, and other elements.

Methods for introducing desired amino acid modifications into antibodies have been established in the art. For example, the library can be constructed by introducing a modification of at least 1 amino acid residue that may be exposed on the surface of the antibody revealing A or B and/or at least 1 amino acid that changes the antigen-binding activity of the antibody according to ion concentration conditions. to make. In addition, if necessary, 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 containing a nucleic acid encoding an ion concentration-dependent antigen-binding domain with an increased isoelectric point value as described above, or an ion concentration-dependent antibody with an increased isoelectric point value as described above. In one embodiment, the vector can be obtained by, for example, vectors described in the following documents: WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/11524 1 , WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227 or WO20 12/093704, each of which is incorporated by reference in its entirety.

In one embodiment, a nucleic acid encoding an embodiment revealing A or B can be operatively cloned (inserted into) an appropriate vector and introduced into a host cell. For example, when using E. coli as a host, vectors include selection vectors, pBluescript vectors (Stratagene), or any of a variety of other commercially available vectors.

In one embodiment, the expression vector may serve as a vector containing nucleic acid directed to reveal A or B. Expression vectors can be used to express polypeptides in vitro, in E. coli, in cultured cells, or in vivo. For example, pBEST vector (Promega) can be used for in vitro expression; pET vector (Invitrogen) can be used for E. coli expression; pME18S-FL3 vector (GenBank Accession No. AB009864) can be used for cultured cell expression; and pME18S vector (Takebe et al. , Mol. Cell Biol. 8:466-472 (1988)) for in vivo expression. DNA can be inserted into the vector using conventional methods, such as ligase reaction using restriction enzyme sites (see Current protocols in Molecular Biology edit. 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 an antigen-binding domain encoding an ion concentration-dependent increase in the isoelectric point value as described above or an ion concentration-dependent increase in the isoelectric point value as described above. sexual antibodies. In one embodiment, the host or host cell can be prepared by methods described in the following documents: 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、WO2012/0162 27 or WO2012/093704, each of which is incorporated by reference in its entirety.

The type of host cells used to reveal A or B is not particularly limited, and host cells include, for example, bacterial cells such as E. coli and various animal cells. Host cells can be appropriately used as production lines for the production and expression of antibodies. Both eukaryotic and prokaryotic cells can be used.

Eukaryotic cells as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells such as CHO (Puck et al., J. Exp. Med. 108:945-956 (1995)), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa , and Vero; amphibious cells such as Xenopus laevis oocytes (Valle et al., Nature 291:338-340 (1981)); and insect cells such as Sf9, Sf21 and Tn5. Recombinant vectors or other vectors can be introduced into host cells using, for example, the calcium phosphate method, the DEAE-dextran method, the method using cationic liposome DOTAP (Boehringer-Mannheim), electroporation, and lipostaining.

Plant cells known as protein production lines include, for example, tobacco (Nicotiana tabacum)-derived cells and duckweed (Lemna minor)-derived cells. Callus tissue can be cultured from these cells to produce antibodies revealing A or B. Fungal cell line protein production systems include production systems using yeast cells, such as cells of the genus Saccharomyces, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; and cells of filamentous fungi, such as Cells of the genus Kojima such as Kojima black. When using prokaryotic cells, bacterial cell line production lines can be used. Bacterial cell line production systems include, for example, production systems using Bacillus subtilis and E. coli.

To produce an A or B-revealing antibody using a host cell, the host cell is transformed with an expression vector containing a nucleic acid encoding an A- or B-revealing antibody and cultured to express the nucleic acid. For example, when animal cells are used as hosts, the culture medium may include, for example, DMEM, MEM, RPMI1640, and IMDM, which may be appropriately combined with serum supplements such as FBS or fetal calf serum (FCS), or the cells may be cultured without serum.

On the other hand, animals or plants can be used in an in vivo production system for the production of antibodies revealing A or B. For example, nucleic acids encoding antibodies revealing A or B can be introduced into such animals or plants to produce the antibodies in vivo, This antibody can then be collected from the animal or plant.

When using animals as hosts, production lines using mammals or insects can be used. Ideal mammals include, but are not limited to, goats, pigs, sheep, mice and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Genetically modified animals may also be used.

In one example, a nucleic acid encoding an antibody revealing A or B is prepared as a fusion gene encoding a polypeptide specifically included in milk, such as goat Beta-casein, and then a polynucleotide fragment containing this fusion gene is injected. Goat embryos, transplanted into female goats. The antibody of interest may be obtained from the milk secreted by a transgenic goat born from a goat that received the embryo or from its offspring. Hormones can be appropriately administered to the genetically modified goats to increase the amount of antibody-containing milk produced by the goats (Ebert et al., Bio/Technology 12:699-702 (1994)).

Insects useful in producing antibodies revealing A or B include, for example, silkworms. When silkworms are used, baculoviruses having polynucleotides encoding the antibodies of interest inserted into the viral genome are used to infect silkworms. Antibodies of interest can be obtained from body fluids of infected silkworms (Susumu et al., Nature 315:592-594 (1985)).

When using plants to produce antibodies that reveal A or B, tobacco can be used. When using tobacco, a recombinant vector obtained by inserting a polynucleotide encoding an antibody of interest into a plant expression vector such as pMON 530 can be introduced into bacteria such as Agrobacterium tumefaciens. The bacteria obtained can be used to infect tobacco, such as Nicotiana tabacum (Ma et al., Eur. J. Immunol. 24:131-138 (1994)), and the desired antibodies are obtained from infected tobacco leaves. Such modified bacteria can also be used to infect duckweed (Lemna minor), and the desired antibodies can be obtained from selected cells of infected duckweed (Cox et al. Nat. Biotechnol. 24(12):1591-1597 (2006) ).

In order to secrete the antibody expressed in the host cell into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, a stable secretion signal can be encapsulated in the polypeptide of interest. Such a signal may be endogenous to the antibody of interest, or may be a heterogeneous signal as is known in the art.

Antibodies revealing A or B produced as described above can be isolated from host cells or within or outside the host (such as culture medium and milk) and refined to substantially pure and homogeneous antibodies. This antibody can be appropriately isolated and purified, for example by appropriately selecting and combining chromatography for column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polypropamide gel electrophoresis , isoelectric focusing, dialysis, recrystallization, etc. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse chromatography, and adsorption chromatography. Such chromatography can be performed, for example, by using liquid chromatography such as HPLC and FPLC. The column used for affinity chromatography can be a Protein A column or a Protein G column. Protein A columns include, for example, Hyper D, POROS, Sepharose F.F. (Pharmacia).

If necessary, the antibody can be modified or the antibody can be treated with an appropriate protein-modifying enzyme to partially delete the peptide before or after antibody purification. For such protein-modifying enzymes, for example, trypsin, chymotrypsin, amide endopeptidase, protein kinase, and glucosidase can be used.

In an alternative embodiment, Disclosure A relates to a method for producing an antibody containing an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, which may include culturing host cells or feeding the host, and from the culture of these cells, host secretions , or other methods known in the art to collect antibodies.

In one embodiment, Disclosure A relates to a production method that may include one or more steps selected from the group consisting of: (a) selecting an antibody that promotes elimination of the antigen from plasma compared to a reference antibody; ( b) Select antibodies with enhanced binding activity to extracellular matrix; (c) Select antibodies with enhanced FcγR-binding activity under neutral pH conditions; (d) Select antibodies with enhanced FcγRIIb-binding activity under neutral pH conditions ; (e) Selecting an antibody that has maintained or increased FcγRIIb binding activity and reduced binding activity to one or more activating FcγRs selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc , FcγRIIIa, FcγRIIIb and FcγRIIa; (f) Select antibodies with enhanced FcRn-binding activity under neutral pH conditions; (g) Select antibodies with pI; (h) Confirm the isoelectric point (pI) of the collected antibodies, and then Select antibodies whose isoelectric point (pI) increases; and (i) select antibodies whose antigen-binding activity changes or increases according to ion concentration conditions.

The reference antibodies here include but are not limited to natural antibodies (such as natural Ig antibodies, preferably natural IgG antibodies) and antibodies before modification (before or during the construction of the antibody library, such as increasing the ion concentration dependence of the isoelectric point value Antibodies or antibodies that confer an ion concentration-dependent increase in the isoelectric point value in front of the antigen-binding domain).

After generating the antibody revealing A, the obtained antibody can be used for antibody pharmacokinetic analysis using, for example, the plasma of mice, rats, rabbits, dogs, monkeys, and humans to select antibodies that have improved antigen elimination from plasma compared with reference antibodies. .

Alternatively, after producing an antibody that reveals A, the obtained antibody and the reference antibody can be compared with respect to the extracellular matrix binding ability using electrochemical electroluminescence or other methods to select antibodies with increased binding to the extracellular matrix.

Alternatively, after producing an antibody that reveals A, the obtained antibody and the reference antibody can be compared with respect to the binding activity for various FcγRs under neutral pH conditions using BIACORE (registered trademark) or other tools to select the binding activity for various FcγRs under neutral pH conditions. Antibodies with increased activity. In this case, various FcγRs may focus on the type of FcγR, such as FcγRIIb. Likewise, antibodies can be selected that maintain or increase FcγRIIb binding activity (under neutral pH conditions) and have reduced binding activity to one or more activating FcγRs selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc, FcγRIIIa , FcγRIIIb and FcγRIIa, etc. In such a case, the FcγR can be a human FcγR.

Or after producing the antibody revealing A, the obtained antibody and the reference antibody can be compared with respect to the FcRn-binding activity under neutral pH conditions using BIACORE or other known techniques to select antibodies with increased FcRn-binding activity under neutral pH conditions. . In this case, the FcRn can be human FcRn.

Alternatively, after the antibody revealing A is produced, the isoelectric point of the obtained antibody can be evaluated by isoelectric focusing or other methods to select antibodies with an increased isoelectric point value compared to the reference antibody. In this case, an antibody whose isoelectric point value increases 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 can be selected; or the isoelectric point Antibodies with an increase in value of 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 producing an antibody revealing A, the obtained antibody can be compared with a reference antibody for binding activity to the desired antigen under low and high ion concentration conditions using BIACORE or other methods to select antibodies whose antigen-binding activity changes or increases according to 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 binding activity is altered or increased can be assessed based on the presence of, for example, the following: (a) altered or increased cellular uptake of the antigen; (b) altered or increased ability to bind to different antigen molecules multiple times; (c) reduced antigen concentration in plasma Change or increase; or (d) Change in plasma retention of antibodies. Or any two or more of these selection methods can optionally be combined.

In an alternative embodiment, disclosure A relates to a method of making or screening an antibody that contains an antigen-binding domain whose antigen-binding activity changes depending on ion concentration conditions and whose pI may be exposed on at least 1 of the antibody surface by modification amino acid residues ("ion concentration-dependent antibody with increased isoelectric point value"). The production method can, for example, optionally appropriately combine the embodiments described in the scope of disclosure A, such as the aforementioned embodiments of the method for manufacturing or screening antibodies for increased isoelectric point values, and for manufacturing or screening calcium ion concentration. Dependent antigen-binding domain or calcium ion concentration-dependent antibody or embodiment of the method of the aforementioned library, the antigen-binding activity of which is higher under high calcium ion concentration conditions than under low calcium ion concentration conditions, and/or is used for manufacturing or screening pH dependence Embodiments of the methods of antigen-binding domains or pH-dependent antibodies or libraries thereof have higher antigen-binding activity under neutral pH conditions than under acidic pH conditions.

In an alternative embodiment, Disclosure A provides a method of manufacturing or screening an antibody containing an antigen-binding domain with increased extracellular matrix binding activity, wherein the antigen-binding activity changes according to ion concentration conditions, and the pI is determined by The modification may increase the exposure of at least 1 amino acid residue on the surface of the antibody ("an ion concentration-dependent antibody with an increased isoelectric point value"). Ion concentration-dependent increases in the isoelectric point value of the antibody can be taken into account in this method. Such a method may be implemented, for example, by appropriately optionally combining relevant embodiments described within the scope of disclosure A, such as embodiments of methods for manufacturing or screening the aforementioned antibodies with increased isoelectric point values, and for manufacturing or Embodiments of methods for screening calcium ion concentration-dependent antigen-binding domains or calcium ion concentration-dependent antibodies or libraries thereof, whose antigen-binding activity is higher under high calcium ion concentration conditions than under low calcium ion concentration conditions, and/or for manufacturing or Embodiments of methods for screening pH-dependent antigen-binding domains or pH-dependent antibodies or libraries thereof, whose antigen-binding activity is higher under neutral pH conditions than under acidic pH conditions. For example, the obtained antibody can be compared with a reference antibody for extracellular matrix binding ability using electrochemical electroluminescence or other known techniques to select antibodies with increased extracellular matrix binding.

The reference antibodies here may include but are not limited to natural antibodies (such as natural Ig antibodies, preferably natural IgG antibodies) and antibodies before modification (before or during the construction of the antibody library, such as the ion concentration before the isoelectric point value increases. dependent antibodies, or antibodies that confer an ion concentration-dependent increase in the isoelectric point value before the antigen-binding domain).

In an alternative embodiment, Disclosure A relates to a method for producing an antibody, the antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, the method comprising the following steps: modifying at least one molecule that may be exposed on the surface of the antibody. Amino acid residues to increase the isoelectric point (pI). In some embodiments, the amino acid residue modification includes a modification selected from the group consisting of: (a) substituting a negatively charged amino acid residue with an uncharged amino acid residue; (b) substituting a negatively charged amino acid residue The negatively charged amino acid residue is 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 1 modified amino acid residue is substituted with histidine. In a further embodiment, the antibody includes a variable region and/or a constant region, and the amino acid residues are modified in the variable region and/or constant region. In a further embodiment, at least 1 amino acid residue modified according to this method is at a position in the CDR or FR selected from the group consisting of: (a) in the FR of the heavy chain variable region. Kabat numbering method 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, Positions 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112; (b) The CDRs of the heavy chain variable region are numbered according to Kabat numbering 31, 61, 62, 63, 64, 65, and 97; (c) 1, 3, 7, 8, 9, 11, 12, 16, 1, 3, 7, 8, 9, 11, 12, 16, in the FR of the light chain variable region according to the Kabat numbering system 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, Positions 80, 81, 85, 100, 103, 105, 106, 107, and 108; and (d) CDRs of the light chain variable region according to Kabat numbering 24, 25, 26, 27, 52, 53, 54 , 55, and 56 bits. In another embodiment, at least 1 amino acid residue modified according to this method is located at one position in the CDR or FR selected from the group consisting of: (a) 8 of the FR of the heavy chain variable region , 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105; (b) Heavy chain variable region 31, 61, 62, 63, 64, 65, and 97 in the CDR; (c) 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, FR of the light chain variable region Positions 77, 79, and 107; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 of the CDR of the light chain variable region. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further includes comparing the KD of the antibody produced by the method against the corresponding antigen at acidic pH (eg, pH 5.8) and neutral pH (eg, pH 7.4). In a further embodiment, the method includes selecting antibodies with a KD (acidic pH range (e.g., pH 5.8))/KD (neutral pH range (e.g., pH 7.4)) of 2 or higher against the antigen. In some embodiments, the method further includes comparing the antigen-binding activity of the antibody produced by the method under high ion concentration (such as hydrogen ion or calcium ion concentration) and low ion concentration conditions. In a further embodiment, the method further includes selecting an antibody with higher antigen-binding activity at a high ion concentration (eg, 2 times) than an antibody with a low ion concentration. In some embodiments, if the ion concentration is a calcium ion concentration, the high calcium ion concentration may be between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM Or choose between 400 μM and 1 mM. It is also suitable to select a concentration between 500 μM and 2.5 mM, which is close to the plasma (blood) concentration of calcium ions in vivo. In some embodiments, the low calcium ion concentration can be between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, or between 1 μM and 5 μM, or between 2 μM and 4 μM. Choose between. It is also advisable to choose a concentration between 1 μM and 5 μM, which is close to the calcium ion concentration in early endosomes in vivo. In some embodiments, the lower limit value of KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 μM Ca)/KD (2 mM Ca)) is 2 or more, 10 Or more or 40 or more, the high limit is 400 or less, 1000 or less or 10000 or less. In some alternative embodiments, the lower limit of kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) (e.g., kd (3 μM Ca)/kd (2 mM Ca)) is a value of 2 or more. More, 5 or more, 10 or more or 30 or more, the high limit is 50 or less, 100 or less or 200 or less. In some embodiments, if the ion 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, from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0 range. The low hydrogen ion concentration is preferably pH 7.4, which is close to the pH in plasma (blood) in vivo, but for convenience of measurement, for example, pH 7.0 can be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) can be selected from pH 4.0 to pH 6.5, from pH 4.5 to pH 6.5, from pH 5.0 to pH 6.5, or from pH 5.5 to pH 6.5. The acidic pH range is preferably pH 5.8, which is connected to the living hydrogen ion concentration in the early endosome, but for convenience of measurement, for example, pH 6.0 can 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, with the high limit being 400 or less, 1,000 or less, or 10,000 or less. In some embodiments, the method further includes comparing the elimination of antigen from plasma before administration of an antibody produced by this method and after administration of a different reference antibody that does not include modifications introduced by this method. In a further embodiment, the method further includes selecting antibodies produced by this method and antibodies that do not contain modifications introduced by this method, for promoting the elimination of the antigen from the plasma (eg, 2-fold). In some embodiments, the method further includes comparing the extracellular matrix binding of the antibody produced by this method with an antibody that is different only and without modifications introduced by this method. In a further embodiment, the method further includes selecting an antibody produced by this method that has increased extracellular matrix binding (e.g., 2 when bound to the antigen) compared to a different antibody that does not include the modification introduced by this method. times). In a further embodiment, the antibody produced according to this method has a pi before (natural antibody (such as a natural Ig antibody, preferably a natural antibody) before modification or modification of at least 1 amino acid residue to increase the pi IgG antibody) or a reference antibody (e.g., an antibody before modification of the antibody or before or during library construction), (substantially) retains this antigen-binding activity. In this case, "(substantially) retaining the antigen-binding activity" may mean that it is at least 50% or more, preferably 60% or more, more preferably 60% or more, compared to the binding activity of the antibody before modification or transformation. 70% or 75% or more, and more preferably 80%, 85%, 90% or 95% or more activity.

In an additional embodiment, Disclosure A relates to a method for producing an antibody, the antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, wherein the method includes modifying at least one molecule that may be exposed on the surface of the antibody constant region. Amino acid residues to increase isoelectric point (pI). In some embodiments, amino acid residue modifications include modifications selected from the group consisting of: (a) substituting a negatively charged amino acid residue with an uncharged amino acid residue; (b) substituting a negatively charged amino acid residue The amino acid residue is a positively charged amino acid residue; and (c) the uncharged amino acid residue is replaced by a positively charged amino acid residue. In some embodiments, at least 1 modified amino acid residue is substituted with histidine. In a further embodiment, the antibody includes a variable region and/or a constant region, and the amino acid residues are modified in the variable region and/or constant region. In a further embodiment, at least 1 amino acid residue modified according to this method is located in the constant region at a position selected from the group consisting of: 196, 253, 254, 256, 258 according to EU numbering ,278,280,281,282,285,286,307,309,311,315,327,330,342,343,345,356,358,359,361,362,373,382,384,385,386 , 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443. In a further embodiment, at least 1 amino acid residue modified according to this method is located in the constant region at a position selected from the group consisting of: 254, 258, 281, 282, 285 according to EU numbering ,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 bits. In another embodiment, at least 1 amino acid residue modified according to this method is located in the constant region at a position selected from the group consisting of: 282, 309, 311, 315, 342 according to EU numbering , 343, 384, 399, 401. In some embodiments, the method further includes comparing the antigen-binding activity of the antibody produced by the method under high ion concentration (such as hydrogen ion or calcium ion concentration) and low ion concentration conditions. In a further embodiment, the method further includes selecting antibodies with higher antigen-binding activity at high ion concentrations than antibodies at low ion concentrations. In some embodiments, the method further includes comparing the elimination of antigen from plasma before administration of an antibody produced by this method and after administration of a different reference antibody that does not include modifications introduced by this method. In a further embodiment, the method further includes selecting antibodies produced by this method and antibodies that do not contain modifications introduced by this method, for promoting the elimination of the antigen from the plasma (eg, 2-fold). In some embodiments, the method further includes comparing the extracellular matrix binding of the antibody produced by this method with an antibody that is different only and without modifications introduced by this method. In a further embodiment, the method further includes selecting an antibody produced by this method that has increased extracellular matrix binding (e.g., 2 when bound to the antigen) compared to a different antibody that does not include the modification introduced by this method. times). In some embodiments, the method includes an antibody made according to the method and a reference antibody including the constant region of a native IgG, directed against the Fc gamma receptor (FcγR) binding activity at neutral pH (eg, pH 7.4). In a further embodiment, the method includes the step of selecting an antibody produced according to the method that has enhanced FcγR binding activity at neutral pH (e.g., pH 7.4) compared to a reference antibody comprising the constant region of a native IgG. In some embodiments, antibodies produced according to this method are selected to have enhanced FcγRIIb binding activity at neutral pH. In some embodiments, the selected antibody produced according to this method has binding activity to one or more activating FcγRs (preferably selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa) and to FcγRIIb , optionally, the FcγRIIb binding activity is maintained or enhanced and the binding activity to activating FcγR is reduced compared to a reference antibody that differs only in that the constant region is that of a native IgG. In some embodiments, the method further includes comparing the FcRn-binding activity under neutral pH conditions of an antibody produced according to this method and a reference antibody that differs only in that the constant region is that of a native IgG. In a further embodiment, the method further includes selecting an antibody produced according to the method that has FcRn-binding activity under neutral pH conditions compared to a reference antibody that differs only in that the constant region is that of a native IgG. increase (e.g. 2 times). In some embodiments, the antibody produced according to this method has a pi before (natural antibody (such as a natural Ig antibody, preferably a natural IgG Antibodies) or reference antibodies (eg, antibodies before modification of the antibody or before or during library construction), (substantially) retain this antigen-binding activity. In this case, "(substantially) retaining the antigen-binding activity" may mean at least 50% or more, preferably 60% or more, and more preferably 70% compared to the binding activity of the antibody before modification or transformation. % or 75% or more, preferably 80%, 85%, 90% or 95% or more activity.

In additional embodiments, the method includes modifying at least one potentially surface-exposed amino acid residue of the variable and constant regions of the antibody to increase the isoelectric point (pI) value. In a further embodiment, at least one amino acid residue modified according to this method is located at one of the positions in the constant region disclosed above. In a further embodiment, at least one amino acid residue modified according to this method is located at one of the positions in the variable region disclosed above. In a further embodiment, at least 1 amino acid residue modified according to this method is located at one of the constant regions disclosed above and at least 1 amino acid residue modified according to this method is located at one of the above disclosed constant regions. A location in the variable region. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further includes comparing the KD of the antibody produced by the method against its corresponding antigen at acidic pH (eg, pH 5.8) and neutral pH (eg, pH 7.4). In a further embodiment, the method includes selecting antibodies having a KD (acidic pH range)/KD (neutral pH range) for the antigen of 2 or higher. In some embodiments, the method further includes comparing the antigen-binding activity of the antibody produced by the method under high ion concentration (such as hydrogen ion or calcium ion concentration) conditions and low ion concentration conditions. In a further embodiment, the method further includes selecting antibodies that have higher antigen-binding activity at high ion concentrations than at low ion concentrations. In some embodiments, if the ion concentration is a calcium ion concentration, the high calcium ion concentration may be selected from the group consisting of between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM or a concentration between 400 μM and 1 mM. A concentration chosen between 500 μM and 2.5 mM is also ideal. In some embodiments, the low calcium ion concentration can be selected from the group consisting of between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, or between 1 μM and 5 μM, or between 2 μM and 20 μM. 4 μM. Preferably, the concentration is selected from 1 μM and 5 μM. In some embodiments, the lower limit of the KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 μM Ca)/KD (2 mM Ca)) value is 2 or more, 10 Or more or 40 or more, the high limit is 400 or less, 1000 or less or 10000 or less. In some alternative embodiments, the lower limit of kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) (e.g., kd (3 μM Ca)/kd (2 mM Ca)) is 2 or more, 5 or more, 10 or more or 30 or more, with a high limit of 50 or less, 100 or less or 200 or less. In some embodiments, if the ion concentration is a hydrogen ion concentration, the low hydrogen ion concentration (neutral pH range) may be from pH 6.7 to pH 10.0, from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0 Choice. The low hydrogen ion concentration is preferably pH 7.4, which is close to the (blood) pH in plasma in vivo. For ease of measurement, for example, pH 7.0 can be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) may be selected from pH 4.0 to pH 6.5, from pH 4.5 to pH 6.5, from pH 5.0 to pH 6.5, or from pH 5.5 to pH 6.5. The acidic pH range can 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, with the high limit being 400 or less, 1,000 or less, or 10,000 or less.

In some embodiments, the method further includes comparing the elimination of antigen from plasma before administration of an antibody produced by this method and after administration of a different reference antibody that does not include modifications introduced by this method. In a further embodiment, the method further includes selecting antibodies produced by this method and antibodies that do not contain modifications introduced by this method, for promoting the elimination of the antigen from the plasma (eg, 2-fold). In some embodiments, the method further includes comparing the extracellular matrix binding of the antibody produced by this method with an antibody that is different only and without modifications introduced by this method. In a further embodiment, the method further includes selecting an antibody produced by this method that has increased extracellular matrix binding (e.g., 5 when complexed with the antigen) compared to a different antibody that does not include modifications introduced by this method. times). In some embodiments, the method further includes comparing the Fc gamma receptor (FcγR) binding activity at neutral pH (e.g., pH 7.4) of the antibody produced by the method and a reference antibody including the constant region of native IgG. In a further embodiment, the method includes selecting an antibody produced according to the method that has increased FcγR binding activity at neutral pH (e.g., pH 7.4) compared to an antibody comprising the constant region of a control native IgG. In some embodiments, selected antibodies produced according to this method have enhanced FcγRIIb binding activity at neutral pH. In some embodiments, the selected antibody produced according to this method has binding activity to one or more activating FcγRs (preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa) and FcγRIIb, And optionally, the FcγRIIb binding activity is maintained or increased, and the binding activity to activating FcγR is reduced. In some embodiments, the method further includes comparing the antibody produced according to this method with the constant region of a natural IgG only in the constant region FcRn binding activity under neutral pH conditions of different reference antibodies. In a further embodiment, the method further includes selecting an antibody produced according to the method that has FcRn-binding activity under neutral pH conditions compared to a reference antibody that differs only in that the constant region is that of a native IgG. increase (e.g. 2 times). The antibody produced according to this method is a natural antibody (such as a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody before modification or at least one amino acid residue is modified to increase the pI. (for example, the antibody before modification or library construction or during construction), (substantially) retains this antigen-binding activity. In this case, "(substantially) retaining the antigen-binding activity" may refer to at least 50% or more, preferably 60% or more, and more preferably 70% compared to the binding activity of the antibody before modification or transformation. % or 75% or more, preferably 80%, 85%, 90% or 95% or more activity.

In an alternative embodiment, Disclosure A relates to an antibody obtained by a method of making Disclosure A or screening for antibodies as described above.

In an alternative embodiment, disclosure A relates to a composition or pharmaceutical composition comprising an antibody of disclosure A above. In one embodiment, the pharmaceutical composition of Disclosure A can be a pharmaceutical composition for accelerating the elimination of antigens from the subject's biological fluid (preferably plasma, etc.) and/or for increasing extracellular matrix binding (if the antibody of Disclosure A is administered A pharmaceutical composition that is administered (applied) to the subject (preferably in vivo). The pharmaceutical composition of disclosure A may optionally contain a pharmaceutically acceptable carrier. Pharmaceutical compositions here generally refer to pharmaceuticals used to treat, prevent, diagnose or examine diseases.

The compositions or pharmaceutical compositions of Disclosure A may be suitably formulated. In some embodiments, it may be used parenterally, such as in the form of a sterile solution or suspension for injection in water or any other pharmaceutically acceptable liquid. The composition can be appropriately formulated with a pharmaceutically acceptable carrier or vehicle into a unit dose generally accepted in pharmaceutical practice. Such pharmaceutically acceptable carriers or media include, but are not limited to, sterile water, physiological saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavors, excipients, carriers, preservatives and binders. agent. The amount of active ingredients in the composition can be adjusted so that the dose falls within an appropriate preset range.

In some embodiments, the composition or pharmaceutical composition of Disclosure A can be administered parenterally. The composition or pharmaceutical composition may be suitably prepared, for example, as an injectable, nasal, transpulmonary, or transdermal composition. The composition or pharmaceutical composition may be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection.

In some embodiments, the present disclosure provides antibodies whose pI is increased (antibodies with increased isoelectric point values) by modifying at least 1 amino acid residue that may be exposed on the surface; methods of producing such antibodies; or the like Such antibodies enhance the elimination of antigens from plasma when administered into a subject. It is understood that the scope of the disclosure A described here and the contents described in the examples can be appropriately applied to such embodiments. In other embodiments, the present disclosure provides antibodies whose pI is reduced by modifying at least 1 amino acid residue that may be exposed on the surface ("an antibody with a reduced isoelectric point value"); methods of producing such antibodies; or the use of such antibodies to improve plasma retention when the antibody system is administered to a subject in vivo. The inventors found that the cellular internalization of antibodies can be enhanced by introducing specific amino acid mutations into specific parts of the amino acid sequence of the constant region to increase the isoelectric point value. Those with ordinary knowledge in this technical field will understand that by introducing amino acids with different side chain charges into the above-mentioned sites, the isoelectric point value is reduced and the cellular internalization of the antibody is inhibited, so that the plasma retention of the antibody can be prolonged. It can be understood that the contents described in the scope of disclosure A and the paired examples can be appropriately applied to such an implementation.

In one embodiment, the present disclosure provides a method for producing a modified antibody that has an extended or reduced half-life in plasma compared to the antibody before modification, the method comprising: (a) modifying a nucleic acid encoding the antibody before modification to change The charge of at least 1 amino acid residue at a position selected from the group consisting of: 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285 according to EU numbering ,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; (b) culturing host cells to express the modified nucleic acid to produce the antibody; and (c) ) Collect produced antibodies from host cell cultures.

An additional embodiment provides a method of extending or shortening the half-life of an antibody in plasma, comprising modifying at least 1 amino acid residue selected from the group consisting of: According to EU numbering law 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 bits.

Such methods may further include comparing the antibody before modification and determining whether the half-life of the collected and/or modified antibody in plasma is extended or shortened.

Changes in charge can be achieved by amino acid substitution. In some embodiments, the substituted amino acid residue may be selected from the group consisting of: amino acid residues of groups (a) and (b), but is not limited thereto: (a) Glu (E ) and Asp (D); and (b) Lys (K), Arg (R) and His (H).

In some embodiments, the antibody can 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.

Reveal B In non-limiting embodiments, Series B is disclosed regarding Fc region variants, their uses, and methods of making them.

Within the scope of disclosures A and B described herein, "Fc region variant" may refer to modification of the Fc region of a natural IgG antibody, for example, by modifying at least one amino acid of the Fc region to another amino acid. , or may refer to an Fc region that is modified by additionally modifying at least one amino acid of the Fc region variant to another amino acid. Here, Fc region variants include not only Fc regions into which amino acid modifications have been introduced, but also include Fc regions containing the same amino acid sequence as the aforementioned Fc region.

In an alternative embodiment, B is disclosed for an Fc region variant that contains an FcRn binding domain containing Ala at position 434; any one of Glu, Arg, Ser, and Lys at position 438; and Glu, Asp and Gln at position 440 (in the scope of disclosure B disclosed here, such Fc region variants are also referred to as "novel Fc region variants" for documentation purposes), in accordance with EU numbering.

Practically, it is revealed that Fc region variants of B can be introduced into almost any antibody (eg, multispecific antibodies such as bispecific antibodies) regardless of the type of target antigen. For example, the Fc region variant shown in Example 20 can be used to produce anti-Factor IXa/Factor :324) as heavy chain and F8ML (SEQ ID NO:325) as light chain]; F8M-F1868mv [F8M-F1868mv1 (SEQ ID NO:326) and F8M-F1868mv2 (SEQ ID NO:327) as heavy chain and F8ML (SEQ ID NO:325) as the light chain]; and F8M-F1927mv [F8M-F1927mv1 (SEQ ID NO:328) and F8M-F1927mv2 (SEQ ID NO:329) as the heavy chain and F8ML (SEQ ID NO:325) as light chain]).

As mentioned above, WO2013/046704 reports that Fc region variants that have introduced mutations that increase FcRn binding under acidic conditions and specific mutations (a representative example is a double residue mutation, according to the EU numbering method Q438R/S440E) show binding to rheumatoid arthritis. factor is significantly reduced. However, WO2013/046704 does not record that the Fc region variant that reduces rheumatoid factor binding due to Q438R/S440E modification has better retention in plasma than antibodies with natural Fc region. Safer and more favorable Fc region variants that improve plasma retention but do not bind to pre-existing ADA are therefore sought. The inventors herein disclose safer and more advantageous Fc region variants that improve plasma retention but do not bind to anti-drug antibodies (pre-existing ADA, etc.). Specifically, it is disclosed here for the first time that unexpectedly, the combination includes amino acid residue mutations (the amino acid at position 434 is replaced by Ala (A) according to EU numbering) and specific two-residue mutations (representative examples are The Fc region variant Q438R/S440E) is suitable for prolonging the retention of antibodies in plasma and maintaining significantly reduced binding to rheumatoid factor.

The novel Fc region variants of Disclosure B disclosed herein therefore provide an advantageous and surprising improvement over the Fc region variants described in WO2013/046704, which is hereby incorporated by reference in its entirety.

In one embodiment, Disclosure B provides novel amino acid substitution combinations of the FcRn binding domain that increase the FcRn binding activity of the antibody in the acidic pH range and in the neutral pH range, especially in the acidic pH range.

In one embodiment, it is disclosed that an Fc region variant of B contains 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 method; and preferably contains Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, in accordance with the EU numbering method. Ideally, the Fc region variant of Disclosure B additionally contains Ile or Leu at position 428, and/or any of Ile, Leu, Val, Thr and Phe at position 436, according to EU numbering. Preferably, the Fc region variant contains Leu at position 428, and/or Val or Thr at position 436, according to EU numbering.

In one embodiment, it is disclosed that the Fc region variant of B can be an Fc region variant of a natural Ig antibody, more preferably an Fc region variant of a natural IgG (IgG1, IgG2, IgG3 or IgG4 type) antibody. The native Fc region is partially described within the scope of disclosure A and B. More specifically, in Disclosure B, the natural Fc region may refer to an unmodified or naturally occurring Fc region, and is preferably an unmodified or naturally occurring Fc region of a natural Ig antibody, in which the amine group of the Fc region Acid residues were left unmodified. The source of the antibody for the Fc region may be Ig, such as IgM or IgG, such as human IgGl, IgG2, IgG3 or IgG4. In one embodiment, it can be human IgG1. A (control) antibody that includes a native Fc region may refer to an antibody that contains an unmodified or naturally occurring Fc region.

Positions 428, 434, 438, and 440 are common in the Fc region of all natural human IgG1, IgG2, IgG3, and IgG4 antibodies. However, at position 436 of the Fc region, natural human IgG1, IgG2 and IgG4 antibodies are all Tyr (Y), and natural human IgG3 antibodies are Phe (F). On the other hand, Stapleton et al. (Nature Comm. 599 (2011) reported that the human IgG3 subtype containing the amino acid substitution R435H according to EU numbering has a plasma half-life comparable to IgG1. Therefore, the inventors also envisioned plasma retention It can be improved by introducing a combination of R435H amino acid substitution and amino acid substitution at position 436 to increase FcRn binding under acidic conditions.

WO2013/046704 also specifically reports the substitution of diamino acid residues according to EU numbering method Q438R/S440E, Q438R/S440D, Q438K/S440E and Q438K/S440D, which when combined with FcRn binding that can increase in acidic conditions, will Significantly reduces rheumatoid factor binding.

Therefore, in a preferred embodiment, the FcRn binding domain of the Fc region variant of disclosure B may include a combination of substituted amino acid positions selected from the group consisting of: (a) According to EU numbering, 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/R4 35H/ F436T/Q438K/ S440E; (p) N434A/R435H/F436T/Q438K/S440D; (q) N434A/R435H/F436V/Q438R/ S440E; (r) N434A/R435H/F436V/Q438R/S440D; (s) N4 34A/ R435H/F436V/Q438K/ S440E; (t) N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v) M428L/N434A/Q438R/S440D; (w) M428L/N4 34A/ Q438K/S440E; (x) M428L/ N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/ Y436T/Q438R/S440D; (aa) M428L/N434A/Y 436T/ Q438K/S440E; (ab) M428L/N434A/ Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad) M428L/N434A/ Y436V/Q438R/S440D; (ae) M428L/N4 34A/ Y436V/Q438K/S440E; (af) M428L/N434A/ Y436V/Q438K/S440D; (ag) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/ S440E; and (ah) L235R/G236R/A327 G/A330S/ P331S/M428L/N434A/Y436T/Q438R/S440E.

In a preferred embodiment, the FcRn binding domain of the Fc region variant of Disclosure B may include a combination of substituted amino acids selected from the group consisting of: (a) According to EU numbering, N434A/Q438R/S440E; (B) N434A/Y436T/Q438R/S440E; (C) N434A/Y436V/Q438R/S440E; (D) M428L/N434A/Q438R/S440E; ) 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.

In one embodiment, it is disclosed that the Fc region variant of B has increased FcRn binding activity under acidic pH conditions compared to the Fc region of native IgG.

Increased FcRn-binding activity (binding affinity) of the FcRn-binding domain over a pH range may correspond to a lower measured FcRn-binding activity (binding affinity) than the measured FcRn-binding activity (binding affinity) of the native FcRn-binding domain. sex) has increased. In this case, KD (native Fc region)/KD (Fc region variant revealing B), which represents the difference in binding activity (binding affinity), can be at least 1.5 times, 2 times, 3 times, 4 times, 5 times , 10 times, 15 times, 20 times, 50 times, 70 times, 80 times, 100 times, 500 times or 1000 times. Such an increase can occur in the acidic pH range and/or in the neutral pH range; but for revealing the mechanism of action of B, an increase in the acidic pH range is more ideal.

In some embodiments, it is revealed that the increase in FcRn-binding activity of the Fc region variant of B in the acidic pH range (for example, at pH 6.0 and 25°C) is greater than that of the Fc region of native IgG, for example, 1.5 times, 2 times, 3 times. Times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 50 times, 75 times, 100 times, 200 times, 500 times, 1000 times or more. In some embodiments, the increase in FcRn binding activity of an Fc region variant in the acidic pH range may be at least 5-fold or at least 10-fold greater than the FcRn binding activity of the Fc region of native IgG.

Manipulating the FcRn binding domain by introducing amino acid substitutions can occasionally reduce antibody stability (WO2007/092772). Proteins with poor stability tend to aggregate easily during storage, and the stability of pharmaceutical proteins is very important in the manufacture of pharmaceutical agents. Therefore, the decrease in stability caused by the substitution of the Fc region will make it difficult to develop stable antibody preparations (WO2007/092772).

The purity of pharmaceutical proteins in the form of monomers and high molecular weight forms is also important for the development of pharmaceutical agents. After purification with Protein A, wild-type IgG1 does not contain significant amounts of high molecular weight species, whereas manipulating the FcRn binding domain by introducing substitutions may produce large amounts of high molecular weight species. In this case, such high molecular weight species must be removed from the drug using purification steps.

Amino acid substitutions in antibodies may have negative effects, such as increasing the immunogenicity of therapeutic antibodies, resulting in cytokine storms and/or the production of antidrug antibodies (ADAs). The clinical utility and efficacy of therapeutic antibodies may be limited by ADAs, which can affect the pharmacokinetics of therapeutic antibodies and sometimes cause serious side effects. There are many factors that influence the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is one of them. Likewise, the presence of pre-existing antibodies against therapeutic antibodies is also a problem. Such pre-existing antibodies are such as rheumatoid factor (RF), which are autoantibodies (antibodies directed against self-proteins) against the Fc portion of the antibody (i.e. IgG). Rheumatoid factor is particularly found in patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis. In patients with arthritis, RF combines with IgG to form immune complexes and contribute to disease progression. Recently, humanized anti-CD4 IgG1 antibodies with the Asn434His mutation were reported to induce significant rheumatoid factor binding (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011)). Detailed studies have confirmed that the Asn434His mutation of human IgG1 will increase the binding of the Fc region of the antibody to rheumatoid factors compared with the parent human IgG1.

RF is a multi-strain autoantibody against human IgG. The RF epitope of the human IgG sequence varies among clones; however, the RF epitope appears to be located at the CH2/CH3 junction and CH3 domain, overlapping with the FcRn-binding epitope. Therefore, mutations that increase FcRn binding activity at neutral pH may also increase binding activity for specific RF colonists.

In the context of Disclosure B, the term "drug-resistant antibody" or "ADA" may refer to an endogenous antibody that has binding activity for an epitope located on a therapeutic antibody and thus can bind to the therapeutic antibody. The term "pre-existing anti-drug antibodies" or "pre-existing ADA" may refer to anti-drug antibodies that are present and detectable in the patient's blood before the therapeutic antibodies are administered to the patient. In some embodiments, the pre-existing ADA is a human antibody. In further embodiments, the pre-existing ADA is rheumatoid factor.

Binding activity of the antibody Fc region (variant) for pre-existing ADA is expressed, for example, by an electrochemiluminescence (ECL) response at acidic pH and/or at neutral pH. The ECL analysis method is described in, for example, Moxness et al. (Clin Chem. 51:1983-1985 (2005)) and Example 6. The analysis can be performed under conditions such as MES buffer and 37°C. The antigen-binding activity of the antibody can be determined using BIACORE (registered trademark) analysis, for example.

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

To the extent disclosure B is described herein, a significant increase in or equivalent binding activity for (pre-existing) ADA may refer to a detected variant of the Fc region of disclosure B or an antibody containing such a variant (pre-existing) ADA Binding activity (binding affinity) (i.e. KD) of a reference antibody containing the reference Fc region variant compared to the measured reference Fc region variant, e.g. increased 0.55 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times , 2.2 times or 2.3 times or more. Such increased binding activity to pre-existing ADA may be observed in individual patients or in groups of patients.

In one embodiment, the terms "patients" or "patients" as used in the context of disclosure B are not limiting and include all persons suffering from a disease treated with a therapeutic antibody. The patient may be a person affected by an autoimmune disease such as joint disease or systemic lupus erythematosus (SLE). Joint diseases can include rheumatoid arthritis.

In one embodiment of Disclosure B, the binding activity to pre-existing ADA is significantly increased in an individual patient, which may refer to the binding of an antibody (e.g., a therapeutic antibody) containing an Fc region variant to pre-existing ADA detected in the patient. Activity that increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or more compared to the binding activity of the reference antibody for pre-existing ADA. Or it can be pointed out that the ECL response of this antibody should be 250 or at least 500 or at least 1000 or at least 2000 or more. Ideally, this increase would be relative to an increase in a reference antibody with an ECL response of less than 500 or 250. Specifically, between the binding activity of the reference antibody to pre-existing ADA and the binding activity of the antibody with Fc region variants, the ECL response should be less than 250 to at least 250, less than 250 to at least 500, less than 500 and 500 or more, less than 500 to 1000 or more, or less than 500 to at least 2000, but not limited.

In one embodiment, increased binding activity to pre-existing ADA may refer to a group of patients that includes (a) increased binding activity to FcRn at acidic pH and (b) increased binding activity to pre-existing ADA at neutral pH. The measured fraction of patients with an ECL response of at least 500 (preferably at least 250) to the antibody to the increased Fc region variant compared to the fraction of patients with an ECL response of at least 500 (preferably at least 250 or more) to the reference antibody Partial improvement, for example, a partial improvement of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the proportion of patients with ECL responses to the reference antibody.

In one embodiment of Disclosure B, reduced binding activity to pre-existing ADA may refer to reduced binding activity (i.e., KD or ECL response) measured for an antibody containing an Fc region variant compared to a reference antibody. Such reductions may be observed in individual patients or in groups of patients. In each patient, a significant reduction in the affinity of the Fc region variant-containing antibody for pre-existing ADA at neutral pH may refer to the binding activity for pre-existing ADA at neutral pH measured in the patient, compared to the measured binding activity for pre-existing ADA at neutral pH. The resulting reference antibody has, for example, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% reduction in binding activity to pre-existing ADA at neutral pH.

Alternatively, in an individual patient, a significant reduction in the binding activity of an antibody containing an Fc region variant to pre-existing ADA may mean that the original ECL response to the antibody is 500 or more (preferably) compared to the ECL response to the reference antibody. 1,000 or more or 2,000 or more) becomes less than 500, preferably less than 250.

In a preferred embodiment, it is disclosed that Fc region variants of B and antibodies containing Fc region variants have low binding activity to pre-existing ADA at neutral pH. Specifically, it is preferred that an antibody containing an Fc region variant revealing B has lower binding activity to pre-existing ADA at neutral pH than a reference antibody containing an Fc region of native IgG that has a lower binding activity to pre-existing ADA at neutral pH (e.g., pH 7.4). The binding activity of ADA may not be significantly increased in the presence of ADA. For pre-existing ADA, low binding activity (binding affinity) or affinity at baseline levels may mean, but is not limited to, an ECL response of less than 500 or less than 250 in an individual patient. Low binding activity to pre-existing ADA in a group of patients may, for example, mean that the ECL response in this group of patients is 90%, preferably 95%, more preferably 98%, less than 500.

It is appropriate to select B-revealing Fc region variants or antibodies containing the same that do not significantly increase the binding activity of (pre-existing) ADA in plasma at neutral pH and have FcRn binding activity at neutral pH and/or acidic pH. increaser. Desirably, the FcRn binding activity increases at acidic pH (eg pH 5.8). In one embodiment, the Fc region variant preferably does not significantly increase the binding activity to ADA under neutral pH conditions (such as pH 7.4) compared to the Fc region of native IgG. ADA may be pre-existing ADA, preferably rheumatoid arthritis. sex factor (RF).

In one embodiment, it is appropriate to disclose that the Fc region variant of B has increased FcRn binding activity under acidic pH conditions compared to the Fc region of native IgG, and as a result, the clearance rate (CL) in plasma is reduced and the retention in plasma is reduced. The time is prolonged or the half-life (t1/2) in plasma is prolonged. This correlation is known in the art.

In one embodiment, it is appropriate to reveal that the Fc region variant of B has increased FcRn-binding activity under acidic pH conditions but does not significantly increase ADA-binding activity under neutral pH conditions compared to the Fc region of natural IgG. As a result, in plasma The clearance rate (CL) is reduced, the residence time in plasma is prolonged or the half-life (t1/2) in plasma is prolonged. The ADA can be pre-existing ADA, preferably rheumatoid factor (RF).

In one embodiment, it is disclosed that the Fc region variant of B is advantageous because its plasma retention is compared to the reference Fc region variant containing the combination of amino acid substitutions N434Y/Y436V/Q438R/S440E according to EU numbering. improved.

Examples 5 to 7 compare two Fc region variants: the Fc region variant F1718 described in WO2013/046704 (Fc region, mutations introduced at 4 locations: N434Y/Y436V/Q438R/S440E), and a novel Fc region variant Plasma retention of F1848m (introduced mutations at 4 sites: N434A/Y436V/Q438R/S440E). The only difference in the amino acid mutations of the two Fc region variants is that according to EU numbering, the introduced amino acid mutation is Y (tyrosine) at position 434 for F1718, and A (alanine) for F1848m. While F1848m showed improved plasma retention compared to native IgG1, F1718 did not show such improved plasma retention (see Example (7-2)). Therefore, it is disclosed that the Fc region variant of B should have improved plasma retention compared to the reference Fc region variant containing the combination of amino acid substitutions N434Y/Y436V/Q438R/S440E according to EU numbering. The experimental results described in Examples (5-2) and (7-3) prove that various Fc region variants, namely F1847m, F1886m, F1889m, and F1927m, have better improvement in plasma retention than F1848m. Therefore, a person with ordinary knowledge in this technical field can know that the Fc region variant of the disclosure B including F1847m, F1886m, F1889m or F1927m and F1848m, compared with the reference Fc region variant including the replacement of N434Y/Y436V/Q438R/S440E, there are Better improvement in plasma retention.

Binding to FcγR or α complement proteins may also have adverse effects (eg inappropriate platelet activation). Fc region variants that do not bind to effector receptors, such as the FcyRIIa receptor, are safer and/or more beneficial. In some embodiments, it is disclosed that Fc region variants of B have only weak effector receptor binding activity or do not bind to effector receptors. Examples of effector receptors include activating FcγRs, particularly FcγRI, FcγRII, and FcγRIII. FcγRI includes FcγRIa, FcγRIb, and FcγRIc, and their subtypes. FcγRII includes FcγRIIa (with 2 subtypes: R131 and H131) and FcγRIIb. FcγRIII includes FcγRIIIa (with 2 subtypes: V158 and F158) and FcγRIIIb (with 2 subtypes: FcγRIIIb-NA1 and FcγRIIIb-NA2). Antibodies that have only weak effector receptor binding activity or do not bind to effector receptors include, for example, antibodies containing a silent Fc region and antibodies without an Fc region (e.g., Fab, F(ab)' ₂ , scFv, sc(Fv ) ₂ and diabodies).

Fc regions that have only weak effector receptor binding activity or do not bind to effector receptors are described, for example, by Strohl et al. (Curr. Op. Biotech. 20(6):685-691 (2009)), particularly including For example, deglycated Fc region (N297A and N297Q) and silent Fc region are used to silence its effector function (or suppress immunity) by manipulating the Fc region (IgG1-L234A/L235A, IgG1-H268Q/A330S/P331S, IgG1 -C226S/ C229S, IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S, IgG4-L2 35A/G237A/E318A and IgG4 -L236E). WO2008/092117 describes antibodies containing silent Fc regions containing the substitutions G236R/L328R, L235G/G236R, N325A/L328R or N325L/L328R according to EU numbering. WO2000/042072 describes an antibody containing a silent Fc region, which includes substitutions at one or more of the following positions: EU233 (position 233 according to EU numbering), EU234, EU235 and EU237. WO2009/011941 describes an antibody containing a silent Fc region, which lacks residues EU231 to EU238. Davis et al. (J. Rheum. 34(11):2204-2210 (2007)) describe antibodies with silent Fc regions, including the substitutions C220S/C226S/C229S/P238S. Shields et al. (J. Biol. Chem. 276(9):6591-6604 (2001)) describe an antibody containing a silent Fc region containing the substitution D265A. Modifications of these amino acid residues can also be appropriately introduced into Fc region variants revealing B.

The expression "weak binding to effector receptors" may mean that the effector receptor binding activity is less than, for example, 95% or less, preferably 90%, compared to native IgG or an antibody containing a native IgG Fc region. 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.

A "silent Fc region" is an Fc region variant that includes one or more amino acid substitutions, insertions, additions, deletions, etc., and has reduced binding to effector receptors compared to the native Fc region. Because the effector receptor binding activity can be significantly reduced, such a silent Fc region will no longer bind to the effector receptor. Silent Fc regions may include, for example, Fc regions 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 EU332. Modifications of these amino acid positions can also be appropriately introduced into Fc region variants revealing B.

In a further embodiment, the silent Fc region has 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 EU332, preferably selected from the group consisting of: EU235, EU237, EU238, EU239, EU270, EU298, EU325, and EU329 The group, wherein, is substituted with an amino acid residue selected from the following:

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, with 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 at position EU297 is preferably substituted 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 should be replaced 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 should be 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 include: EU235 replaced by Lys or Arg, EU237 replaced by Lys or Arg, EU238 replaced by Lys or Arg, EU239 replaced by Lys or Arg, EU270 replaced by Phe, EU298 replaced by Gly , substituted by Gly at EU325, or substituted by Lys or Arg at EU329. More preferably, the silent Fc region may include: EU235 substituted by arginine or EU239 substituted by lysine. It is better for the silent Fc area to include L235R/S239K instead. Modifications of these amino acid residues can also be appropriately introduced into Fc region variants revealing B.

In one embodiment, antibodies comprising Fc region variants of disclosure B have only weak complement protein binding activity or do not bind to complement proteins. In some embodiments, the complement protein is Clq. In some embodiments, weak complement protein binding activity refers to a complement protein binding activity that is reduced by 10-fold or more, 50-fold or more compared to the complement protein binding activity of native IgG or antibodies including the native IgG Fc region. More or 100 times or more. The complement protein binding activity of the Fc region can be reduced using amino acid modifications such as amino acid substitutions, insertions, additions or deletions.

In one embodiment, the (human) FcRn binding activity of the Fc region variant of Disclosure B or the antibody containing the Fc region variant can be evaluated in the same manner as above for its (human) FcRn binding activity in the neutral pH range and/or in the acidic pH range. .

In one embodiment, a method of modifying an antibody constant region to create Fc region variants revealing B can be based, for example, on evaluating the structural isotypes of several constant regions (IgG1, IgG2, IgG3, and IgG4) to select antigens in the acidic pH range. Constructive isoforms with reduced binding activity and/or increased dissociation rates in the acidic pH range. Alternative approaches may be based on the introduction of amino acids that are substituted for the amino acid sequence of the native IgG structural isotype to reduce the antigen-binding activity in the acidic pH range (e.g., pH 5.8) and/or to increase the dissociation rate in the acidic pH range. The hinge region sequence of the antibody constant region is significantly different among different structural isotypes (IgG1, IgG2, IgG3, and IgG4). The differences in the amino acid sequences of the hinge region significantly affect the antigen-binding activity. Therefore, structural isoforms with reduced antigen-binding activity in the acidic pH range and/or increased dissociation rates in the acidic pH range can be selected by selecting appropriate structural isoforms based on the type of antigen or epitope. And because the difference in the amino acid sequence of the hinge region has a significant impact on the antigen-binding activity, the amino acid substitution of the amino acid sequence of the natural structurally homologous amino acid sequence can be located in the hinge region.

In an alternative embodiment, Disclosure B provides the use of an antibody containing an Fc region variant of Disclosure B as described above to accelerate the release of an antibody that has been internalized into cells in an antigen-bound form into an antigen-free form to the outside of the cell. Here, "the antibody that has been internalized into the cell in the antigen-binding form is released out of the cell in the antigen-free form" does not necessarily mean that the antibody that has been internalized in the cell in the antigen-binding form is completely released in the antigen-free form. Extracellular. Compared with before modification of the FcRn-binding domain (for example, before increasing the FcRn-binding activity of the antibody in the acidic pH range), it is acceptable for some antibodies to be completely released outside the cell in the form of antigen-free form. It is preferred that the antibody released outside the cell maintains its antigen-binding activity.

The term "ability to eliminate antigen from plasma" or equivalent terms may refer to the ability to eliminate antigen from plasma when the antibody is administered or secreted in vivo. Therefore, "the antibody has an increased ability to eliminate antigens from plasma" can mean that if the antibody is administered, the rate of antigen elimination from plasma is increased compared to before modifying its FcRn-binding domain. The increased plasma antigen-eliminating activity of an antibody can be assessed, for example, by administering a soluble antigen and the antibody in vivo and measuring the soluble antigen concentration in the plasma after administration. The soluble antigen can be an antibody-bound antigen or an antigen without antibody binding, and the concentration can be determined by "antibody-bound antigen concentration in plasma" and "antibody-unbound antigen concentration in plasma" respectively. The latter is synonymous with "free antigen concentration in plasma". "Total antigen concentration in plasma" may refer to the sum of the concentration of antigen bound to antibodies and the concentration of antigen without bound antibodies.

In an alternative embodiment, Disclosure B provides a method of extending the plasma residence time of an antibody containing an Fc region variant of Disclosure B. Natural human IgG can bind to FcRn from non-human animals. For example, because native human IgG binds to mouse FcRn more strongly than human FcRn (Ober et al., Intl. Immunol. 13(12):1551-1559 (2001)), this antibody was administered to mice to evaluate Measure the properties of this antibody. Or, for example, mice whose own FcRn gene is disrupted but whose human FcRn gene is replaced and expressed as transgenic mice (Roopenian et al., Meth. Mol. Biol. 602:93-104 (2010)) are also suitable for evaluating this antibody.

Within the scope of disclosures A and B described here, one can determine the plasma concentration of free antigen not bound to the antibody, or the ratio of the free antigen concentration to the total antigen concentration (e.g., Ng et al., Pharm. Res. 23 (1):95-103 (2006)). Alternatively, if the antigen displays a specific function in vivo, whether the antigen is bound to an antibody (antagonist molecule) that will neutralize the function of the antigen can be evaluated by testing whether the function of the antigen is neutralized. Whether the antigen function is neutralized can be assessed by measuring specific in vivo markers that reflect the antigen function. Whether the antigen is bound 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 special limitations on the measurement. For example, it can determine the concentration of free antigen in plasma, determine the ratio of free antigen in plasma to the total amount of antigen in plasma, and measure labeling in vivo; however, the measurement should be within a certain period after antibody administration. implemented later. In the context of Disclosure B, "after a certain period after the antibody is administered" is not particularly limited. This period can be appropriately determined by a person with ordinary knowledge in the technical field based on the nature of the administered antibody, including, for example, 1 after the antibody is administered. days, 3 days, 7 days, 14 days or 28 days. The term "plasma antigen concentration" as used herein may refer to the "total antigen concentration in plasma," which is the sum of the concentration of antigen bound to antibodies and the concentration of antigen not bound to antibodies, or to the "concentration of free antigen in plasma," which is the concentration of antigen without bound antibodies. The antigen concentration of the antibody.

The molar ratio of antigen to antibody can be calculated using the formula C = A/B. The value A is the molar concentration of the antigen at each time point. The value B is the molar concentration of the antibody at each time point. The value C is the molar concentration of the antibody at each time point. The molar concentration of antigen per molar concentration of antibody (mol ratio of antigen/antibody).

The smaller the C value, the higher the efficiency of each antibody in eliminating the antigen, and the larger the C value, the lower the efficiency of each antibody in eliminating the antigen.

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

The reduction in total antigen concentration or antigen/antibody molar ratio in plasma can be assessed using methods known in the art, such as those described in Examples 6, 8, and 13 of WO2011/122011. More specifically, if the antibody of interest in Disclosure B does not cross-react with the mouse paired antigen, the mouse strain 32 or 276 can be transduced using the human FcRn gene based on the steady-state antigen in the antigen-antibody coinjection model or fusion model ( Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010)) to evaluate. When the antibody cross-reacts with the mouse antigen, it can be evaluated by simply injecting the antibody into the human FcRn transgenic mouse strain 32 or 276 (Jackson Laboratories). In the coinjection model, a mixture of antibody and antigen is administered to mice. In the steady-state antigen fusion model, a fusion pump filled with antigen solution is transplanted into mice to achieve a constant concentration of antigen in the plasma, and then the antibody is 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 effects of B-revealing antibodies, total or free antigen concentrations or antigen/antibody ratios in plasma can be measured at 2, 4, 7, 14, 28, 56, or 84 days after administration. Erbi. In other words, in order to evaluate the properties of this antibody, the total or free antigen concentration or antigen/antibody ratio in the plasma can be measured 2 days, 4 days, 7 days, 14 days, 28 days, 56 days, or 84 days after administration. molar ratio to determine the concentration of antigen in plasma over time. Whether the antigen concentration or antigen/antibody molar ratio in plasma is reduced due to this antibody can be determined by assessing such reduction at one or more time points as described above.

To assess the short-term effects of B-revealing antibodies, total or free antigen concentrations or antigen/antibody ratios in plasma can be measured at 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration. Erbi. In other words, to assess the properties of the antibody, the total or free antigen concentration or moles of antigen/antibody in the plasma can be measured at 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration. The ratio determines the short-term plasma antigen concentration. When it is difficult to determine plasma retention in humans, predictions of plasma retention can be based on mice (such as normal mice, human antigen-expressing gene transgenic mice or human FcRn-expressing gene transgenic mice) or monkeys (such as Malay monkey).

In an alternative embodiment, Disclosure B is directed to an antibody comprising an Fc region variant of Disclosure B as described above. Various embodiments of this antibody are within the scope of disclosures A and B described herein and may be used without violating the general technical knowledge in the art unless inconsistent with the context.

In one embodiment, antibodies comprising Fc region variants of Disclosure B are useful as therapeutic antibodies for the treatment of patients with autoimmune diseases, graft rejection (graft-compatible host disease), other inflammatory diseases, or allergic diseases. Patient, as documented in WO2013/046704.

In one embodiment, antibodies comprising Fc region variants of Disclosure B may have modified sugar chains. Antibodies with modified sugar chains may include, for example, antibodies with modified glycation (WO99/54342), antibodies lacking fucose (WO00/61739, WO02/31140, WO2006/067847, WO2006/067913), and antibodies with Antibody against the divided sugar chain of GlcNAc (WO02/79255). In one embodiment, the antibody can be deglycated. In some embodiments, the antibody includes, for example, a mutation in the glycation site of the heavy chain to inhibit glycation at this site, as described in WO2005/03175. Such unglycated antibodies can be produced by modifying the glycated site of the heavy chain, i.e., introducing the N297Q or N297A substitution according to EU numbering, and expressing the protein in appropriate host cells.

In an alternative embodiment, disclosure B relates to a composition or a pharmaceutical composition comprising an antibody containing such an Fc region variant. The various embodiments of the compositions or pharmaceutical compositions described in the scope of disclosures A and B can be applied without violating general technical knowledge in the technical field unless inconsistent with the context. Such compositions can be used to enhance plasma retention in a subject when an antibody system of Disclosure B is administered to the subject.

In an alternative embodiment, B is disclosed to be directed to a nucleic acid encoding an Fc region variant or an antibody containing an Fc region variant. Various embodiments of nucleic acids within the scope of disclosures A and B described herein may be applied without violating general technical knowledge in the art, unless inconsistent with the context. Or reveal that B is related to the vector containing the nucleic acid. Various embodiments within the scope of disclosures A and B described herein can be applied without violating general technical knowledge in the technical field, unless inconsistent with the context. Or reveal that B is related to the host or host cell containing the antibody. Various embodiments within the scope of disclosures A and B described herein can be applied without violating general technical knowledge in the technical field, unless inconsistent with the context.

In an alternative embodiment, disclosure B relates to a method for producing an Fc region variant comprising an FcRn binding domain or an antibody containing the Fc region variant, comprising culturing the above host cells or growing the above host, from the cell culture, the host The secreted material collects the Fc region variant or an antibody containing the Fc region variant. In this case, Disclosure B may include a manufacturing method, which optionally further includes one or more of the following steps: (a) Selecting an Fc region variant that has enhanced FcRn-binding activity under acidic pH conditions compared to the Fc region of native IgG. body; (b) Select Fc region variants whose binding activity for (pre-existing) ADA does not significantly increase under neutral pH conditions compared to the Fc region of native IgG; (c) Select Fc region variants whose binding activity for (pre-existing) ADA is not significantly improved under neutral pH conditions; increased plasma retention compared to the Fc region of native IgG; and (d) selecting antibodies that include an Fc region variant that promotes elimination of the antigen from plasma compared to a reference antibody that includes the Fc region of native IgG. Eliminate antigens.

From the perspective of evaluating the plasma retention of the Fc region variant of Disclosure B, it is not limited. It is appropriate that the antibody containing the Fc region variant produced by Disclosure B and the "reference antibody containing the Fc region of the natural IgG" are identical to each other, only The exception is the Fc area for comparison. The FcRn can be human FcRn.

For example, after producing an antibody including an Fc region variant revealing B, the antibody and a reference antibody including an IgG Fc region, which may be natural, can be used to use BIACORE (registered trademark) for FcRn-binding activity under acidic pH conditions (e.g., pH 5.8). Or other known techniques for comparison to select Fc region variants with increased FcRn binding activity under acidic pH conditions or antibodies containing the Fc region variants.

Or, for example, after making an antibody that includes an Fc region variant that reveals B, this antibody and a reference antibody that includes a native IgG Fc region can be used to measure ADA-binding activity under neutral pH conditions by electrochemiluminescence (ECL). Or compare with known techniques to select Fc region variants or antibodies containing the Fc region variants that do not significantly increase the ADA binding activity under neutral pH conditions.

Alternatively, for example, after producing an antibody including an Fc region variant revealing B, this antibody and a reference antibody including a native IgG Fc region can be borrowed from, for example, mouse, rat, rabbit, dog, monkey or human plasma. Antibody pharmacokinetic testing is performed to select Fc region variants or antibodies containing the Fc region variants that have been shown to improve plasma retention in subjects.

Alternatively, for example, antibodies comprising Fc region variants revealing B can be produced by combining the antibody with a reference antibody comprising a native IgG Fc region by using plasma from, for example, mice, rats, rabbits, dogs, monkeys or humans. Antibody pharmacokinetic testing is performed to select Fc region variants that enhance antigen elimination from plasma or antibodies containing such Fc region variants.

Alternatively, for example, the above-mentioned selection methods may be appropriately combined as necessary.

In one embodiment, disclosure B relates to a method of producing an Fc region variant, an FcRn binding domain-containing antibody, or an antibody containing such a variant, the method comprising substituting amino acids such that the obtained Fc region variant or antibody contains This variant of this antibody includes 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 an additional embodiment, such methods include substituting amino acids such that the resulting Fc region variant or antibody containing the antibody further includes, according to EU numbering, Ile or Leu at position 428 and/or Ile, Leu, Val, Thr or Phe at position 436. In another embodiment, the amino acid substitution into the Fc region variant obtained according to this method or the antibody containing this variant further includes Leu at position 428 and/or Val or Thr at position 436 according to EU numbering.

In one embodiment, Disclosure B relates to a method for producing an Fc region variant comprising an FcRn binding domain or an antibody containing such a variant, the method comprising substituting amino acids such that the obtained Fc region variant or containing such variant Variant antibodies according to EU numbering include Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440. In an additional embodiment, such methods include substituting amino acids such that the resulting Fc region variant or antibody containing the variant further includes Ile or Leu at position 428 and/or Ile, Leu according to EU numbering , Val, Thr or Phe at position 436. In another embodiment, the amino acid substituted according to this method to obtain an Fc region variant according to the present invention or an antibody containing this variant further includes Leu at position 428 and/or Val or Thr at position 436 according to EU numbering. .

In one embodiment, such methods include replacing all amino acids at positions 434, 438, and 440 with Ala; Glu, Arg, Ser, or Lys; and Glu, Asp, or Gln. In an additional embodiment, the method includes substituting amino acids such that the resulting Fc region variant or an antibody containing the variant further includes Ile or Leu at position 428 and/or Ile, Leu, Val, Thr or Phe at position 436. In yet another embodiment, the amino acid substitution to the Fc region variant obtained or the antibody containing the variant according to this method further includes Leu at position 428 and/or Val or Thr at position 436 according to EU numbering.

In an alternative embodiment, Disclosure B is directed to an Fc region variant or an antibody containing the Fc region variant, and is obtained by any of the methods described above for Disclosure B.

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

In some embodiments, the Fc domain (variant) in step (a) is suitably a human IgG Fc domain (variant). Furthermore, in order to increase the FcRn-binding activity at acidic pH and reduce the (pre-existing) ADA-binding activity in the neutral pH range (e.g., pH 7.4), the Fc region (variant) should contain a member selected from the group consisting of: Combinations of amino acid substitutions: (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; 440e; (J ) N434A/Y436V/Q438R/S440D; (k) N434A/Y436V/ Q438K/S440E; (l) N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/ S440E; (n) N434A/R 435H /F436T/Q438R/S440D; (o) N434A/R435H/F436T/Q438K/ S440E; (p) N434A/R435H/F436T/Q438K/S440D; (q) N434A/R435H/F436V/Q438R/ S440E; (r) N 434A /R435H/F436V/Q438R/S440D; (s) N434A/R435H/F436V/Q438K/ S440E; (t) N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v) M 428L /N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x) M428L/ N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/ Y 436T /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/Y436 T/Q438R/ S440E; and (ah) L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

This method may optionally further include: (c) Evaluating whether the antibody includes an Fc region variant that reduces the (pre-existing) ADA binding activity of the antibody compared to the antibody that reduces the binding activity of the reference antibody.

Alternatively, this method can be used as a method to enhance the release of an antibody that has been internalized into cells in an antigen-bound form into an antigen-free form out of the cell without significantly increasing the (existing) ADA-binding activity of the antibody at neutral pH.

Reveal C Disclosure C also relates to anti-IL-8 antibodies, nucleic acids encoding such antibodies, pharmaceutical compositions containing such antibodies, methods of producing such antibodies, and uses of such antibodies to treat IL-8-related diseases, as detailed below narrate. The meanings of the terms given below apply to the entire disclosure C and are not inconsistent with the common technical knowledge of those with ordinary skill in the technical field and the implementations known to those with ordinary skill in the technical field.

I. Reveal the definition within the scope of C Within the definition disclosed in C, "acidic pH" means a pH 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 one embodiment, the term acidic pH refers to pH 5.8.

Within the definition disclosed in the scope of C, "neutral pH" may be a pH 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 one embodiment, the term neutral pH refers to pH 7.4.

The term "IL-8" used in Disclosure C, unless otherwise specified, refers to any vertebrate, primate (such as humans, Malay monkeys, rhesus monkeys) and other mammals (such as dogs and rabbits) Comes with natural IL-8. The term "IL-8" includes full-length IL-8, unprocessed IL-8, and any form of IL-8 resulting from intracellular processing. The term "IL-8" also includes derivatives of native IL-8, such as cleavage variants or dual variants. An example of the amino acid sequence of human IL-8 is shown in SEQ ID NO:66.

The terms "anti-IL-8 antibody" and "antibody that binds to IL-8" refer to antibodies that bind IL-8 with sufficient affinity to be useful as diagnostic and/or therapeutic agents that target IL-8. .

In one embodiment, the binding of an anti-IL-8 antibody to unrelated non-IL-8 proteins is, for example, less than about 10% of the binding of the antibody to IL-8.

The term "affinity" as used in the context of Disclosure C generally refers to the total strength 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 specified, the term "binding affinity" as used in the context of Disclosure C refers to intrinsic binding affinity, reflecting a 1:1 interaction between the members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Binding affinity can be measured using methods known in the art, including those included in the description of the category disclosing C.

In certain embodiments, the dissociation constant (KD) of an antibody that binds to IL-8 can be, for example, ≤1000 nM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less , from 10 ^-8 M to 10 ^-13 M, from 10 ^-9 M to 10 ^-13 M).

The term "antibody" used in the context of Disclosure C is used in a broad sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. That’s it.

"Binds to the same epitope" as a reference antibody means an antibody that blocks the binding of a control antigen to its antigen by, for example, 50%, 60%, 70% or 80% or more; and conversely, a reference antibody blocks the binding of a control antigen to its antigen The antibody binds, for example, 50%, 60%, 70% or 80% or more to its antigen. Exemplary competition analysis methods may be used here without limitation.

A "chimeric antibody" is an antibody in which part of the heavy and/or light chain is derived from one specific source or species and the other parts are derived from a different source or species.

A "humanized" antibody refers to a chimeric antibody that includes amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody may include substantially at least 1, and typically 2 variable regions, wherein all (or substantially all) of the HVRs (e.g., CDRs) correspond to the non-human antibody, and all (or Substantially all) of the FRs correspond to human antibodies. Humanized antibodies may optionally include at least a portion of an antibody constant region derived from a human antibody.

The term "monoclonal antibody" as used in the context of Disclosure C refers to an antibody obtained from a population of antibodies that is substantially homogeneous, that is, the individual antibodies that make up the population are identical and/or bind to the same epitope, except where there is possible variation. Antibodies, for example, contain naturally occurring mutations or variants resulting from the manufacture of monoclonal antibodies, which are generally present in trace amounts. In contrast, a polyclonal antibody preparation generally includes different antibodies directed to different epitopes (epitopes), and in a monoclonal antibody preparation each monoclonal antibody is directed to a single epitope on the antigen. Therefore, the modifier "single strain" represents the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and does not mean that a specific method is required to produce the antibody. For example, monoclonal antibodies used according to disclosure C can be produced using various techniques, including but not limited to fusion tumor methods, recombinant DNA methods, phage display methods, and methods of genetically transforming animals including all or part of human immunoglobulin gene loci. , such methods and other methods for producing monoclonal antibodies will be described here.

Within the context of Disclosure C, "natural antibodies" refer to immunoglobulin molecules of various naturally occurring structures. In one embodiment, a natural IgG antibody is, for example, a heterotetrad glycoprotein of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. In order from N-to C-terminal, each heavy chain has a variable region (VH), also called variable heavy chain domain or heavy chain variable domain, followed by three certain domains (CH1, CH2, and CH3 ). Likewise, in order from N-to C-terminus, each light chain has a variable region (VL), called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Antibody light chains can be assigned to either of two types based on the amino acid sequence of their invariant domain, namely kappa (κ) and lambda (λ). Such invariant domains used by this disclosure C include any reported paratype (dual type) or any subcategorical/constructive isotype. Heavy chain constant regions include, but are not limited to, constant region antibodies of native IgG (IgG1, IgG2, IgG3, and IgG4). Known IgG1 partner genes include, for example, IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, and IGHG1*05 (see imgt.org), any of which can be used as a natural human IgG1 sequence. Invariant domain sequences can be from a single allele or subcategory/architectural isotype or from multiple alleles or subcategories/architecture isotypes. Specifically, such antibodies include, but are not limited to, antibodies in which CH1 is derived from IGHG1*01, and CH2 and CH3 are derived from IGHG1*02 and IGHG1*01.

"Effector function" as described within the context of Disclosure C refers to the biological activity attributable to the Fc region of an antibody that varies with the structural isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (e.g., B cell receptors) ) down-regulation; and B cell activation, but not limited to this.

The term "Fc region" used to describe the category of C is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The term includes native Fc regions and variant Fc regions. The natural Fc region refers to the Fc region of a natural antibody.

In one embodiment, the human IgG heavy chain Fc region extends from the amino acid residue of Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not exist. Unless otherwise specified in the context of Disclosure C, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The "framework" or "FR" described in the scope of disclosure C refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4 in VH (or VL).

The "human common framework" recorded in the scope of disclosure C is the framework of the most commonly occurring amino acid residues in the selected human immunoglobulin VL or VH framework sequence. Typically, human immunoglobulin VL or VH sequences are selected from a subpopulation of variable domain sequences. In general, sequence subgrouping follows that of Kabat et al., Sequences of proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

In one embodiment, the subgroup for VL is subgroup κI, as described by Kabat et al., supra. In one embodiment, the subgroup for VH is subgroup III, as described by Kabat et al., supra.

The "acceptor human framework" used to disclose the scope of C is a framework that includes the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. Acceptor human frameworks "from" a human immunoglobulin framework or a human consensus framework may include the same amino acid sequence thereof or may include substitutions of existing amino acid sequences. 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 receptor human framework and the VL human immunoglobulin framework sequence or human consensus framework sequence are identical.

Within the context of the disclosure C, the term "variable region" or "invariant domain" refers to the domain of the antibody heavy or light chain involved in binding the antibody to the antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL respectively) generally have similar structures. Each domain includes four conserved framework regions (FR) and three hypervariable regions (HVRs) (see Kindt et al., Kuby Immunology, ^6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity but is not limited thereto. Alternatively, antibodies that bind to specific antigens can use VH or VL domains from antibodies to screen for complementary VL or VH domains for isolation, see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term "highly variable region" or "HVR" as used in the context of the description disclosing C refers to sequences that are highly variable ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("Complementarity Determining Regions" or "CDRs"). "High variability loops") and/or regions of an antibody variable domain that contain antigen contact residues ("antigen contact points"). Generally, antibodies include 6 hypervariable regions: 3 in VH (H1, H2, H3) and 3 in VL (L1, L2, L3).

Not limited to this, here, HVR is for example: (a) High variability loop, 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) CDR, 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., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact point, where 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) combination (a), (b) and/or (c), including 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 specified, HVR and other variable region residues (such as FR residues) are numbered according to Kabat et al., as mentioned above.

In the context of the records revealing C, the "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cattle, sheep, cats, dogs and horses), primates (such as humans and non-human primates such as monkeys), rabbits and rodents (such as mice and rats) . In certain embodiments, the "individual" is a human.

Within the context of the record revealing C, an "isolated" antibody system has been separated from components of the natural environment. In some embodiments, the antibody has been purified, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC) to a purity greater than 95% or 99 %. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

Within the context of disclosure C, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are present in the cells that normally contain the nucleic acid molecule but exist outside the chromosome or at a location on the chromosome that is different from its native chromosomal location.

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

Within the context of the disclosure C, the terms "host cell", "host cell strain" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells . Host cells include "transformants" and "transformed cells", including cells to be transformed and their descendants regardless of their generation number. Progeny do not necessarily have identical nucleic acid content to their parent cells and may include mutations. This also includes screening or selecting mutant progeny that have the same function or biological activity as the original transformed cells.

In the context of the record disclosing C, the term "vector" refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. The term includes vectors that self-replicate nucleic acid constructs, as well as vectors that are introduced into a host cell and incorporate its genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such a vehicle is called an "expression vehicle".

Within the context of the record disclosed in Disclosure C, the term "package insert" is used to refer to the instructions custom-made included in the commercial package of a medicinal product containing information on indications, usage, dosage, administration, combination Information on treatments, contraindications, and/or warnings regarding the use of such medicinal products.

Within the scope of the disclosure C, the "percentage of amino acid sequence identity (%)" with respect to a control polypeptide sequence is defined as the difference between the amino acid residues in the candidate sequence and the amino acid residues in the control polypeptide sequence. Percent identity of the sequences after alignment, gaps may be introduced as necessary to achieve maximum percent sequence identity, and any conservative substitutions will not be considered as part of the sequence identity. In order to determine the ranking of the percentage of amino acid sequence identity, various methods within the ability of those of ordinary skill in the art can be used, such as 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 the appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximal alignment over the full length of the aligned sequences. For this purpose, values for % amino acid sequence identity are generated, for example, using the comparison computer program ALIGN-2. ALIGN-2 is produced by Genentech, Inc., the source code and user documentation have been submitted to the U.S. Copyright Office, Washington D.C., 20559, and U.S. Copyright Registration No. TXU510087 has been registered. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. ALIGN-2 programs should use UNIX (Registered Trademark) operating system encoding, including Digital UNIX (Registered Trademark) V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

When using ALIGN-2 to compare amino acid sequences, a given amino acid sequence A has or includes a given amino acid sequence B (it can also be said that a given amino acid sequence A has or includes a given amino acid sequence B The amino acid sequence identity %) is calculated in the following way: 100 times the score X/Y, where X is the same match for A and B when aligned using the sequence alignment program ALIGN-2 Amino acid residue fraction, Y is the total number of amino acid residues in B. It will be understood that if 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 is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values were obtained using the ALIGN-2 computer program as demonstrated within the ranges reported in Disclosure C.

Within the scope of disclosure C, "pharmaceutical composition" generally refers to pharmaceuticals used to treat, prevent, examine or diagnose diseases. "Pharmaceutically acceptable carrier" refers to the ingredients other than the active ingredients in the pharmaceutical composition, which are non-toxic to the subject. Such pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, and preservatives.

"Treatment" as used in the context of the disclosure C (and its grammatical variations such as "treat" or "treating") refers to clinical intervention intended to alter the natural course of the individual to be treated. Such clinical intervention may be prophylactic or performed during a clinical episode. The desired effects of treatment include but are not limited to preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological results of the disease, preventing metastasis, slowing down the progression of the disease, improving or alleviating the disease state, and alleviating or improving the prognosis. In one embodiment, C-revealing antibodies may be used to slow the progression of a disease or disorder.

Within the context of disclosure C, an "effective amount" of an antibody or pharmaceutical composition is an amount that is effective if used at the dose and for the period of time necessary to achieve the desired therapeutic or preventive result.

II. Compositions and Methods In one embodiment, disclosure C is based on the use of an anti-IL-8 antibody with pH-dependent affinity for IL-8 as a pharmaceutical composition. C-disclosing antibodies are useful, for example, in the diagnosis or treatment of diseases in which IL-8 is present in excess.

A. Examples of anti-IL-8 antibodies In one embodiment, it is disclosed that C provides an anti-IL-8 antibody with pH-dependent affinity for IL-8.

In one embodiment, it is disclosed that C provides an anti-IL-8 antibody with pH-dependent affinity for IL-8, including at least 1, 2, 3, 4, 5, 6 in the following amino acid sequence , 7 or 8 amino acid substituted sequences: (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2, including the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3, including the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1, including the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2, including SEQ ID NO: The amino acid sequence of SEQ ID NO:71; and (f) HVR-L3, including the amino acid sequence of SEQ ID NO:72.

In another embodiment, Disclosure C provides an anti-IL-8 antibody with pH-dependent affinity for IL-8, comprising at least 1 amino acid substitution in at least one of the following amino acid sequences: (a) HVR-H1 , including the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2, including the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3, including the amino acid sequence of SEQ ID NO: 69 ; (d) HVR-L1, including the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2, including the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3, including SEQ ID NO. Amino acid sequence of NO: 72.

Unless otherwise specified, the amino acid may be substituted with any other amino acid. In one embodiment, the anti-IL-8 antibody of Disclosure C includes one or more amino acid substitutions at a position selected from the group consisting of: (a) at position 1 of the sequence of SEQ ID NO:67 Aspartic acid; (b) tyrosine at position 2 of the sequence of SEQ ID NO:67; (c) tyrosine at position 3 of the sequence of SEQ ID NO:67; (d) tyrosine at position 3 of the sequence of SEQ ID NO:67 :Leucine at position 4 of the sequence of SEQ ID NO:67; (e) Serine at position 5 of the sequence of SEQ ID NO:67; (f) Leucine at position 1 of the sequence of SEQ ID NO:68; (g) Isoleucine at position 2 of the sequence of SEQ ID NO:68; (h) Arginine at position 3 of the sequence of SEQ ID NO:68; (i) Isoleucine at position 3 of the sequence of SEQ ID NO:68 Asparagine at position 4 of SEQ ID NO: 68; (j) Lysine at position 5 of the sequence of SEQ ID NO: 68; (k) Alanine at position 6 of the sequence of SEQ ID NO: 68; (l) Asparagine at position 7 of the sequence of SEQ ID NO:68; (m) Glycine at position 8 of the sequence of SEQ ID NO:68; (n) Glycine at position 9 of the sequence of SEQ ID NO:68 tyrosine at position; (o) threonine at position 10 of the sequence of SEQ ID NO:68; (p) arginine at position 11 of the sequence of SEQ ID NO:68; (q) arginine at position 11 of the sequence of SEQ ID NO:68 Glutamic acid at position 12 of the sequence of NO:68; (r) Tyrosine at position 13 of the sequence of SEQ ID NO:68; (s) Serine at position 14 of the sequence of SEQ ID NO:68 ; (t) alanine at position 15 of the sequence of SEQ ID NO:68; (u) serine at position 16 of the sequence of SEQ ID NO:68; (v) amino acid at position 16 of the sequence of SEQ ID NO:68 Valine at position 17; (w) Lysine at position 18 of the sequence of SEQ ID NO:68; (x) Glycine at position 19 of the sequence of SEQ ID NO:68; (y) SEQ. Glutamic acid at position 1 of the sequence of SEQ ID NO:69; (z) Aspartic acid at position 2 of the sequence of SEQ ID NO:69; (aa) Glutamic acid at position 3 of the sequence of SEQ ID NO:69 Tyrosine; (ab) arginine at position 4 of the sequence of SEQ ID NO:69; (ac) tyrosine at position 5 of the sequence of SEQ ID NO:69; (ad) tyrosine at position 5 of the sequence of SEQ ID NO:69; Aspartic acid at position 6 of the sequence 69; (ae) Valine at position 7 of the sequence of SEQ ID NO: 69; (af) Glutamic acid at position 8 of the sequence of SEQ ID NO: 69; (ag) Leucine at position 9 of the sequence of SEQ ID NO:69; (ah) Alanine at position 10 of the sequence of SEQ ID NO:69; (ai) Leucine at position 11 of the sequence of SEQ ID NO:69 tyrosine at position; (aj) arginine at position 1 of the sequence of SEQ ID NO:70; (ak) alanine at position 2 of the sequence of SEQ ID NO:70; (al) alanine at position 2 of the sequence of SEQ ID NO:70 :Serine at position 3 of the sequence of SEQ ID NO:70; (am) Glutamic acid at position 4 of the sequence of SEQ ID NO:70; (an) Isoleucine at position 5 of the sequence of SEQ ID NO:70 ; (ao) isoleucine at position 6 of SEQ ID NO:70; (ap) tyrosine at position 7 of SEQ ID NO:70; (aq) tyrosine at position 7 of SEQ ID NO:70 Serine at position 8 of the sequence; (ar) Tyrosine at position 9 of the sequence of SEQ ID NO:70; (as) Leucine at position 10 of the sequence of SEQ ID NO:70; (at) Alanine at position 11 of the sequence of SEQ ID NO:70; (au) Asparagine at position 1 of the sequence of SEQ ID NO:71; (av) Position 2 of the sequence of SEQ ID NO:71 alanine; (aw) lysine at position 3 of the sequence of SEQ ID NO:71; (ax) threonine at position 4 of the sequence of SEQ ID NO:71; (ay) threonine at position 4 of the sequence of SEQ ID NO:71; Leucine at position 5 of the sequence of SEQ ID NO:71; (az) Alanine at position 6 of the sequence of SEQ ID NO:71; (ba) Aspartic acid at position 7 of the sequence of SEQ ID NO:71; (az) bb) Glutamic acid at position 1 of the sequence of SEQ ID NO:72; (bc) Histidine at position 2 of the sequence of SEQ ID NO:72; (bd) Glutamic acid at position 2 of the sequence of SEQ ID NO:72 Histidine at position 3; (be) Phenylalanine at position 4 of the sequence of SEQ ID NO:72; (bf) Glycine at position 5 of the sequence of SEQ ID NO:72; (bg) Glycine at position 5 of the sequence of SEQ ID NO:72 Phenylalanine at position 6 of the sequence of NO:72; (bh) Proline at position 7 of the sequence of SEQ ID NO:72; (bi) Arginine at position 8 of the sequence of SEQ ID NO:72; and (bj) threonine at position 9 of the sequence of SEQ ID NO:72.

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

In one embodiment, the anti-IL-8 antibody of C is disclosed to include a combination of amino acid substitutions at a position selected from the group consisting of: (a) alanine at position 6 of the sequence of SEQ ID NO: 68 ; (b) Glycine at position 8 of the sequence of SEQ ID NO:68; (c) Tyrosine at position 9 of the sequence of SEQ ID NO:68; (d) Sequence of SEQ ID NO:68 arginine at position 11; and (e) tyrosine at position 3 of the sequence of SEQ ID NO:69.

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

In one embodiment, the anti-IL-8 antibody of Disclosure C includes amino acid substitutions at the following positions: (a) alanine at position 6 of the sequence of SEQ ID NO:68; (b) alanine at position 6 of SEQ ID NO:68 Glycine at position 8 of the sequence of SEQ ID NO: 68; (c) Tyrosine at position 9 of the sequence of SEQ ID NO: 68; (d) Arginine at position 11 of the sequence of SEQ ID NO: 68; and ( e) Tyrosine at position 3 of the sequence of SEQ ID NO:69.

In one embodiment, the anti-IL-8 antibody disclosed in C includes: (a) at position 6 of the sequence of SEQ ID NO:68, replacing alanine with aspartic acid; (b) at position 6 of SEQ ID NO:68 At position 11 of the sequence, arginine is replaced by proline; and (c) at position 3 of the sequence of SEQ ID NO: 69, tyrosine is replaced by histidine.

In one embodiment, the anti-IL-8 antibody disclosed in C includes: (a) at position 8 of the sequence of SEQ ID NO:68, replacing glycine with tyrosine; and (b) at position 8 of SEQ ID NO:68 Position 9 of the sequence replaces tyrosine with histamine.

In one embodiment, the anti-IL-8 antibody disclosed in C includes: (a) at position 6 of the sequence of SEQ ID NO:68, replacing alanine with aspartic acid; (b) at position 6 of SEQ ID NO:68 At position 8 of the sequence, glycine is replaced by tyrosine; (c) At position 9 of the sequence of SEQ ID NO:68, tyrosine is replaced by histamine; (d) At position 9 of the sequence of SEQ ID NO:68, tyrosine is replaced by histamine; (d) At position 9 of the sequence of SEQ ID NO:68, at position 11, arginine is replaced by proline; and (e) at position 3 of the sequence of SEQ ID NO:69, tyrosine is replaced by histidine.

In one embodiment, the anti-IL-8 antibody disclosed in C includes HVR-H2, including the amino acid sequence of SEQ ID NO: 73.

In one embodiment, the anti-IL-8 antibody of Disclosure C includes HVR-H3, including the amino acid sequence of SEQ ID NO: 74.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes HVR-H1 containing the amino acid sequence of SEQ ID NO: 67, HVR-H2 containing the amino acid sequence of SEQ ID NO: 73, and HVR-H2 containing the amino acid sequence of SEQ ID NO: 73. Amino acid sequence of ID NO: HVR-H3 of 74.

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

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

In one embodiment, the anti-IL-8 antibody of Disclosure C includes amino acid substitutions at the following positions: (a) aspartate at position 1 of the sequence of SEQ ID NO:71; (b) at position 1 of SEQ ID NO:71 Leucine at position 5 of the sequence of NO:71; and (c) Glutamic acid at position 1 of the sequence of SEQ ID NO:72.

In one embodiment, the anti-IL-8 antibody of Disclosure C includes amino acid substitutions at: (a) serine at position 8 of the sequence of SEQ ID NO:70; (b) serine at position 8 of SEQ ID NO:70; asparagine at position 1 of the sequence of SEQ ID NO:71; (c) leucine at position 5 of the sequence of SEQ ID NO:71; and (d) gluten at position 1 of the sequence of SEQ ID NO:72 amino acids.

In one embodiment, the anti-IL-8 antibody disclosed in C includes: (a) at position 1 of the sequence of SEQ ID NO:71, replacing aspartic acid with lysine; (b) in SEQ ID NO: In position 5 of the sequence of SEQ ID NO:71, leucine is replaced by histidine acid; and (c) In position 1 of the sequence of SEQ ID NO:72, glutamic acid is replaced by lysine acid.

In one embodiment, the anti-IL-8 antibody disclosed in C includes: (a) at position 8 of the sequence of SEQ ID NO:70, replacing serine with glutamic acid; (b) at position 8 of SEQ ID NO:71 At position 1 of the sequence, aspartic acid is replaced by lysine acid; (c) At position 5 of the sequence of SEQ ID NO:71, leucine is replaced by histidine acid; and (d) at position 5 of SEQ ID NO:72 Position 1 of the sequence replaces glutamine with lysine.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes HVR-L2 containing the amino acid sequence of SEQ ID NO: 75.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes HVR-L3 containing the amino acid sequence of SEQ ID NO: 76.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes HVR-L1 containing the amino acid sequence of SEQ ID NO: 70, HVR-L2 containing the amino acid sequence of SEQ ID NO: 75, and HVR-L2 containing the amino acid sequence of SEQ ID NO: 75. Amino acid sequence of NO: 76 HVR-L3.

In one embodiment, the anti-IL-8 antibody of Disclosure C includes amino acid substitutions at the following positions: (a) alanine at position 55 of the sequence of SEQ ID NO:77; (b) alanine at position 55 of SEQ ID NO:77 Glycine at position 57 of the sequence of SEQ ID NO:77; (c) Tyrosine at position 58 of the sequence of SEQ ID NO:77; (d) Arginine at position 60 of the sequence of SEQ ID NO:77; (e ) glutamine at position 84 of the sequence of SEQ ID NO:77; (f) serine at position 87 of the sequence of SEQ ID NO:77; and (g) serine at position 87 of the sequence of SEQ ID NO:77 Tyrosine at position 103.

In one embodiment, the anti-IL-8 antibody disclosed in C includes: (a) at position 55 of the sequence of SEQ ID NO:77, replacing alanine with aspartic acid; (b) at position 55 of SEQ ID NO:77 At position 57 of the sequence, glycine is replaced by tyrosine acid; (c) At position 58 of the sequence of SEQ ID NO:77, tyrosine is replaced by histamine; (d) At position 58 of the sequence of SEQ ID NO:77, tyrosine is replaced by histamine; (d) At position 58 of the sequence of SEQ ID NO:77, tyrosine is substituted At position 60, arginine is replaced by proline; (e) At position 84 of the sequence of SEQ ID NO:77, glutamine is replaced by threonine; (f) At position 87 of the sequence of SEQ ID NO:77 at position 103 of the sequence of SEQ ID NO: 77, replacing tyrosine with histidine.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 78.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 79.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:78; and a light chain variable region containing the amino group of SEQ ID NO:79 acid sequence. The anti-IL-8 antibody including a heavy chain variable region containing the amino acid sequence of SEQ ID NO:78 and a light chain variable region containing the amino acid sequence of SEQ ID NO:79 can be pH-dependent. Anti-IL-8 antibodies that bind to IL-8 in a sexual manner. The anti-IL-8 antibody including a heavy chain variable region containing the amino acid sequence of SEQ ID NO:78 and a light chain variable region containing the amino acid sequence of SEQ ID NO:79 can be stably in Anti-IL-8 antibodies that maintain IL-8 neutralizing activity in vivo (eg, in plasma). The anti-IL-8 antibody including a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 78 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 79 can be a hypoimmune Original antibodies.

In an alternative aspect, it is disclosed that the anti-IL-8 antibodies of C also include those that have pH-dependent affinity for IL-8 and contain at least 1 amino acid substitution in at least any of the following amino acid sequences : (a) HVR-H1, containing the amino acid sequence of SEQ ID NO:102; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO:103; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: : the amino acid sequence of SEQ ID NO: 104; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 105; (e) HVR-L2, containing the amino acid sequence of SEQ ID NO: 106; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:107.

In an alternative aspect, it is disclosed that the anti-IL-8 antibodies of C also include those that have pH-dependent affinity for IL-8 and contain at least 1 amino acid substitution in at least any of the following amino acid sequences : (a) HVR-H1, containing the amino acid sequence of SEQ ID NO: 108; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO: 109; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO : the amino acid sequence of SEQ ID NO: 110; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2, containing the amino acid sequence of SEQ NO: 112; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:113.

In an alternative aspect, it is disclosed that the anti-IL-8 antibodies of C also include those that have pH-dependent affinity for IL-8 and contain at least 1 amino acid substitution in at least any of the following amino acid sequences : (a) HVR-H1, containing the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO: 115; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: : the amino acid sequence of SEQ ID NO: 116; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 117; (e) HVR-L2, containing the amino acid sequence of SEQ ID NO: 118; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:119.

In an alternative aspect, it is disclosed that the anti-IL-8 antibodies of C also include those that have pH-dependent affinity for IL-8 and contain at least 1 amino acid substitution in at least any of the following amino acid sequences : (a) HVR-H1, containing the amino acid sequence of SEQ ID NO:120; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO:121; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: : the amino acid sequence of SEQ ID NO: 122; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2, containing the amino acid sequence of SEQ ID NO: 124; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:125.

In an alternative aspect, it is disclosed that the anti-IL-8 antibodies of C also include those that have pH-dependent affinity for IL-8 and contain at least 1 amino acid substitution in at least any of the following amino acid sequences : (a) HVR-H1, containing the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: : the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2, containing the amino acid sequence of SEQ ID NO: 130; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:131.

In an alternative aspect, it is disclosed that the anti-IL-8 antibodies of C also include those that have pH-dependent affinity for IL-8 and contain at least 1 amino acid substitution in at least any of the following amino acid sequences : (a) HVR-H1, containing the amino acid sequence of SEQ ID NO:132; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO:133; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: : the amino acid sequence of SEQ ID NO: 134; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 135; (e) HVR-L2, containing the amino acid sequence of SEQ ID NO: 136; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:137.

In an alternative aspect, it is disclosed that the anti-IL-8 antibodies of C also include those that have pH-dependent affinity for IL-8 and contain at least 1 amino acid substitution in at least any of the following amino acid sequences : (a) HVR-H1, containing the amino acid sequence of SEQ ID NO:138; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO:139; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: : the amino acid sequence of SEQ ID NO: 140; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 141; (e) HVR-L2, containing the amino acid sequence of SEQ ID NO: 142; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:143.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C has IL-8 neutralizing activity. IL-8 neutralizing activity refers to the activity of inhibiting the biological activity of IL-8, or may refer to the activity of inhibiting the receptor binding of IL-8.

In an alternative aspect, the anti-IL-8 antibody of C is disclosed to be an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. In the context of Disclosure C, an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner refers to an antibody that has a higher binding affinity for IL-8 at acidic pH than for IL-8 at neutral pH. The binding affinity of -8 is reduced. For example, pH-dependent anti-IL-8 antibodies include antibodies that have higher affinity for IL-8 at neutral pH than at acidic pH. In one embodiment, it is disclosed that the affinity of the anti-IL-8 antibody of C for IL-8 at neutral pH is at least 2 times, 3 times, 5 times, 10 times, 15 times greater than the affinity at acidic pH. 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times , 200 times, 400 times, 1,000 times, 10,000 times or more. Binding affinity can be measured using, but not limited to, surface plasmon resonance methods (such as BIACORE (registered trademark)). The association rate constant (kon) and dissociation rate constant (koff) can be calculated based on a simple one-to-one Langmuir binding model by simultaneously fitting the association and dissociation sensorgrams using BIACORE (registered trademark) T200 Evaluation Software (GE Healthcare). The equilibrium dissociation constant (KD) is calculated as the ratio koff/kon. In order to screen for antibodies whose binding affinity changes depending on pH, ELISA, kinetic exclusion analysis (KinExA ^™ ), etc., and surface plasmon resonance methods (such as BIACORE (registered trademark)) can be used, without particular limitation. pH-dependent IL-8 binding ability refers to the property of binding to IL-8 in a pH-dependent manner. At the same time, whether an antibody can bind to IL-8 multiple times can be evaluated according to the method described in WO2009/125825.

In one embodiment, it is preferred that the anti-IL-8 antibody of Disclosure C has a small dissociation constant (KD) for IL-8 at neutral pH. In one embodiment, the dissociation constant of the antibody disclosed as C against IL-8 at neutral pH is, for example, 0.3 nM or less but is not limited thereto. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at neutral pH is, for example, 0.1 nM or less but is not limited thereto. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at neutral pH is, for example, 0.03 nM or less but is not limited thereto.

In one embodiment, it is preferred that the anti-IL-8 antibody of Disclosure C has a small dissociation constant (KD) for IL-8 at pH 7.4. In one embodiment, it is disclosed that the dissociation constant (KD) of the antibody of C against IL-8 at pH 7.4 is, for example, 0.3 nM or less but is not limited thereto. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at pH 7.4 is, for example, 0.1 nM or less but is not limited thereto. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at pH 7.4 is, for example, 0.03 nM or less but is not limited thereto.

In one embodiment, it is preferred that the anti-IL-8 antibody of Disclosure C has a large dissociation constant (KD) for IL-8 at acidic pH. In one embodiment, it is disclosed that the dissociation constant of the antibody of C against IL-8 at acidic pH is, for example, 3 nM or more but is not limited thereto. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at acidic pH is, for example, 10 nM or more but is not limited thereto. In one embodiment, it is disclosed that the dissociation constant of the antibody of C against IL-8 at acidic pH is, for example, 30 nM or more but is not limited thereto.

In one embodiment, it is preferred that the anti-IL-8 antibody of Disclosure C has a large dissociation constant (KD) for IL-8 at pH 5.8. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at pH 5.8 is, for example, 3 nM or more but is not limited thereto. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at pH 5.8 is, for example, 10 nM or more but is not limited thereto. In one embodiment, the dissociation constant of the antibody disclosed in C against IL-8 at pH 5.8 is, for example, 30 nM or more but is not limited thereto.

In one embodiment, it is preferred that the anti-IL-8 antibody of Disclosure C has a binding affinity for IL-8 that is greater at neutral pH than at acidic pH.

In one embodiment, it is disclosed that the dissociation constant ratio between acidic pH and neutral pH of the anti-IL-8 antibody of C, ie [KD (acidic pH)/KD (neutral pH)], is, for example, 30 or more But it is not limited to this. In one embodiment, it is disclosed that the dissociation constant ratio between acidic pH and neutral pH of the anti-IL-8 antibody of C, ie [KD (acidic pH)/KD (neutral pH)], is, for example, 100 or more , such as 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 not limited to this.

In one embodiment, the dissociation constant ratio between pH 5.8 and pH 7.4 of the anti-IL-8 antibody disclosed in C, that is, [KD (pH 5.8)/KD (pH 7.4)] is 30 or more but is not limited to this. In one embodiment, it is disclosed that the dissociation constant ratio of the anti-IL-8 antibody of C between pH 5.8 and pH 7.4, ie [KD (pH 5.8)/KD (pH 7.4)], is, for example, 100 or more, for example, 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 it is not limited to this.

In one embodiment, it is preferred that the anti-IL-8 antibody revealing C has a large dissociation rate constant (koff) at acidic pH. In one embodiment, the dissociation rate constant of the antibody disclosing C at acidic pH is, for example, 0.003 (1/s) or more but is not limited thereto. In one embodiment, the dissociation rate constant of the antibody disclosing C at acidic pH is, for example, 0.005 (1/s) or more but is not limited thereto. In one embodiment, the dissociation rate constant of the antibody disclosing C at acidic pH is, for example, 0.01 (1/s) or more but is not limited thereto.

In one embodiment, it is preferred that the anti-IL-8 antibody of Disclosure C has a large dissociation rate constant (koff) at pH 5.8. In one embodiment, the dissociation rate constant of the antibody disclosing C at pH 5.8 is, for example, 0.003 (1/s) or more but is not limited thereto. In one embodiment, the dissociation rate constant of the antibody disclosing C at pH 5.8 is, for example, 0.005 (1/s) or more but is not limited thereto. In one embodiment, the dissociation rate constant of the antibody disclosing C at pH 5.8 is, for example, 0.01 (1/s) or more but is not limited thereto.

In one embodiment, it is advantageous to disclose that the anti-IL-8 antibody of C stably maintains IL-8 neutralizing activity in solution (eg, PBS). Whether the activity is stably maintained in the solution can be evaluated by measuring the IL-8 neutralizing activity of the C-revealing antibody added to the solution before and after storage at a specific temperature for a specific period. In one embodiment, the storage period is, for example, 1, 2, 3 or 4 weeks but is not limited thereto. In one embodiment, the storage temperature is, for example, 25°C, 30°C, 35°C, 40°C or 50°C but is not limited thereto. In one embodiment, the storage temperature is, for example, 40°C but is not limited thereto; and the storage period is, for example, 2 weeks but is not limited thereto. In one embodiment, the storage temperature is, for example, 50°C but is not limited thereto; and the storage period is, for example, 1 week but is not limited thereto.

In one embodiment, it is advantageous to disclose that the anti-IL-8 antibody of C stably maintains IL-8 neutralizing activity in vivo (eg, plasma). Whether the activity is stably maintained in vivo can be evaluated by measuring the IL-8 neutralizing activity of the C-revealing antibody added to the plasma of animals (eg, mice) or humans before and after storage at a specific temperature for a specific period. In one embodiment, the storage period is, for example, 1, 2, 3 or 4 weeks but is not limited thereto. In one embodiment, the storage temperature is, for example, 25°C, 30°C, 35°C or 40°C but is not limited thereto. In one embodiment, the storage temperature is, for example, 40°C but is not limited thereto; and the storage period is, for example, 2 weeks but is not limited thereto.

In one embodiment, it is disclosed that the cellular uptake rate of the anti-IL-8 antibody of C is greater when the antibody and IL-8 form a complex outside the cell (eg, plasma) than when the antibody is alone. IL-8-revealing C antibodies enter cells more easily when complexed with IL-8 than when not complexed with IL-8.

In one embodiment, it is preferred that the immunogenicity of the anti-IL-8 antibody revealing C is predicted to be reduced in the human host. "Low immunogenicity" may mean, but is not limited to, for example, that when a sufficient amount of anti-IL-8 antibody is administered for a sufficient period of time to achieve a therapeutic effect, no immune response is induced in at least half or more individuals. Inducing an immune response may include the production of anti-drug antibodies. "Low drug-resistant antibody production" and "low immunogenicity" are used interchangeably. Human immunogenicity levels were assessed using T cell epitope prediction programs. Such T cell epitope prediction programs include Epibase (Lonza), iTope/TCED (Antitope), EpiMatrix (EpiVax), etc. EpiMatrix is a system for predicting the immunogenicity of sequences of proteins of interest. It automatically designs the sequence of peptide fragments by cutting the amino acid sequence of the protein to be analyzed for immunogenicity, and then calculates its binding to eight major MHC classes. II ability to pair genes (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301 and DRB1*1501) (De Groot et al., Clin. Immunol. 131 (2):189-201 (2009)). The modified amino acid sequence of the amino acid sequence of the anti-IL-8 antibody can be analyzed using the above-mentioned T cell epitope prediction program to design a sequence with reduced immunogenicity. Preferred sites for amino acid modifications that reduce the immunogenicity of the anti-IL-8 antibody disclosed in C include, but are not limited to, the heavy chain sequence of the anti-IL-8 antibody shown in SEQ ID NO: 78, according to Kabat The amino acid at position 81 and/or 82b of the numbering method.

In one embodiment, Disclosure C provides a method for improving elimination of IL-8 from an individual compared to use of a reference antibody, comprising administering to the individual an anti-IL-8 antibody of Disclosure C. In one embodiment, an anti-IL-8 antibody of C is disclosed for use in enhancing the elimination of IL-8 from an individual compared to the use of a reference antibody. In one embodiment, Disclosure C is directed to the use of an anti-IL-8 antibody of Disclosure C for enhanced elimination of IL-8 from an individual compared to use of a reference antibody. 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 to enhance elimination of IL-8 from a living body compared to use of a reference antibody. In one embodiment, Disclosure C relates to a pharmaceutical composition comprising an anti-IL-8 antibody of Disclosure C that enhances elimination of IL-8 from a living subject compared to use of a reference antibody. In one embodiment, Disclosure C is directed to a method of increasing elimination of IL-8 from a living subject compared to use of a reference antibody, comprising administering to a subject an anti-IL-8 antibody of Disclosure C. In the embodiment of Disclosure C, the reference antibody refers to the anti-IL-8 antibody before modification in order to obtain the antibody of Disclosure C, or an antibody with strong IL-8 binding affinity at both acidic and neutral pH. The reference antibody may be an antibody containing the amino acid sequences of SEQ ID NO: 83 and 84 or SEQ ID NO: 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 to IL-8 and then binds to the extracellular matrix. In one embodiment, Disclosure C relates to the use of the anti-IL-8 antibody of Disclosure C for manufacturing a pharmaceutical composition, characterized in that the anti-IL-8 antibody of Disclosure C binds to IL-8 and then binds 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 includes the heavy chain variable region of any of the above embodiments and the light chain variable region of any of the above embodiments. In one embodiment, the antibody of Disclosure C includes the heavy chain variable region of SEQ ID NO: 78 and the light chain variable region of SEQ ID NO: 79, and may include post-translational modifications of the sequences thereof.

In another aspect, an anti-IL-8 antibody according to any of the above embodiments may include, alone or in combination, the characteristics described in Sections 1 to 7 below.

1. Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided in Disclosure C can be chimeric antibodies. Some chimeric antibodies are described in, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984). In one example, a chimeric antibody may include a non-human variable region (eg, a variable region 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. Generally, non-human antibody systems are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable regions, wherein the HVRs such as CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody may be substituted with residues from the corresponding non-human antibody (eg, the antibody from which the HVR residues are derived), for example, to maintain or improve antibody specificity or affinity.

Humanized antibodies and preparation methods are reviewed in, for example, Almagro et al., Front. Biosci. 13:1619-1633 (2008), and documented in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al ., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (documented exclusively Sex-determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (documenting "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (documenting "FR drag"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (documenting "guidance for FR drag") selection method).

Human frame regions that can be used for humanization include, but are not limited to, frame regions selected using "best-fit" methods (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); or a framework region derived from the consensus sequence of a specific subgroup of human antibodies in the heavy chain variable region (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. , J. Immunol., 151:2623 (1993)); and framework regions 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 may be antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv 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 of scFv fragments, see for example Pluckthun, in 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.

Bifunctional antibodies are antibody fragments with two antigen-binding sites and can 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) ). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single domain antibodies are antibody fragments that include 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, eg, U.S. Patent No. 6,248,516 B1). Antibody fragments can be produced using a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or bacteriophage), as described within the context of disclosure C.

3. Human Antibodies In certain embodiments, the antibodies provided in Disclosure C can be human antibodies. Human antibodies can be prepared using various techniques known in the art. General terms for human antibodies as described in Van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies can be produced by administering immunogens to modified genetically modified animals to produce fully human antibodies or intact antibodies with human variable regions that respond to an antigenic challenge. Such animals generally contain all or part of the human immunoglobulin gene locus, which is obtained by replacing the animal (non-human) immunoglobulin gene locus and making it extrachromosomal or randomly integrated into the animal's chromosome. In such transgenic mice, the animal (non-human) immunoglobulin gene locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also US Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE ^™ technology; US Patent No. 5,770,429 for HUMAB ^™ technology; US Patent No. 7,041,870 for KM MOUSE ^™ technology; and US Patent Application Publication No. US 2007/0061900 for VELOCIMOUSE ^™ technology. .

The human variable regions of intact antibodies produced by such animals can be further modified by combining different human constant regions. Human antibodies can also be prepared using fusion tumor-based methods. Human myeloma and mouse-human heterogeneous myeloma cell lines for the production of human monoclonal antibodies are described, for example, 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 using human B cell fusion tumors are described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 for the production of monoclonal human IgM antibodies from fusionoma cell lines, and for example, those described in Ni, Xiandai Mianyixue, 26(4):265-268 (2006) Methods for human-human fusion tumors. Human fusion tumor technology (ternary fusion tumor technology) is also documented in Vollmers et al., Histol. And Histopath. 20(3):927-937 (2005) and Vollmers et al., Methods and Findings 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. Such a sequence of variable fields can be combined with any desired invariant fields. Techniques for selecting human antibodies from antibody libraries are as follows.

4. Antibodies derived from libraries Antibodies revealing C can be isolated by screening combinatorial libraries for antibodies with the desired activity. For example, various methods exist for generating phage display libraries and screening libraries for antibodies possessing desired binding properties. Such an approach is described in Hoogenboom et al., in Meth. Mol. Biol. 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and for example, McCafferty et al., Nature 348:552-554 (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. Reviewed in , J. Immunol. Meth. 284(1-2):119-132 (2004).

In some phage display methods, libraries of VH and VL coding sequences can be cloned separately using polymerase chain reaction (PCR) and randomly recombined in the phage library. The obtained phage library was screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically present antibody fragments, either as single chain Fv (scFv) fragments or Fab fragments.

Alternatively, unchanged stocks can be cloned (e.g. from humans) to provide a single source of antibodies against a broad range of non-self and self antigens without the need for any immunization, as in Griffiths et al., EMBO J. 12:725-734 (1993 ) recorded.

Finally, one can also select unrearranged V-gene segments from stem cells, use PCR primers containing random sequences to encode the highly variable CDR3 region, and complete the rearrangement in vitro to synthesize the unchanged construct. Libraries (see below and Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992); patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373; and U.S. Application Publication No. 2005 /0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. Here, antibodies or antibody fragments isolated from the human antibody library are regarded as human antibodies or human antibody fragments.

5. Multispecific antibodies In certain embodiments, antibodies provided in accordance with Disclosure C can be, for example, multispecific antibodies, such as bispecific antibodies. Multispecific antibody systems target monoclonal antibodies with binding specificity 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 localize cytotoxic agents to cells expressing IL-8. Bispecific antibodies can be in the form of full-length antibodies or antibody fragments.

Techniques for producing multispecific antibodies include, but are not limited to, recombinant two immunoglobulin heavy chain-light chain pairs that co-express 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. Patent No. 5,731,168). Multispecific antibodies can be prepared by cross-linking 2 or more antibodies or fragments using electrostatic steering effects to prepare Fc-heterodimer molecules (WO 2009/089004A1) (US Patent No. 4,676,980; and Brennan et al ., Science 229:81 (1985)), by using leucine zippers to create bispecific antibodies (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)), by using "Bifunctional antibody" technology to produce bispecific antibody fragments (Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), by using single-chain Fv (scFv) dyads (Gruber et al., J. Immunol. 152:5368 (1994)) or other methods. The preparation of trispecific antibodies is described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

Engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies", are also included here (see, eg, US 2006/0025576).

Within the context of disclosure C, such antibodies or antibody fragments also include "dual-acting Fab" or "DAF", including an antigen-binding site that binds to IL-8 and a different antigen (see, for example, US 2008/0069820) .

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, deletion, and/or insertion and/or substitution of residues in the amino acid sequence of the antibody. The final construct may include any combination of deletions, insertions, and substitutions, provided that the final construct is an antibody having the desired properties disclosed in the context of C.

In one embodiment, it is disclosed that C provides an antibody variant with one or more amino acid substitutions. Such substitution sites can be anywhere in the antibody. Conservatively substituted amino acids are shown in Table 10, titled "Conservative Substitutions." Commonly substituted amino acids that result in more substantial changes are shown in Table 10 under the heading "General Substitutions" and are further documented in the reference to the Amino Acid Side Chain Category.

[Table 10]

Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidity: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain alignment: Gly, Pro; and (6) Aromaticity: Trp, Tyr, Phe.

Non-conservative substitutions would cause a member of one of these classes to change into another class.

Amino acid insertions include fusions ranging from 1, 2, or 3 to 100 or more residues at the N-terminus and/or C-terminus of a polypeptide, and insertion of one or more amino acid residues into a sequence. Antibodies with such terminal insertions include, for example, antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include those derived from N- or C-terminal fusions of the antibody to enzymes (e.g., ADEPT) or peptides that increase the plasma half-life of the antibody.

7. Glycation variants In one embodiment, the antibody provided in accordance with Disclosure C can be a glycated antibody. Glycation sites can be created or removed by changing the amino acid sequence by adding or deleting them from the antibody.

When the antibody includes an Fc region, the attached sugar chains may vary. Unaltered antibodies produced by animal cells generally contain branched, bibranched oligosaccharides that are N-linked to Asn297 in the CH2 domain of the Fc region (see Wright et al. TIBTECH 15:26-32 (1997)). Such oligosaccharides include, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose, GlcNAc attached to the "stem" of the bibranched oligosaccharide structure. In one embodiment, it is disclosed that the oligosaccharides of the antibody C are modified to create antibody variants with specific improved properties.

8. Fc region variants In one embodiment, one or more amino acid modifications are introduced into the Fc region of an antibody provided in accordance with Disclosure C to generate Fc region variants. Fc region variants include modifications (eg, substitutions) of 1, 2, 3, or more amino acids in the native human Fc region sequence (eg, the Fc region of human IgG1, IgG2, IgG3, or IgG4).

The anti-IL-8 antibody disclosed in C may include an Fc region having at least one of the following five properties, but is not limited to: (a) Binding affinity for FcRn relative to the native Fc region at acidic pH. The binding affinity of the FcRn for the Fc region is increased; (b) The binding affinity of the Fc region for existing ADA is decreased relative to the binding affinity of the native Fc region for existing ADA; (c) The binding affinity for the existing ADA is decreased relative to the native Fc region the plasma half-life of the Fc region is increased; (d) the plasma clearance rate of the Fc region is decreased relative to the plasma clearance rate of the native Fc region; and (e) the plasma clearance rate of the Fc region is decreased relative to the native Fc region targeting effector receptors The binding affinity of the Fc region for the effector receptor is reduced. In some embodiments, the Fc region has 2, 3, or 4 of the properties listed above. In one embodiment, Fc region variants include those with increased FcRn binding affinity at acidic pH. Fc region variants with increased FcRn binding affinity include, but are not limited to, FcRn binding affinity increased by up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold compared to the FcRn binding affinity of an antibody containing a native IgG Fc region. 15x, 20x, 30x, 50x or 100x Fc region variants.

In one embodiment, Fc region variants include safe and beneficial Fc region variants that do not bind to existing ADA and have improved plasma retention. As used in the context of Disclosure C, the term "ADA" refers to an endogenous antibody that has binding affinity for the epitope on the therapeutic antibody. As used in the context of Disclosure C, the term "existing ADA" refers to detectable anti-drug antibodies present in the patient's blood prior to administration of therapeutic antibodies to the patient. Pre-existing ADA includes rheumatoid factor. Fc region variants with low binding affinity for existing ADA include, but are not limited to, ADA binding affinity reduced to 1/10 or less, 1/ 50 or less or 1/100 or less Fc region variant.

In one embodiment, Fc region variants include Fc region variants that have low binding affinity for complement proteins or do not bind to complement proteins. Complement proteins include C1q. Fc region variants with low binding affinity for complement proteins include, but are not limited to, those with binding affinity for complement proteins reduced by 1/10 or less compared to the complement protein binding affinity of antibodies containing native IgG Fc regions. , 1/50 or less or 1/100 or less.

In one embodiment, Fc region variants include Fc region variants that have low binding affinity for effector receptors or have no binding affinity for effector receptors. Effector receptors include, but are not limited to, FcyRI, FcyRII, and FcyRIII. FcγRI includes, but is not limited to, FcγRIa, FcγRIb, and FcγRIc, and subtypes thereof. FcγRII includes but is not limited to FcγRIIa (with 2 subtypes: R131 and H131) and FcγRIIb. FcγRIII includes but is not limited to FcγRIIIa (with 2 subtypes: V158 and F158) and FcγRIIIb (with 2 subtypes: FcγRIIIb-NA1 and FcγRIIIb-NA2). Fc region variants with low binding affinity for the effector receptor include, but are not limited to, those that have binding affinity for the effector receptor reduced to at least 1/1 compared to the binding affinity of an antibody containing a native IgG Fc region. 10 or less, 1/50 or less, or 1/100 or less Fc region variants.

In one embodiment, the Fc region variant includes an Fc region with one or more amino acid substitutions at any position selected from the following positions according to EU numbering: 235, 236, 239 compared to the native Fc region. , 327, 330, 331, 428, 434, 436, 438, and 440 bits.

In one embodiment, the Fc region variant includes one or more amino acid substitutions at positions 235, 236, 239, 428, 434, 436, 438, and 440 according to EU numbering compared to the native Fc region. Fc area.

In one embodiment, the Fc region variant includes one or more at positions 235, 236, 327, 330, 331, 428, 434, 436, 438, and 440 according to EU numbering compared to the native Fc region. Amino acid substituted Fc region.

In one embodiment, the Fc region variant includes an Fc region substituted with one or more amino acids selected from the group consisting of: L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R , and S440E.

In one embodiment, Fc region variants include Fc regions containing amino acid substitutions M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, Fc region variants include Fc regions containing amino acid substitutions L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, Fc region variants include Fc regions containing amino acid substitutions L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the anti-IL-8 antibody of Disclosure C includes the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82. The anti-IL-8 antibody including 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 that binds to IL-8 in a pH-dependent manner. antibody. The anti-IL-8 antibody including 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 (eg, plasma). The anti-IL-8 antibody including 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. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may comprise the FcRn binding affinity at acidic pH compared to the native Fc region , an Fc region with increased FcRn binding affinity at acidic pH (e.g., pH 5.8). The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may comprise a binding affinity compared to the native Fc region for existing ADA, Targets the Fc region with reduced binding affinity for existing ADA. The anti-IL-8 antibody including the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may include an Fc region with an extended half-life in plasma compared to the native Fc region. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO: 80 and/or the amino acid sequence of SEQ ID NO: 82 may comprise a binding affinity for the effector receptor compared to the native Fc region. Sexually reduced Fc region. In yet another embodiment, the anti-IL-8 antibody includes all 7 properties in combination with any 2, 3, 4, 5, and 6 of the above properties.

In one embodiment, the anti-IL-8 antibody of Disclosure C includes the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82. The anti-IL-8 antibody including 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 that binds to IL-8 in a pH-dependent manner. antibody. The anti-IL-8 antibody including 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 (for example, in plasma). . The anti-IL-8 antibody including 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. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may comprise an FcRn binding affinity at acidic pH compared to the native Fc region. The FcRn binding affinity of the Fc region is increased. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may have a binding affinity compared to the native Fc region for existing ADA, Targets the Fc region with reduced binding affinity for existing ADA. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO: 81 and/or the amino acid sequence of SEQ ID NO: 82 may comprise an Fc region with an extended half-life in plasma compared to the native Fc region. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may comprise a binding affinity for the effector receptor compared to the native Fc region. Sexually reduced Fc region. In yet another embodiment, the anti-IL-8 antibody includes all 7 properties in combination with any 2, 3, 4, 5, and 6 of the above properties.

In certain embodiments, Disclosure C includes an antibody variant that possesses some, but not all, effector functions. This antibody variant may be an ideal candidate when certain effector functions (such as complement and ADCC) are not required or can be deleted. In vitro and/or in vivo cytotoxicity assays known in the art can be routinely performed to confirm reduction/total loss of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to identify antibodies that lack FcγR binding (and therefore ADCC activity) ability but retain FcRn binding ability.

Primary culture cells targeting intermediary ADCC and NK cells express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcRs expressed on heme-producing cells are summarized in Table 3 on page 464 of Ravetch et al., Annu. Rev. Immunol. 9:457-492 (1991). Nonlimiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom. et al., Proc. Natl Acad. Sci. USA 83:7059-7063 (1986), Hellstrom et al. al., Proc. Natl Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 and Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, nonradioactive isotope assays may be used to assess effector cell function (see, e.g., ACTI(TM) nonradioactive cytotoxic assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ^TM nonradioactive cytotoxic assay (Promega, Madison, CA)). WI)). Effector cells useful in such analyzes include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.

Alternatively or additionally, the ADCC activity of antibody variants of interest can be assessed in vivo, for example in animal models, such as Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998) revealed. A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISA described in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Meth. 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance/half-life can be performed using methods known in the art (see, eg, Petkova et al., Intl. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include one or more substitutions of Fc region residues at positions 238, 265, 269, 270, 297, 327, or 329 (U.S. Patent No. 6,737,056). Such Fc region variants include Fc region variants with 2 or more substitutions at residues 265, 269, 270, 297 or 327, including those in which the residues at positions 265 and 297 are alanine, which is called "DANA" "Fc region variants (U.S. Patent No. 7,332,581).

Antibody variants with improved or reduced binding to FcR groups are described below. (See, eg, US Patent No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

Antibodies with increased half-life in blood and improved FcRn binding at acidic pH are described in. US2005/0014934. The antibodies described include one or more substitutions in the Fc region that improve the binding of the Fc region to FcRn. Such Fc region variants include those in the Fc region selected from the group consisting of 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382 Substitution of one or more of positions 413, 424 or 434, such as substitution of position 434 of the Fc region (US Patent No. 7,371,826).

Other examples of Fc region variants are also found in Duncan et al., Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94/29351.

9. Antibody derivatives In certain embodiments, the antibodies provided by Disclosure C can be further modified to contain additional non-protein structures that are known and readily available in the art. Structures suitable for derivatizing such antibodies include, but are not limited to, water-soluble polymers. Examples of water-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-tris[ethyl]ane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), poly(n- Vinylpyrrolidone) polyethylene glycol, propylpropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl alcohol and its mixture. Polyethylene glycol propionaldehyde has advantages in industrialization because it is stable in water.

The polymer can be of any molecular weight and can be branched or unbranched. If there is more than one polymer, the number of polymers attached to the antibody can be different and they can be the same or different molecules. Generally speaking, the number and/or type of polymers used for derivatization, if the antibody derivative is used in a defined therapy, can be determined based on, but not limited to, the specific properties or functions of the antibody to be improved.

In another embodiment, conjugates of C-disclosing anti-IL-8 antibodies and non-protein structures can be provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein structure is, for example, a carbon nanotube (see, for example, Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including, but not limited to, harmless to humans but capable of heating the non-protein structure to a temperature that will kill cells in proximity to the antibody-non-protein structure.

B. Recombinant methods and compositions Anti-IL-8 revealing C antibodies can be produced using, for example, the recombinant methods and compositions described in US Pat. No. 4,816,567. One embodiment provides an isolated nucleic acid encoding an anti-IL-8 antibody as presented in Disclosure C. Such a nucleic acid may encode an amino acid sequence that includes the VL of the antibody and/or an amino acid sequence that includes the VH of the antibody (eg, the light chain and/or the heavy chain of the antibody). In yet another embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In one embodiment, a host cell comprising such a nucleic acid is provided. In one embodiment, the host cell includes (e.g., has been transformed): (1) a vector including nucleic acids encoding the VL of the antibody and the VH of the antibody, or (2) a first vector including the VL encoding an antibody The nucleic acid; and the second vector, including the nucleic acid encoding the VH of the antibody.

In one embodiment, the host is eukaryotic (e.g., Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, SP20 cells)).

In one embodiment, a method for producing an anti-IL-8 antibody revealing C is provided, the method comprising: culturing a host cell including a nucleic acid encoding the anti-IL-8 antibody as described above under suitable conditions for expressing the antibody, and The antibody is optionally recovered (eg from the host cell or host cell culture medium).

To recombinantly produce anti-IL-8 antibodies, for example, as described above, nucleic acids encoding the antibodies are isolated and inserted into one or more vectors for further selection and/or expression of host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that bind specifically to the nucleic acid encoding the heavy and light chains of the antibody).

Host cells suitable for colonization or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells described within the context of disclosure C. For example, antibodies can be produced in bacteria, especially when glycation and Fc effector functions are not required. For expression of antibody fragments and peptides against bacteria, see, for example, U.S. Patent 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 antibodies can be isolated from the bacterial cell paste in the soluble fraction and further purified.

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable hosts for colonization or expression of vectors encoding antibodies, including fungi and yeast strains with "humanized" glycation pathways capable of producing parts of or Antibodies with fully human glycation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Host cells suitable for expressing glycated antibodies can also originate from multicellular organisms (invertebrate and vertebrate organisms). Examples of invertebrate cells include plant and insect cells. Without special limitation, a combination of baculovirus and insect cells can be used to transfect Spodoptera frugiperda cells, and many baculovirus strains have been identified.

Plant cell cultures can be used as hosts. See U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing PL Antibody™ technology for producing antibodies in genetically modified plants).

Vertebrate animal cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension are useful. Other useful mammalian host cells include the monkey kidney CV1 strain, which is transformed by SV40 (COS-7); the human embryonic kidney cell strain (293 cells, described in Graham et al., J. Gen Virol. 36:59 (1977) )); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, documented in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); Africa Green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); Mouse mammary tumor (MMT 060562); TRI cells, as described in 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 not limited. For a review of 8AD6 other mammalian host cell lines 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 C-revealing antibody produced by culturing a host cell carrying a nucleic acid encoding the antibody as described above under conditions suitable for antibody expression can be isolated from within or outside the host cell (culture medium, milk) and refined to substantially Pure homogeneous antibodies. The isolation/purification method generally used for purifying polypeptides can be appropriately used to isolate and purify the antibody; however, this method is not limited to the above example. Antibodies can be prepared by, for example, appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization for appropriate separation and purification, but not limited. Chromatography includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed phase chromatography and adsorption chromatography. Such chromatography can be performed using liquid chromatography such as HPLC and FPLC. Columns used in 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), etc.

In view of the characteristics of anti-IL-8 antibodies, such as the above-mentioned increased extracellular matrix binding activity and promotion of cellular uptake complex, Disclosure C provides a method of selecting antibodies with increased extracellular matrix binding activity and antibodies with enhanced cellular uptake complex. In one embodiment, Disclosure C provides a method for producing an anti-IL-8 antibody that includes a variable region that binds IL-8 in a pH-dependent manner, comprising the following steps: (a) Evaluating anti-IL-8 Binding of the antibody to the extracellular matrix; (b) selecting an anti-IL-8 antibody that binds strongly to the extracellular matrix; (c) culturing a host that includes a vector carrying a nucleic acid encoding the antibody; and (d) growing a monomer from the culture medium away from this antibody.

The combination with the extracellular matrix can be performed by any method without limitation, as long as it is known to those with ordinary knowledge in the technical field. For example, an ELISA system can be used to detect the binding between an antibody and an extracellular matrix, in which the antibody is added to an extracellular matrix-immobilized plate and a labeled antibody against the antibody is added. Alternatively, for example, the analysis can use an electrochemiluminescence (ECL) method, in which a mixture of the antibody and the ruthenium antibody is added to an extracellular matrix-immobilized plate, and the binding of the antibody to the extracellular matrix is assessed based on the electrochemiluminescence of ruthenium. .

The anti-IL-8 antibody used to assess extracellular matrix binders in step (a) can be the antibody itself or in contact with IL-8. "Selecting an anti-IL-8 antibody that strongly binds to the extracellular matrix" in step (b) refers to the selected anti-IL-8 antibody that, when evaluating extracellular matrix binding, is based on representing the extracellular matrix and this antibody. -The binding value of IL-8 antibody is higher than the value representing the binding of extracellular matrix and reference antibody, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times or more; but the ratio is not particularly limited to the above example. Conditions other than the presence of IL-8 should be the same as the procedure for assessing the binding of anti-IL-8 antibodies to the extracellular matrix. The control anti-IL-8 antibody used to compare several modified anti-IL-8 antibodies can be an unmodified anti-IL-8 antibody. In this case, other conditions should be the same except for the presence of IL-8. Specifically, in one embodiment, disclosure C includes selecting an antibody with a higher value representing extracellular matrix binding from a plurality of anti-IL-8 antibodies that have not been contacted with IL-8. In another embodiment, disclosure C includes selecting an antibody with a higher value representing extracellular matrix binding from among several anti-IL-8 antibodies in contact with IL-8. In an alternative embodiment, "selecting an anti-IL-8 antibody that binds strongly to the extracellular matrix" of step (b) means that when assessing extracellular matrix binding, it can be based on the presence of IL-8, Antibodies are selected based on their ability to bind to the extracellular matrix. A value representing the ratio of the extracellular matrix binding of the anti-IL-8 antibody that is in contact with IL-8 relative to the extracellular matrix binding of the anti-IL-8 antibody that is not in contact with IL-8 is, for example, 2 to 1000. The value of this ratio can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

C. Analysis Anti-IL-8 antibodies provided within the scope of disclosure C can be identified, screened or characterized with respect to their physical/chemical properties or biological activity using various methods known in the art.

1. Combining assays and other assays in one aspect, the antibody disclosed in C can be targeted for its antigen-binding activity using known methods, such as ELISA, Western blotting, kinetic exclusion assay (KinExA ^TM ) and surface plasmonics Resonance is evaluated using, for example, a BIACORE (GE Healthcare) device.

In one embodiment, binding affinity can be evaluated using BIACORE T200 (GE Healthcare) in the following manner. An appropriate amount of capture protein (such as Protein A/G (PIERCE)) is immobilized on the sensor chip CM4 (GE Healthcare) using the amine coupling method, so that the antibody of interest is captured. Then inject the diluted antigen solution and running buffer (as a reference solution: for example, 0.05% tween20, 20 mM ACES, 150 mM NaCl, pH 7.4) to allow the antigen molecules to interact with the antibodies captured on the sensor chip. Regenerate the sensor wafer using 10 mM glycine HCl solution (pH 1.5). Measurement is performed at a predetermined temperature (eg, 37°C, 25°C, or 20°C). The association rate constant kon (1/Ms) and the dissociation rate constant koff (1/s) are both kinetic parameters and are calculated from the obtained induction pattern. The KD (M) of an antibody against this antigen is calculated based on these constants. Each parameter was calculated using BIACORE T200 Evaluation Software (GE Healthcare).

In one embodiment, IL-8 can be quantified as follows. An anti-human IL-8 antibody including a mouse IgG constant region is immobilized on the plate, and a solution including IL-8 bound to the humanized anti-IL-8 antibody that does not compete with the anti-human IL-8 antibody is provided, Add to immobilization plate. After stirring, biotinylated anti-human Igκ light chain antibody was added and allowed to react for a period of time. Then SULFO-Tag-labeled streptavidin is added and allowed to react for a period of time. Analysis buffer was then added and immediately measured with SECTOR Imager 2400 (Meso Scale Discovery).

2. Activity analysis method In one aspect, an assay for identifying biologically active anti-IL-8 antibodies is provided. Biological activities include, for example, IL-8 neutralizing activity and activity to block IL-8 signaling. Disclosure C also provides antibodies having such biological activity in vivo and/or in vitro.

In one embodiment, the method for determining the IL-8 neutralization level is not particularly limited, and the following method can also be used. PathHunter ^TM CHO-K1 CXCR2 Beta-Arrestin Cell Line (DiscoveRx, Cat.# 93-0202C2) is an artificial cell line created to express human CXCR2, which is known as the human IL-8 receptor, and through human IL-8 It emits chemical electroluminescence when receiving a signal. When human IL-8 is added to the culture medium of cells, the cells then emit chemiluminescence in a manner that is dependent on the concentration of added human IL-8. When human IL-8 and anti-human IL-8 antibodies are added to the culture medium in combination, the chemiluminescence of the cells is reduced or undetectable compared to when this antibody is not added. The reason is that the anti-human IL-8 antibody 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 stronger the level of chemiluminescence. Therefore, the human IL-8 neutralizing activity of anti-human IL-8 antibodies can be evaluated by examining the above differences.

D. Pharmaceutical formulas Pharmaceutical formulations including anti-IL-8 antibodies within the scope described in Disclosure C can be prepared by mixing an anti-IL-8 antibody of a desired degree of purity with one or more optional pharmaceutically acceptable carriers. They may be prepared in the form of dry formulations or aqueous solution formulations (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, histidine, and other organic acids; antioxidants, including Ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butylphenol or benzyl alcohol ; Phenyl 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, and Aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars such as sucrose, mannitol, seaweed Sugar or sorbitol; salt-forming counterions, such as sodium; metal complexes (such as Zn-protein complexes); and/or nonionic surfactants, such as TWEEN ^™ , PLURONICS ^™ or polyethylene glycol (PEG).

Pharmaceutically acceptable carriers also include enteric drug dispersants, such as soluble hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX ^TM , Baxter International, Inc .). Some examples of sHASEGP, and methods of use including rHuPH20, are described in US Application Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more glycosaminoglycanases, such as chondroitinase.

An example of a freeze-dried antibody formulation is described in U.S. Patent No. 6,267,958. Aqueous antibody formulations are documented in US Patent No. 6,171,586 and WO2006/044908; the WO2006/044908 formulation includes a histidine-acetate buffer.

Formulations within the scope of disclosure C may also include more than one active ingredient necessary for the treatment of specific indications, which should have complementary activities without being detrimental to each other. Such active ingredients are preferably combined in an amount effective for the purpose.

The active ingredients can be encapsulated in microcapsules, for example prepared by coacervation technology or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in a colloidal drug delivery system (such as microlipids body, albumin microspheres, microemulsifiers, nanoparticles and nanocapsules) or macroemulsifiers. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained release preparations can be prepared. Suitable examples of sustained release preparations include a semipermeable matrix containing a solid hydrophobic polymer of the anti-IL8 antibody of Disclosure C, wherein the matrix is in the form of a shaped article, such as a film or microcapsule.

Formulations for in vivo administration are generally sterile. Sterilization can easily be achieved by filtration through a sterile filter membrane.

E. Treatment Methods and Compositions In some embodiments, it is disclosed that the anti-IL-8 antibody system provided by C is used in a method of treatment.

In one aspect, anti-IL-8 antibodies for use as pharmaceutical compositions are provided. In an alternative aspect, anti-IL-8 antibodies are provided for treating diseases in which excess IL-8 is present. In one embodiment, anti-IL-8 antibodies are provided for use in methods of treating diseases in which excess IL-8 is present. In one embodiment, Disclosure C provides a method for treating a subject in which excess IL-8 is present (eg, a disease resulting from the presence of excess IL-8), comprising administering to the subject an effective amount of anti-IL-8 antibody. In another embodiment, Disclosure C provides anti-IL-8 antibodies for use in such methods. In one embodiment, disclosure C relates to a pharmaceutical composition comprising an effective amount of an anti-IL-8 antibody for treating diseases in which IL-8 is excessively present. In one embodiment, Disclosure C relates to the use of anti-IL-8 antibodies in the manufacture of pharmaceutical compositions for treating diseases in which excess IL-8 is present. In one embodiment, disclosure C relates to the use of an effective amount of an anti-IL-8 antibody to treat a disease in which excess IL-8 is present. Diseases in which IL-8 is present in excess include, but are not limited to, inflammatory skin diseases such as inflammatory keratitis (psoriasis, etc.), 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; inflammatory liver diseases such as hepatitis B, hepatitis B, alcoholic hepatitis, and drug-induced allergies Hepatitis; Inflammatory kidney diseases such as glomerulonephritis; Inflammatory respiratory diseases such as bronchitis and asthma; Chronic inflammatory vascular diseases such as atherosclerosis; Multiple sclerosis; Oral ulcers; Vocal fold inflammation; Artificial organs/artificial vasculature Inflammation caused; malignancies such as ovarian, lung, prostate, stomach, breast, melanoma, head and neck, and kidney cancer; sepsis caused by infection; cystic fibrosis; pulmonary fibrosis; and acute lung injury.

In an alternative embodiment, Disclosure C provides anti-IL-8 antibodies for inhibiting the accumulation of biologically active IL-8. "Inhibiting accumulation of IL-8" can be achieved by preventing the amount of IL-8 existing in the body from increasing or decreasing the amount of IL-8 existing in the body. In one embodiment, Disclosure C provides an anti-IL-8 antibody for inhibiting the accumulation of IL-8 in an individual to inhibit the accumulation of biologically active IL-8. "IL-8 present in vivo" as used herein may refer to IL-8 complexed with an anti-IL-8 antibody or free IL-8; or, to total IL-8. Here, "exist in vivo" may mean "secreted into cells outside the body." In one embodiment, Disclosure C provides a method of inhibiting the accumulation of biologically active IL-8, comprising the step of administering an effective amount of an anti-IL-8 antibody to a subject. In one embodiment, disclosure C relates to a pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, including an effective amount of an anti-IL-8 antibody. In one embodiment, disclosure C relates to the use of anti-IL-8 antibodies to produce pharmaceutical compositions for inhibiting the accumulation of biologically active IL-8. In one embodiment, disclosure C relates to using an effective amount of an anti-IL-8 antibody to inhibit the accumulation of biologically active IL-8. In one embodiment, it is disclosed that the anti-IL-8 antibody of C inhibits IL-8 accumulation compared to an anti-IL-8 antibody without pH-dependent binding activity. In the above embodiments, the "individual" is preferably a human.

In an alternative embodiment, C is disclosed to provide an anti-IL-8 antibody for inhibiting angiogenesis (eg, neoangiogenesis). In one embodiment, Disclosure C provides a method of inhibiting angiogenesis in an individual, comprising administering an effective amount of an anti-IL-8 antibody to the antibody, and provides an anti-IL-8 antibody for use in the method. In one embodiment, it is disclosed that C relates to a pharmaceutical composition for inhibiting angiogenesis, including an effective amount of an anti-IL-8 antibody. In one embodiment, Disclosure C relates to the use of anti-IL-8 antibodies to produce pharmaceutical compositions for inhibiting angiogenesis. In one embodiment, disclosure C relates to using an effective amount of an anti-IL-8 antibody to inhibit angiogenesis in the above embodiment, and the "individual" is preferably a human.

In an alternative aspect, C is disclosed to provide an anti-IL-8 antibody for inhibiting the promotion of neutrophil migration. In one embodiment, Disclosure C provides a method of inhibiting neutrophil migration promotion in an individual, comprising administering an effective amount of an anti-IL-8 antibody to the antibody; and provides anti-IL for use in the method. -8 antibodies. In one embodiment, it is disclosed that C relates to a pharmaceutical composition for inhibiting the promotion of neutrophil migration in an individual. In one embodiment, Disclosure C relates to the use of an anti-IL-8 antibody to manufacture a pharmaceutical composition that inhibits the promotion of neutrophil migration in an individual. In one embodiment, disclosure C relates to using an effective amount of an anti-IL-8 antibody to inhibit neutrophil migration promotion in an individual. In the above embodiments, the "individual" is preferably a human.

In an alternative embodiment, Disclosure C provides a pharmaceutical composition comprising an anti-IL-8 antibody provided herein, for example, for use in any method of treatment. In one embodiment, a pharmaceutical composition includes the anti-IL-8 antibody provided in Disclosure C and a pharmaceutically acceptable carrier.

C-disclosing antibodies can be used alone or in combination with other agents in therapy. For example, an antibody revealing C can be co-administered with at least 1 additional therapeutic agent.

The C-disclosing antibody (and any additional treatment) may be administered using any suitable method, including parenterally, intrapulmonary, and intranasal, and if local treatment is desired, focal administration may be performed. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, such as injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or long-term. Various administration procedures include, but are not limited to, single or multiple administrations at various time points, bolus administration, and intermittent infusion.

Antibodies that reveal C should be formulated, administered, and administered in accordance with good medical practice. Factors considered in this context include the specific disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the pharmaceutical composition, the procedure of administration, and other prescribers of the drug. Knowing factors. The antibody need not, but optionally, be formulated with one or more agents currently used in the prevention or treatment of the condition of concern.

Effective amounts of such other agents will depend on the amount of antibody present in the formulation, the type of condition or treatment, and other factors discussed above. Usually administered at the same dose and via the same route as stated in the disclosure C, or at a dose ranging from 1 to 99% of the disclosure C, or any dose deemed experimentally/clinically appropriate. and any path.

For the prevention or treatment of a disease, the appropriate dosage of an antibody disclosed in C (either 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 progression of the disease, whether this The antibody variant system is administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for administration to patients once or as a series of treatments. Depending on the type and severity of the disease, the initial dosage of the antibody to the patient can be approximately 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg), such as a single administration or several separate administrations. Give or continue infusion. Typical daily dosages range from about 1 mg/kg to 100 mg/kg or more, depending on the factors noted above. With repeated administration over several days or longer, depending on the condition, treatment is generally continued until the desired disease symptom suppression effect is demonstrated. Typical dosages of antibodies range, for example, from about 0.05 mg/kg to about 10 mg/kg. Thus one or more doses of, for example, about 0.5 mg/kg, such as 2.0 mg/kg, such as 4.0 mg/kg, or such as 10 mg/kg (or any combination) may be administered to the patient. Such administration may be intermittently, such as every week or every three weeks (eg, such that the patient receives about 2 to about 20 doses or about 6 doses of the antibody). A higher loading dose may be administered initially, followed by one or more lower doses; however, other dosing regimens are useful. The progress of therapy can be easily monitored using well-known techniques and analytics.

F. Manufactured items In another aspect of the disclosure, the present disclosure provides articles of manufacture, including materials useful in treating, preventing, and/or diagnosing the above-mentioned disorders. Such articles of manufacture include containers, labels, or drug instructions on or associated with the container. Suitable containers include, for example, bottles, vials, and intravenous solution bags. Containers can be made of various materials, such as glass or plastic. Such containers may hold compositions, either by themselves or with other compositions useful for treating, preventing, and/or diagnosing conditions, and may also have sterile access holes (for example, the container may be an intravenous solution bag or may have a hole that can be penetrated by a hypodermic needle). small glass bottles with caps). At least one active ingredient in the composition is an antibody revealing C. The label or package insert indicates that the composition is intended to treat the selected condition.

Additionally, articles of manufacture may include: (a) a first container including a composition containing an antibody revealing C; and (b) a second container including a composition containing an additional cytotoxic agent or a different therapeutic agent. The article of manufacture of the embodiment disclosed in C may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture further includes, for example, a second (or third) container, which includes a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. The article of manufacture may further include other materials as desired from a marketing or user perspective, including other buffers, filters, needles and syringes.

A person with ordinary skill in this technical field will know based on the common technical knowledge in this technical field that the present disclosure includes all partial or complete combinations of one or more of the complete embodiments described herein, unless technically incompatible.

Reveal A, B or C All technical background documents mentioned herein are incorporated by reference.

As used herein, the word "and/or" is understood to include combinations of "and/or" before and after "and/or", including combinations of terms appropriately connected with this word.

Although various elements described herein are labeled, for example, 1st, 2nd, 3rd, 4th, etc., it should be understood that the elements are not limited to these terms. These terms are only used to distinguish elements, and it should be understood that without departing from the scope of revealing A, B, and C, for example, the first element can be called the second element, and similarly, the second element can be called the first element.

Unless expressly stated otherwise or unless inconsistent with the context, any expression in the singular includes the plural, and any expression in the plural includes the singular.

The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the disclosure. Unless otherwise specified, all terms used herein (including technical and scientific terms) are to be interpreted with the meaning commonly understood by a person with ordinary knowledge in the technical field to disclose A, B and C, and shall not be interpreted in an ideal or excessive manner. Formal explanation of ideas.

As used herein, unless the context clearly indicates otherwise, "including" is intended to indicate the presence of the recited item (members, steps, elements, quantities, etc.); and the term does not exclude the presence of other items (members, steps, elements, quantities, etc.) .

The embodiments of disclosure A, B, and C are described with reference to the drawings, which may be exaggerated for simplicity.

Unless otherwise contradicted by context, numerical values used herein are to be understood as specific ranges within the ordinary technical knowledge of one of ordinary skill in the art. For example, the expression "1 mg" is understood to mean "about 1 mg", which may have some deviations. For example, the expression "items 1 to 5" is to be understood specifically and individually as "item 1, item 2, item 3, item 4, item 5" unless it is inconsistent with the context.

Hereinafter, A, B, and C will be specifically disclosed using Embodiments 1 to 4 and 21 to 23, Embodiments 5 to 7, 19 and 20, and Embodiments 8 to 19 respectively, but are not intended to be limited thereto. It is to be understood that various other embodiments may be implemented within the general description given above. [Example] [Example 1]

Production of pH-dependent human IL-6 receptor-binding human antibodies with increased isoelectric point values The Fv4-IgG1 disclosed in WO2009/125825 is an antibody that binds to human IL-6 receptor in a pH-dependent manner, and includes VH3-IgG1 (SEQ ID NO:24) as a heavy chain and VL3-CK (SEQ ID NO:32) as a light chain. In order to increase the isoelectric point value of Fv4-IgG1, amino acid substitutions were introduced into the variable region of Fv4-IgG1, which reduced the number of negatively charged amino acids (such as aspartic acid and glutamic acid) while increasing the band The number of positively charged amino acids (such as arginine and lysine). Specifically, VH3 (High_pI)-IgG1 (SEQ ID NO: 25) can be produced by substituting glutamic acid at position 16 of heavy chain VH3-IgG1 with glutamic acid at position 43 according to Kabat numbering. The glutamic acid at position 64 was substituted with arginine, the glutamic acid at position 64 was substituted with lysine, and the glutamic acid at position 105 was substituted with glutamic acid to increase the isoelectric point value of the heavy chain. Similarly, VL3(High_pI)-CK (SEQ ID NO:33) can be produced by replacing the serine at position 18 of the light chain VL3-CK with arginine and gluten at position 24 according to Kabat numbering. Arginine was substituted for arginine, glutamic acid at position 45 was substituted for lysine, glutamic acid at position 79 was substituted for glutamic acid, and glutamic acid at position 107 was substituted for lysine, so that light The isoelectric point value of the chain increases. When substitution was introduced at position 79 of VL3-CK, modifications involving the substitution of alanine at position 80 for proline and alanine at position 83 for isoleucine were simultaneously introduced, even if not to increase the isoelectric point value.

The following antibody system was produced according to the method of Reference Example 2: (a) Low_pI-IgG1, including VH3-IgG1 as the heavy chain and VL3-CK as the light chain; (b) Middle_pI-IgG1, including VH3-IgG1 as the heavy chain and VL3 (High_pI)-CK as the light chain; and (c) High_pI-IgG1, including VH3(High_pI)-IgG1 as the heavy chain and VL3(High_pI)-CK as the light chain.

Methods known in the art are then used (see, for example, Skoog et al., Trends Analyt. Chem. 5(4): 82-83 (1986)) using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) for each Theoretical isoelectric point values of the antibodies produced were calculated. The side chains of cysteine in this antibody molecule are assumed to be used to form disulfide bonds, and the contribution of cysteine side chains to pKa is excluded from the calculation.

The calculated theoretical isoelectric point values are shown in Table 3. The theoretical isoelectric point value of Low_pI-IgG1 is 6.39, while Middle_pI-IgG1 and High_pI-IgG1 are 8.70 and 9.30 respectively, showing that the theoretical isoelectric point value increases in a stepwise manner.

WO2011/122011 reveals the FcRn-mediated uptake of Fv4-IgG1-F11 (hereinafter referred to as Low_pI-F11) and Fv4-IgG1-F939 (hereinafter referred to as Low_pI-F939) into cells by introducing amino acid substitutions into Fv4-IgG1 It enhances the Fc region and imparts FcRn binding ability under neutral pH conditions. WO2013/125667 also revealed that the FcγR-mediated uptake of Fv4-IgG1-F1180 (hereinafter referred to as Low_pI-F1180) into cells is enhanced by introducing amino acids to be substituted into the Fc region of Fv4-IgG1 to enhance the neutral pH condition. FcγR binding ability. At the same time, amino acid modifications that enhance the plasma retention of this antibody by increasing FcRn binding under the acidic pH conditions of endosomes were introduced into Fv4-IgG1-F1180. The antibody systems shown below were produced by increasing the isoelectric point of antibodies containing these novel Fc region variants.

Specifically, VH3-IgG1-F11 (SEQ ID NO: 30) and VH3-IgG1-F939 (SEQ ID NO: 26) of WO2011/122011, and VH3-IgG1-F1180 (SEQ ID NO: 28) According to the Kabat numbering method, the glutamic acid at position 16 is replaced by glutamic acid, the glutamic acid at position 43 is replaced by arginine, the glutamic acid at position 64 is replaced by lysine, and the glutamic acid at position 105 is replaced by lysine. Glutamic acid was substituted with glutamic acid to produce 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), as a heavy chain with an increased isoelectric point value.

The following antibody system was produced using these heavy chains according to the method of Reference Example 2: (1) Low_pI-F939, including VH3-IgG1-F939 as the heavy chain and VL3-CK as the light chain; (2) Middle_pI-F939, including VH3( High_pI)-F939 as heavy chain and VL3-CK as light chain; (3) High_pI-F939, including VH3(High_pI)-F939 as heavy chain and VL3(High_pI)-CK as light chain; (4) Low_pI-F1180, Including VH3-IgG1-F1180 as the heavy chain and VL3-CK as the light chain; (5) Middle_pI-F1180, including VH3-IgG1-F1180 as the heavy chain and VL3(High_pI)-CK as the light chain; (6) High_pI-F1180 , including VH3(High_pI)-F1180 as the heavy chain and VL3(High_pI)-CK as the light chain; (7) Low_pI-F11, including VH3-IgG1-F11 as the heavy chain and VL3-CK as the light chain; and (8) High_pI-F11 includes VH3(High_pI)-F11 as the heavy chain and VL3(High_pI)-CK as the light chain.

Then, the theoretical isoelectric point value of the produced antibody was calculated using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) by a method similar to the above. The calculated theoretical isoelectric point values are shown in Table 3. In all novel antibodies containing Fc region variants, the theoretical isoelectric point values gradually increase in the order of Low_pI, Middle_pI and High_pI.

[table 3] [Example 2]

Antigen elimination effect of antibodies showing an increase in the isoelectric point value of pH-dependent binding (2-1) In vivo analysis method of pH-dependent human IL-6 receptor-binding antibody with adjusted isoelectric point In vivo analysis was performed as follows using various pH-dependent human IL-6 receptor-binding antibodies produced in Example 1: Low_pI-IgG1, High_pI-IgG1, Low_pI-F939, Middle_pI-F939, High_pI-F939, Low_pI-F1180 , Middle_pI-F1180 and High_pI-F1180.

Soluble human IL-6 receptor (also known as "hsIL-6R"), anti-human IL-6 receptor antibody and human immunoglobulin preparation Sanglopor prepared according to the method of Reference Example 3 were administered to human FcRn at the same time Genetically transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice, Jackson Laboratories; Methods Mol. Biol. 602: 93-104 (2010)) were then evaluated for soluble human IL-6 receptor In vivo dynamics of the body. A mixed solution containing soluble human IL-6 receptor, anti-human IL-6 receptor antibody and Sanglopor (concentrations of 5 μg/mL, 0.1 mg/mL, and 100 mg/mL each) was administered with 10 mL via the tail vein. /kg administered once. Since the amount of anti-human IL-6 receptor antibody is in sufficient excess compared to soluble human IL-6 receptor, it is assumed that almost all of the soluble human IL-6 receptor has bound to this antibody. Blood was collected at 15 minutes, 7 hours, 1 day, 2 days, 3 days, and 7 days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 min to obtain plasma. The separated plasma was stored frozen at -20°C or lower until measurement.

(2-2) Measurement of soluble human IL-6 receptor concentration in plasma using electrochemiluminescence method The concentration of soluble human IL-6 receptor in mouse plasma was measured by electrochemiluminescence method. Samples of soluble human IL-6 receptor were adjusted to concentrations of 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg/mL for calibration curve preparation, and mouse plasma assays diluted 50-fold or more were prepared respectively. sample. The sample and monoclonal anti-human IL-6R antibody (R&D) labeled with ruthenium-SULFO-TAG NHS Ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D), and human IL-6 Receptor-binding antibody Tocilizumab (CAS number: 375823-41-9) was mixed, and then reacted at 37°C overnight. Adjust the final concentration of tocilizumab to 333 μg/mL. The reaction solution was then dispensed into Streptavidin Gold Multi-ARRAY plates (Meso Scale Discovery). After reacting at room temperature for another hour, the reaction solution was washed. Read Buffer T(x4) (Meso Scale Discovery) was then immediately allocated to the plate and measurements were performed using SECTOR Imager 2400 (Meso Scale Discovery). The soluble human IL-6 receptor concentration was calculated based on the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

The changes in the soluble human IL-6 receptor concentration in the plasma of human FcRn gene transgenic mice after intravenous administration are shown in Figures 1, 2, and 3. Figure 1 shows the situation of the natural IgG1 constant region. The isoelectric point value of the variable region increases, and the antigen elimination effect is enhanced. Figure 2 shows that the antibody (F939) that has been endowed with the ability to bind to FcRn under neutral pH conditions increases the isoelectric point value of the variable region and enhances the antigen elimination effect. Figure 3 shows an antibody (F1180) with enhanced FcγR binding ability under neutral pH conditions. The isoelectric point value of the variable region increases and the antigen elimination effect is enhanced.

In all cases, it was shown that by increasing the isoelectric point value of the antibody, the rate of antigen elimination by the pH-dependent binding antibody can be accelerated. It has also been shown that by further increasing the binding ability to FcRn or FcγR under neutral pH conditions, the rate of antigen elimination can be further accelerated compared to when only the pH-dependent binding antibody increases the isoelectric point value (compare Figure 1 and Figures 2 and 3).

(2-3) In vivo infusion analysis method of pH-dependent human IL-6 receptor-binding antibody with adjusted isoelectric point value The following in vivo analysis methods were performed using various pH-dependent human IL-6 receptor-binding antibodies prepared in Example 1: Low_pI-IgG1, High_pI-IgG1, Low_pI-F11 and High_pI-F11.

An infusion pump containing soluble human IL-6 receptor (MINI-OSMOTIC PUMP model 2004; alzet) was transplanted subcutaneously into human FcRn gene transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice, Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) on the back of a model animal to maintain a constant plasma concentration of soluble human IL-6 receptor. Anti-human IL-6 receptor antibodies are administered to model animals, and the in vivo kinetics of the antibodies are evaluated after administration.

Specifically, monoclonal anti-mouse CD4 antibodies obtained according to known methods were injected into the tail vein at a single dose of 20 mg/kg to inhibit neutralization of soluble human IL-6 receptors that may be produced autologously in mice. antibody. An infusion pump containing 92.8 μg/ml soluble human IL-6 receptor was then implanted subcutaneously on the back of the mice. Three days after transplantation of the infusion pump, anti-human IL-6 receptor antibody was administered into the tail vein as a single dose at 1 mg/kg. 15 minutes, 7 hours, 1 day, 2 days, 3 or 4 days, 6 or 7 days, 13 or 14 days, 20 or 21 days, and 27 or 28 days after administration of anti-human IL-6 receptor antibody day, collect blood from mice. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 min to obtain plasma. The separated plasma was stored frozen at -20°C or lower until measurement.

(2-4) Measurement of plasma hsIL-6R concentration using electrochemiluminescence method The concentration of hsIL-6R in mouse plasma was measured by electrochemiluminescence method. hsIL-6R samples were adjusted to concentrations of 250, 125, 62.5, 31.25, 15.61, 7.81 or 3.90 pg/mL for calibration curve preparation, and mouse plasma analysis samples diluted 50 times or more were prepared respectively. The sample was mixed with ruthenium-SULFO-TAG NHS Ester (Meso Scale Discovery)-labeled monoclonal anti-human IL-6R antibody (R&D), biotinylated anti-human IL-6R antibody (R&D), and tocilizumab, and then React overnight at 37°C. Adjust the final concentration of tocilizumab to 333 μg/mL. The reaction solution was then dispensed into Streptavidin Gold Multi-ARRAY plates (Meso Scale Discovery). After reacting at room temperature for another hour, the reaction solution was washed. Read Buffer T(x4) (Meso Scale Discovery) was then immediately allocated to the plate and measurements were performed using SECTOR Imager 2400 (Meso Scale Discovery). hsIL-6R concentration was calculated based on the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

The measured changes in human IL-6 receptor concentration are shown in Figure 4. For both the antibody whose Fc region belongs to native IgG1 (High_pI-IgG1) and the antibody containing a novel Fc region variant (High_pI-F11) whose binding to FcRn is enhanced under neutral pH conditions, compared with In the case of low isoelectric point antibodies (also called "Low_pI") and high isoelectric point antibodies (also called "High_pI"), the plasma soluble human IL-6 receptor concentration is reduced.

Without being bound by a specific theory, the results obtained in these experiments can also be explained as follows: when the Fc region of the administered antibody belongs to a natural IgG antibody, the uptake into cells is thought to be mainly due to non-specific uptake (pinocytosis). happen. Here, the cell membrane is negatively charged, so the higher the isoelectric point value of the administered antibody-antigen complex (that is, the charge of the entire molecule is biased towards positive charge), the easier it is for the complex to approach the cell membrane, and the easier it is for non-specific uptake to occur. When an antibody and an antigen with an increased isoelectric point value form a complex, the overall isoelectric point value of the complex is also increased compared to the complex formed by the original antibody and antigen; therefore, cellular uptake can be increased. Therefore, by increasing the isoelectric point value of an antibody that exhibits pH-dependent antigen binding, the speed or rate of elimination of the antigen from the plasma can be further accelerated, and the antigen concentration in the plasma can be maintained at a lower level.

In these embodiments, the isoelectric point of the antibody is increased by introducing amino acid substitutions that reduce the number of negatively charged amino acids and/or increase the number of positively charged amino acids that may be exposed on the surface of the antibody molecule, in the antibody variable region. One of ordinary skill in the art will appreciate that the effect obtained due to such an isoelectric increase is not primarily (or substantially) dependent on the target antigen type or the amino acid sequence making up the antibody, but is expected to be dependent on, etc. Electric point value. For example, WO2007/114319 and WO2009/041643 describe the following matters in general terms.

The molecular weight of IgG antibodies is large enough, so its main metabolic pathway does not involve renal excretion. IgG antibodies with an Fc are known to be recycled in a salvage pathway via FcRn expressed by cells including endothelial cells of blood vessels, and therefore have a long half-life. IgG antibodies are thought to be metabolized primarily in endothelial cells. Specifically, it is believed that IgG that enters endothelial cells non-specifically is recovered by binding to FcRn, whereas molecules that cannot bind FcRn are metabolized. IgGs with reduced FcRn binding ability have shorter half-lives. On the contrary, the blood half-life can be extended by increasing their binding ability to FcRn. As a result, the aforementioned method of controlling IgG dynamics in blood involves modifying Fc to change the binding ability to FcRn; however, the operating examples of WO2007/114319 (mainly the technology of substituting amino acids in the FR region) are different from those of WO2009/041643 (Mainly a technology that substitutes amino acids in the CDR region) shows that regardless of the type of antigen, by modifying the isoelectric point of the variable region of the antibody, the blood half-life can be controlled without modifying the Fc. The rate of nonspecific uptake of IgG antibodies into endothelial cells is thought to depend on physiochemical Coulombic interactions between the negatively charged cell surface and the IgG antibodies. Therefore, reducing (increasing) the isoelectric point value of IgG antibodies thereby reducing (increasing) the Coulomb interaction is considered to reduce (increase) its non-specific uptake into endothelial cells, thus reducing (increasing) its metabolism in endothelial cells, thereby reducing (increasing) its metabolism in endothelial cells. Ability to control plasma pharmacokinetics. Because the Coulombic interaction between endothelial cells and the negatively charged cell surface is a physiochemical interaction, it is believed that this interaction does not depend primarily on the amino acid sequence of the antibody itself. Therefore, the method for controlling plasma pharmacokinetics provided here can be applied not only to specific antibodies, but also to any polypeptide containing the variable region of an antibody. The decrease (increase) of the Coulomb interaction here refers to the decrease (increase) of the Coulomb force expressed as attraction, and/or the increase (decrease) of the Coulomb force expressed as repulsion.

The amino acid substitution used to achieve the above can be a single amino acid substitution or a combination of multiple amino acid substitutions. In some embodiments, methods are provided to introduce a single amino acid substitution or a combination of multiple amino acid substitutions into positions exposed on the surface of the antibody molecule. Alternatively, the multiple amino acid substitutions introduced may be configured close to each other. The inventors found that, for example, when a positively charged amino acid (preferably arginine or lysine) is used to replace an amino acid that may be exposed on the surface of an antibody molecule, or when a pre-existing positively charged amino acid (preferably arginine or lysine) is used, arginine or lysine), it is advisable to further substitute positively charged amino acids for those amino acids that are conformationally close (in some cases, even one or more amino acids buried within the antibody molecule). of one or more amino acids to obtain a state in which local positive charges are clustered at close positions in the configuration. The definition of "configurationally close position" here is not particularly limited, but may, for example, mean that a single amino acid substitution or the introduction of multiple amino acid substitutions are within 20 angstroms, preferably 15 angstroms, or more preferably 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 the amino acid substitution is at a close position, can be determined by known methods such as X-ray crystallography.

In this way, it should be noted that the isoelectric point value is an indicator representing the overall charge of the molecule, and the charges buried in the antibody molecule and the charges on the surface of the antibody molecule are treated indiscriminately. The inventor also envisages that by broadly and comprehensively Taking into account the charge effect to produce antibody molecules, not only the isoelectric point but also the surface charge and the local clustering of charges on the antibody molecules, can further accelerate the elimination of antigens from plasma, and even maintain the concentration of this antigen in the plasma at lower level.

Receptors such as FcRn or FcγR are expressed on cell membranes, and it is believed that antibodies with increased affinity for FcRn or FcγR under neutral pH conditions are mainly taken up into cells via these Fc receptors. The cell membrane is negatively charged, so when the isoelectric point is high (the overall charge of the molecule shifts to positive), the administered antibody-antigen complex is easier to access the cell membrane, and uptake via Fc receptors is more likely to occur. Therefore, antibodies with increased affinity for FcRn or FcγR and an increased isoelectric point value under neutral pH conditions will also show increased uptake into cells via Fc receptors when forming complexes with antigens. Therefore, for antibodies that bind to antigen in a pH-dependent manner and have increased affinity for FcRn or FcγR at neutral pH, the rate of antigen elimination from plasma can be accelerated by increasing the isoelectric point, and the plasma antigen concentration can maintained at a low level. [Example 3]

Assessment of extracellular matrix binding of pH-dependent binding antibodies with increased isoelectric point values (3-1) Evaluate extracellular matrix binding ability The following experiments were performed to evaluate the impact of conferring pH-dependent antigen-binding properties on antibodies and further modifying the isoelectric point on extracellular matrix binding capabilities.

In a method similar to Example 1, three types of antibodies with different isoelectric points were produced as antibodies showing pH-dependent binding to IL-6 receptors: Low_pI-IgG1, Middle_pI-IgG1 and High_pI-IgG1. Produced respectively according to the method of Reference Example 2: Low_pI(NPH)-IgG1, including H54 (SEQ ID NO:34) and L28 (SEQ ID NO:35); and High_pI(NPH)-IgG1 recorded in WO2009125825, including H( WT) (SEQ ID NO:36) and L(WT) (SEQ ID NO:37) as general antibodies that do not show pH-dependent binding to the IL-6 receptor.

In a manner similar to Example 1, the theoretical isoelectric point values were calculated for these antibodies and are shown in Table 4. Antibodies that did not show pH-dependent binding to the IL-6 receptor were also shown to have increased isoelectric point values, similar to antibodies that showed pH-dependent binding.

[Table 4]

(3-2) The electrochemiluminescence (ECL) method was used to evaluate the binding of antibodies to extracellular matrix. The extracellular matrix (BD Matrigel Basement Membrane Matrix; manufactured by BD) was diluted to 2 mg/mL using TBS (Takara). Distribute the diluted extracellular matrix into a MULTI-ARRAY 96-well plate, high-binding, bare plate (Meso Scale Discovery: manufactured by MSD), 5 μL per well, and fix at 4°C overnight. Then dispense 20mM ACES buffer at pH 7.4 containing 150mM NaCl, 0.05% Tween 20, 0.5% BSA, and 0.01% NaN ₃ into the plate for blocking. The antibody to be evaluated was diluted to 30, 10, and 3 μg/mL using 20 mM ACES buffer (ACES-T buffer) containing 150 mM NaCl, 0.05% Tween 20, and 0.01% NaN ₃ pH 7.4, and then used 150 mM NaCl, 0.01% Tween 20, 0.1% BSA, and 0.01% NaN ₃ were diluted in 20 mM ACES buffer (dilution buffer), pH 7.4, to give final concentrations of 10, 3.3, and 1 μg/mL. Add the diluted antibody solution to the plate from which the blocking solution has been removed and shake at room temperature for 1 hour. The antibody solution was removed, ACES-T buffer containing 0.25% glutaraldehyde was added, and after letting it stand for 10 minutes, the plate was washed with DPBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween 20. The antibody system for ECL detection was prepared using Sulfo-Tag NHS Ester (manufactured by MSD) and sulfo-labeled goat anti-human IgG (gamma) (manufactured by Zymed Laboratories). Dilute the antibody for detection to 1 μg/mL in the dilution buffer, add it to the plate, and then shake in the dark at room temperature for 1 hour. Remove the antibody for detection and add a 2-fold dilution solution prepared by diluting MSD Read Buffer T (4x) (manufactured by MSD) with ultrapure water; then measure the luminescence amount with SECTOR Imager 2400 (manufactured by MSD).

The results are shown in Figure 5. Antibodies that exhibit pH-dependent binding, as well as antibodies that do not, increase binding to the extracellular matrix by increasing their isoelectric point. Furthermore, unexpectedly, in antibodies that bind pH-dependent antigens, increasing the isoelectric point has a significant effect on improving extracellular matrix binding. In other words, antibodies that bind to the antigen in a pH-dependent manner and have a high pI (High_pI-IgG1) were found to have the strongest affinity for the extracellular matrix.

Without being bound to a particular theory, the results obtained from such experiments may also be interpreted as follows. Introduction of histidine modifications into antibody variable regions is known to be a method of conferring pH-dependent antigen-binding properties to antibodies (see, eg, WO2009/125825). Histidine acid has an imidazole group in the side chain and is uncharged at neutral to alkaline pH conditions, but is known to be positively charged at acidic pH conditions. Taking advantage of this property of histidine, by introducing histidine into the antibody variable region, especially the CDR close to the site that interacts with the antigen, the charge environment of the site that interacts with the antigen can be changed between neutral and acidic pH conditions. and configuration environment. Such antibodies can be expected to have antigen affinity that changes in a pH-dependent manner. One of ordinary skill in the art will understand that the effect obtained by such introduction of histamine does not depend primarily (or substantially) on the type of target antigen or the sequence of amino acids making up the antibody, but rather on the histamine Acid introduction site or number of introduced histidine residues.

WO2009/041643 is described in general terms as follows: protein-protein interactions are composed of hydrophobic interactions, electrostatic interactions and hydrogen bonds. The strength of this type of binding can usually be measured using binding constants (affinity) or apparent Binding constant (avidity) expression. pH-dependent binding, the change in binding strength between neutral pH conditions (e.g., pH 7.4) and acidic pH conditions (e.g., pH 5.5 to pH 6.0), is dependent on naturally occurring protein-protein interactions. For example, the binding between the aforementioned IgG molecule and FcRn, which is known to be the salvage receptor of 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. The pKa of the histidine residue is close to 6.0 to 6.5, so the proton dissociation state of the histidine residue changes between neutral and acidic pH conditions. More specifically, the histidine residue is uncharged and neutral under neutral pH conditions and acts as a hydrogen atom acceptor; while under acidic pH conditions it is positively charged and acts as a hydrogen atom donor. Furthermore, as in the above-mentioned IgG molecule-FcRn interaction, histidine residues present on the IgG molecule are reported to be involved in pH-dependent binding (Martin et al., Mol. Cell. 7(4):867-877 (2001) )).

Therefore, replacing the amino acid residues involved in protein-protein interaction with histidine residues or introducing histidine into the interaction site can confer pH dependence on the protein-protein interaction. A similar approach has been used for protein-protein interactions between antibodies and antigens; some have successfully obtained antibody mutations that reduce antigen affinity under acidic pH conditions by introducing histidine into the CDR sequence of anti-protein lysozyme antibodies. body (Ito et al., FEBS Lett. 309(1):85-88 (1992)). In addition, it is reported that an antibody specifically binds to the antigen at the low pH of cancer tissue and weakly binds to the corresponding antigen under neutral pH conditions because histidine is introduced into the CDR sequence (WO2003/105757).

The amino acid residue introduced to increase the isoelectric point value is preferably lysine, arginine or histidine with positively charged side chains. The standard pKa of these amino acid side chains: lysine is 10.5, arginine is 12.5, and histidine is 6.0 (Skoog et al., Trends Anal. Chem. 5(4):82-83 (1986) ). According to the acid-base equilibrium theory known in this technical field, these pKa values mean that in a solution with pH 10.5, 50% of the lysine acid side chains are positively charged and the remaining 50% are uncharged. As the pH of the solution increases, the positively charged proportion of the lysine side chain decreases. In a solution with a pH value of 11.5, which is higher than the pKa value of lysine 1, the positively charged proportion becomes about 9%. On the other hand, as the pH of the solution decreases, the proportion of positive charges increases. In a solution with a pH value of 9.5, which is lower than the pKa value of 1 of lysine, the proportion of positive charges becomes about 91%. The same theory applies to arginine and histamine. More specifically, in a neutral pH solution (eg, pH 7.0), almost 100% of lysine or arginine is positively charged, and about 9% of histidine is positively charged. Therefore, although histidine in neutral pH conditions is positively charged, the degree is lower than that of lysine or arginine. Therefore, lysine and arginine are considered to be more suitable for introduction to increase the isoelectric point. value of amino acids. According to Holash et al. (Proc. Natl. Acad. Sci. 99(17):11393-11398 (2002), although the introduction of modifications that increase the isoelectric point is considered to be effective in increasing extracellular matrix binding, due to the above reasons, Substitution by introducing lysine or arginine is considered more suitable than substitution by introducing histidine.

As previously revealed, by combining the introduction of histidine to confer pH-dependent antigen-binding properties and the modification of the antibody to increase its isoelectric point, a surprising synergistic increase in the affinity of the antibody for the extracellular matrix is possible. In fact, the isoelectric point of High_pI-IgG1 is 9.30 and that of High_pI(NPH)-IgG1 is 9.35, so the isoelectric point value of High_pI-IgG1 is slightly lower. However, the actual affinity of High_pI-IgG1 to the extracellular matrix is significantly higher. The affinity shown for the extracellular matrix cannot necessarily be explained by the isoelectric point level alone, but can be said to show a synergistic effect by the combination of the introduction of modifications that increase the isoelectric point and the histidine modification. This result is a surprising and unanticipated phenomenon.

However, we must note that the pKa of the amino acid side chain of the protein will be greatly affected by the surrounding environment, and will not always meet the above theoretical pKa value. More specifically, the above description is presented here based on general scientific theory; but one can easily speculate that there will be many exceptions for actual proteins. For example, Hayes et al., J. Biol. Chem. 250(18):7461-7472 (1975) recorded that when the pKa of histidine contained in muscle protein was determined experimentally, the value was concentrated at about 6.0, but the report was at 5.37 ~8.05 change. Naturally, histidine acids with high pKa are mostly positively charged at neutral pH conditions. Therefore, the above theory does not negate the effect of increasing the isoelectric point value of introduced histamine and the true three-dimensional structure of this protein. Just like the modification of lysine or arginine, the amino acid modification of histine is likely to have the effect of increasing the isoelectric point. [Example 4]

Utilizing an amino acid substitution in the constant region to increase the isoelectric point Methods have been described for increasing the isoelectric point value of an antibody that binds to antigen in a pH-dependent manner by introducing amino acid substitutions into the antibody variable region. In addition, the method of increasing the isoelectric point value of an antibody can also be carried out by implementing as few as one amino acid substitution in the constant region of the antibody.

The method of adding an amino acid substitution to the constant region of the antibody to increase the isoelectric point is not particularly limited. For example, it can be implemented according to the method described in WO2014/145159. In the case of a variable region, amino acid substitutions introduced into the constant region are preferably designed to reduce the number of negatively charged amino acids (such as aspartic acid or glutamic acid) and to increase the number of positively charged amino acids (such as arginine or lysine).

Without limitation, the position where the amino acid substitution is introduced into the constant region is preferably a position where the amino acid side chain is likely to be exposed on the surface of the antibody molecule. Ideal examples include methods that introduce combinations of multiple amino acid substitutions at positions exposed on the surface of the antibody molecule. Or the positions where the plurality of amino acid substitutions are introduced are preferably such that they are close to each other in configuration. In addition, it is not limited, but the introduced multiple amino acid substitutions are preferably substituted with positively charged amino acids, so that in some cases they cause multiple positive charges to be in a state of close configuration. The "conformationally close position" is not particularly limited, but may, for example, mean that one or more amino acids are substituted and introduced into each other within, for example, 20 Angstroms, preferably within 15 Angstroms, and more preferably within 10 Angstroms. Whether the amino acid substitution site of interest is a position exposed on the surface of the antibody molecule or whether multiple amino acid substitution positions are in close proximity can be evaluated using known methods such as X-ray crystallography.

In addition, methods for conferring multiple positive charges on structurally close positions include, in addition to the above methods, methods using amino acids that are originally positively charged in the 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) arginine at position 121 according to EU numbering ,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 lysine at position 439. By performing substitution with positively charged amino acids at sites that are conformationally close to these positively charged amino acids, it is possible to confer multiple positive charges at positions that are conformationally close to each other.

(4-1) Production of pH-dependent anti-IgE binding antibodies The following three antibody systems were produced according to the method of Reference Example 2 as pH-dependent anti-human IgE antibodies: (1) Ab1, a conventional antibody, including Ab1H (SEQ ID NO:38) as the heavy chain and Ab1L (SEQ ID NO:38) NO:39) as the light chain; (2) Ab2, which is a conventional antibody, including 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 includes Ab3H (SEQ ID NO:42) as a heavy chain and Ab3L (SEQ ID NO:43) as a light chain.

(4-2) Evaluation of pH dependence of human IgE binding The affinity of Ab1 for human IgE at pH 7.4 and pH 5.8 was evaluated in the following manner. Kinetic analysis of human IgE and Abl was performed using BIACORE T100 (GE Healthcare). The measurement system uses the following 2 buffers as running buffers: (1) 1.2 mM CaCl ₂ /0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl ₂ /0.05% tween 20 , 20 mM ACES, 150 mM NaCl, pH 5.8.

An appropriate amount of Protein A/G (ACTIGEN) was immobilized on Sensor chip CM4 (GE Healthcare) using the amine coupling method to capture the antibody of interest. Human IgE and the antibodies captured on the sensor chip are then interacted by injecting a dilute IgE solution with running buffer (as a control solution). For the running buffer, use one of the above buffers (1) and (2), and dilute human IgE using each buffer. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 25°C. The KD (M) of human IgE for each antibody was calculated based on the kinetic parameters calculated from the sensorgram obtained by measurement, the association rate constant ka (1/Ms) and the dissociation rate constant kd (1/s). BIACORE T100 Evaluation Software (GE Healthcare) was used to calculate each parameter.

The affinity of Ab2 and Ab3 for human IgE at pH 7.4 and pH 5.8 was evaluated as follows. The binding activity of anti-hlgE antibody to hlgE (dissociation constant KD (M)) was performed using BIACORE T200 (GE Healthcare). The measurement system uses the following 2 buffers as running buffers: (1) 1.2 mM CaCl ₂ /0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl ₂ /0.05% tween 20 , 20 mM ACES, 150 mM NaCl, pH 5.8.

The addition of biotin to a peptide derived from the chemically synthesized human glypican 3 (also known as GPC3) protein (with the amino acid sequence (VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO: 44))) ("biotinylated GPC3 peptide" will be utilized ) was added to the Sensor chip SA (GE Healthcare), and fixed on the chip using the affinity between streptavidin and biotin. Inject the appropriate concentration hIgE, and fix it on the chip by capturing the biotinylated GPC3 peptide. Inject an appropriate concentration of anti-hlgE antibody as the analyte, and allow it to interact with hIgE on the sensor chip. Then regenerate the sensor chip and inject 10 mM glycine-HCl at pH 1.5. All measurements were performed at 37°C. The association rate constant ka (1/Ms) and the dissociation rate constant kd (1/s) were determined by using BIACORE T200 Evaluation Software (GE Healthcare) Curve fitting was performed to analyze the measurement results for calculation, and the dissociation constant KD (M) was calculated based on the above values.

The results are presented in Table 5. All antibodies, Ab1, Ab2 and Ab3, showed pH-dependent binding to human IgE, and their affinity under acidic pH conditions (pH 5.8) was significantly higher than their affinity under neutral pH conditions (pH 7.4). weak. Therefore, administration of these antibodies to living animals is expected to accelerate the elimination of human IgE as the antigen.

[table 5]

The theoretical isoelectric point values of Ab1-Ab3 calculated in a similar manner to Example 1 are shown in Table 6.

[Table 6]

(4-3) Production of antibodies with increased isoelectric point through single amino acid modification in the constant region Abl prepared in Example (4-1) is an antibody having natural human IgG1 as a constant region. Ab1H-P600 is modified by modifying the Fc region of Ab1H of the heavy chain of Ab1 by replacing proline at position 238 according to EU numbering with aspartic acid and serine at position 298 according to EU numbering. The acid was substituted with alanine to prepare. In addition, various Fc variant systems were produced according to the method of Reference Example 2, by introducing various single amino acids indicated in Tables 7-1 and 7-2 respectively into the Fc region of Ab1H-P600. For all Fc variants, AbIL (SEQ ID NO:39) was used as light chain. The affinity of these antibodies for hFcγRII2b was comparable to the P600 variant (data not shown).

[Table 7-1]

[Table 7-2]

(4-4) Human FcγRIIb binding assay using BIACORE using a novel antibody containing Fc region variant, between soluble human FcγRIIb (also known as "hFcγRIIb") and antigen-antibody complexes containing Fc region variant The combined analysis method was performed using BIACORE (registered trademark) T200 (GE Healthcare). A His-tagged molecular version of soluble hFcγRIIb is produced using methods known in the art. An appropriate amount of anti-His antibody was immobilized on Sensor chip CM5 (GE Healthcare) using the amine coupling method using His capture kit (GE Healthcare) to capture hFcγRIIb. The antibody-antigen complex is then injected with running buffer (as a reference solution), allowing it to interact with hFcγRIIb captured on the sensor chip. Use 20 mM N-(2-acetamide)-2-aminoethanesulfonic acid, pH 7.4, 150 mM NaCl, 1.2 mM CaCl ₂ , and 0.05% (w/v) Tween 20 as the running buffer, and use respectively buffer to dilute soluble hFcγRIIb. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 25°C. The analysis is based on calculated binding (RU) from the measured sensorgrams and shows the relative value when the binding amount of P600 is defined as 1.00. To calculate parameters, BIACORE (registered trademark) T100 Evaluation Software (GE Healthcare) was used. The results are shown in Tables 7-1 and 7-2 (see the "BIACORE" column of the table) and Figure 6. Several Fc variants show increased affinity for hFcγRIIb immobilized on BIACORE (registered trademark) sensor chips.

While not bound to a particular theory, this result can be explained as follows. It is known that the BIACORE (registered trademark) sensor chip is negatively charged, so the charged state can be considered to be similar to the surface of a cell membrane. More specifically, the binding of the antigen-antibody complex to hFcγRIIb immobilized on the negatively charged BIACORE sensor chip is presumed to be similar to the binding of the antigen-antibody complex to hFcγRIIb present on the negatively charged cell membrane surface.

In an antibody prepared by introducing a modification that increases the isoelectric point into the Fc region, the Fc region (constant region) is more positively charged than an antibody before the modification is introduced. Therefore, it can be considered that the Coulomb interaction between the Fc region (positive electricity) and the surface of the sensing chip (negative electricity) is strengthened by the modification by increasing the isoelectric point. It is also expected that this effect will also occur on the surface of negatively charged cell membranes; therefore, it is also expected to show the effect of accelerating the speed or rate of uptake into cells in vivo.

From the above results, it is believed that compared to hFcγRIIb bound to Ab1H-P600, the ratio of hFcγRIIb bound to the variant is about 1.2 times or more, indicating that the antibody has a strong charge effect on the binding of hFcγRIIb on the sensor chip. Therefore, modifications expected to produce a charging effect include, for example, EU numbering 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 position modification. Preferred modifications are at positions 282, 309, 311, 315, 345, 356, 359, 361, 362, 385, 386, 387, 389, 399, 418, 419 or 443. The amino acid substitution introduced at such a position is preferably arginine or lysine. Other amino acid mutations for which such a charge effect can be expected include, for example, glutamic acid at position 430 according to EU numbering. The amino acid introduced at position 430 is preferably substituted with positively charged arginine or lysine, or preferably with glycine or threonine in the uncharged residue.

(4-5) Uptake of antibodies containing Fc region variants by hFcγRIIb-expressing cells To assess the rate of intracellular uptake into hFcyRIIb-expressing cell lines using the novel antibodies produced containing Fc region variants, the following analysis was performed.

An MDCK (Madin-Darby canine kidney) cell line constitutively expressing hFcγRIIb was produced using a known method. Using these cells, intracellular uptake of antigen-antibody complexes was assessed. Specifically, pHrodoRed (Life Technologies) was used to calibrate human IgE (antigen) according to established experimental procedures, and the antigen-antibody complex was formed in a culture medium with an antibody concentration of 10.8 mg/mL and an antigen concentration of 12.5 mg/mL. . The culture medium containing the antigen-antibody complex was added to the culture plate of the above-mentioned MDCK cells that inherently express hFcγRIIb, incubated for 1 hour, and then the fluorescence intensity of the antigen taken into the cells was quantified using InCell Analyzer 6000 (GE Healthcare). The amount of antigen ingested is expressed relative to the P600 value, which is defined as 1.00.

The results are shown in Tables 7-1 and 7-2 (see the "Imaging" column of the table) and Figure 7. Strong fluorescence from this antigen in cells was observed with some Fc variants.

While not being bound by a particular theory, this result can be explained as follows: Antigens and antibodies added to the cell culture medium form antigen-antibody complexes in the culture medium. Through the antibody Fc region, this antigen-antibody complex binds to hFcγRIIb expressed on the cell membrane and is taken up into cells in a receptor-dependent manner. The Ab1 used in this experiment is an antibody that binds to the antigen in a pH-dependent manner; therefore, the antibody can be dissociated from the antigen. Because the dissociated antigen is labeled with pHrodoRed as described earlier, it fluoresces in endosomes. Therefore, if the fluorescence intensity in the cells is stronger than that in the control group, it is considered to mean that the uptake of the antigen-antibody complex into the cells occurs faster or more frequently.

Here, if the fluorescence intensity of the antigen entering cells of this variant is about 1.05 times or more compared to the fluorescence intensity of Ab1H-P600, it is considered that there is a charge effect on the antigen entering the cells. Compared with the fluorescence intensity of Ab1H-P600, if the fluorescence intensity of the antigen entering the cells of this variant is about 1.5 times or more, it is considered that there is a strong charge effect on the antigen entering the cells. Therefore, the above results show that by introducing a modification that increases the isoelectric point at an appropriate position in the Fc region, uptake into cells can be accelerated compared to before the modification. Amino acid modification positions showing such effects are, for example, 253, 254, 256, 258, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 358, 384, 385, 387, 399, 400, 421, 433 or 434 bits. Ideally, 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 such a position is preferably arginine or lysine. It is not limited. In order to increase the isoelectric point of the antibody, the amino acid substitution position introduced into the constant region is, for example, the amino acid residue at position 285 according to EU numbering. Or other amino acid substitutions may include amino acid substitution of the amino acid residue at position 399 according to EU numbering. [Example 5]

Fc variants produced in acidic pH conditions with enhanced FcRn binding to improve retention in plasma Under acidic pH conditions in endosomes, IgG antibodies that have entered cells are known to return to the plasma by binding to FcRn. Therefore, IgG antibodies generally have a longer plasma half-life than proteins that are not bound to FcRn. Taking advantage of this property, methods are known to enhance antibody plasma retention by introducing amino acid modifications into the Fc region of the antibody to increase FcRn affinity under acidic pH conditions. Specifically, methods for improving the plasma retention of antibodies by increasing FcRn affinity under acidic pH conditions through amino acid modification are known, such as M252Y/S254T/T256E (YTE) modification (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)), M428L/N434S (LS) modification (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)) and N434H modification (Zheng et al., Clinical Pharmacology & Therapeutics 89(2):283-290 (2011)).

On the other hand, as mentioned above, Fc variants with increased FcRn affinity under acidic pH conditions are known to display undesirable affinity for rheumatoid factor (RF) (WO2013/046704). Therefore, the following assay was performed to create Fc variants that have reduced or substantially no binding to rheumatoid factor and may improve plasma retention.

(5-1) Production of novel antibodies containing Fc region variants Fc variants with increased FcRn affinity under acidic pH conditions include, for example, known modifications, YTE, LS or N434H, and some novel Fc variants (F1847m, F1848m, F1886m, F1889m, F1927m, with F1168m).

According to the method of Reference Example 1, a sequence encoding a heavy chain in which amino acid modification was introduced into the Fc region of the heavy chain of Fv4-IgG1 (VH3-IgG1m), which is an anti-human IL-6 receptor, was produced. antibody. The following antibodies were produced using these heavy chains according to the method of Reference Example 2: (a) Fv4-IgG1, including VH3-IgG1m (SEQ ID NO: 46) as the heavy chain and VL3-CK as the light chain; (b) Fv4- YTE, including VH3-YTE (SEQ ID NO:47) as the heavy chain and VL3-CK as the light chain; (c) Fv4-LS, including VH3-LS (SEQ ID NO:48) as the heavy chain and VL3-CK as the light chain Light chain; (d) Fv4-N434H, including VH3-N434H (SEQ ID NO:49) as heavy chain and VL3-CK as light chain; (e) Fv4-F1847m, including VH3-F1847m (SEQ ID NO:50) as heavy chain and VL3-CK as light chain; (f) Fv4-F1848m, including VH3-F1848m (SEQ ID NO:51) as heavy chain and VL3-CK as light chain; (g) Fv4-F1886m, including VH3- F1886m (SEQ ID NO:52) as the heavy chain and VL3-CK as the light chain; (h) Fv4-F1889m, including VH3-F1889m (SEQ ID NO:53) as the heavy chain and VL3-CK as the light chain; (i ) Fv4-F1927m, including VH3-F1927m (SEQ ID NO:54) as the heavy chain and VL3-CK as the light chain; and (j) Fv4-F1168m, including VH3-F1168m (SEQ ID NO:55) as the heavy chain and VL3-CK serves as the light chain.

(5-2) Kinetic analysis of human FcRn binding An antibody containing VH3-IgG1m or the above variant as the heavy chain and L(WT) (SEQ ID NO: 37) as the light chain was produced according to the method of Reference Example 2, and the binding activity to human FcRn was evaluated in the following manner.

Kinetic analysis of human FcRn with each antibody was performed using BIACORE T100 (GE Healthcare). An appropriate amount of Protein L (ACTIGEN) was immobilized on Sensor chip CM4 (GE Healthcare) using the amine coupling method to capture the antibody of interest. Human FcRn and the antibodies captured on the sensor chip are then allowed to interact by injecting a dilute FcRn solution with running buffer (as a reference solution). For the running buffer, use 50 mM sodium phosphate, 150 mM NaCl, and 0.05% (w/v) Tween 20, pH 6.0. Each buffer is also used to dilute the FcRn. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 25°C. The KD (M) of human FcRn for each antibody was calculated based on the kinetic parameters calculated from the sensorgram obtained by measurement, the association rate constant ka (1/Ms) and the dissociation rate constant kd (1/s). Each parameter was calculated using BIACORE T100 Evaluation Software (GE Healthcare).

The results are shown in Table 8.

[Table 8] [Example 6]

Assessment of the affinity of antibodies containing Fc region variants for rheumatoid factor with enhanced FcRn binding under acidic pH conditions Antidrug antibodies (ADAs) affect the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes cause serious side effects; therefore, the clinical utilization and efficacy of therapeutic antibodies may be limited by the production of ADAs. There are many factors that affect the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is one of them. In addition, ADA present in the patient before the therapeutic antibody is administered (also known as "pre-existing ADA") may present similar problems. Specifically, in therapeutic antibodies for patients with autoimmune diseases, such as rheumatoid arthritis (RA), rheumatoid factor (RF), an autoantibody against human IgG, may cause " There are pre-existing ADA" issues. Recently, humanized anti-CD4 IgG1 antibodies with the N434H (Asn434His) mutation were reported to induce significant rheumatoid factor binding (Zheng et al., Clinical Pharmacology & Therapeutics 89(2):283-290 (2011)). Detailed studies have confirmed that the N434H mutation in human IgG1 increases the binding of rheumatoid factors in the Fc region to antibodies compared to the parent human IgG1.

Rheumatoid factor is a multi-strain autoantibody against human IgG. Its epitope in human IgG varies depending on the clone, and seems to be located at the CH2/CH3 junction. The CH3 domain may bind to FcRn for epitope. base overlap. Therefore, mutations that increase binding activity (binding affinity) to FcRn may increase binding activity (binding affinity) to specific selectogenes of rheumatoid factor.

In fact, regarding Fc that has increased affinity for FcRn at acidic pH or neutral pH, not only N434H modification, but also many other amino acid modifications are known to also increase the binding of the Fc to rheumatoid factors (WO2013 /046704).

On the other hand, WO2013/046704 reveals some amino acid modifications that selectively inhibit the affinity to rheumatoid factors without affecting the affinity to FcRn. Among them, a combination of two amino acid mutations is pointed out, Namely Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D. Therefore, it is disclosed here for the first time that the introduction of Q438R/S440E has a new increased affinity under acidic pH conditions to examine whether the binding to rheumatoid factors will be reduced.

(6-1) Rheumatoid factor binding assay for antibodies containing Fc region variants Binding assays to rheumatoid factor were performed using electrochemiluminescence (ECL) at pH 7.4 using individual sera from 30 RA patients (Proteogenex). Mix 50-fold diluted serum samples, biotinylated test antibodies (1 μg/mL), and SULFO-TAG NHS Ester (Meso Scale Discovery)-labeled test antibodies (1 μg/mL), and incubate at room temperature. Breed hours. Afterwards, this mixture was added to a streptavidin-coated MULTI-ARRAY 96-well plate (Meso Scale Discovery), and the plate was incubated at room temperature for 2 hours and then washed. Immediately after adding Read Buffer T (x4) (Meso Scale Discovery) to each well, the plate was placed on a SECTOR imager 2400 Reader (Meso Scale Discovery), and chemiluminescence was measured.

The results of the analysis are shown in Figures 8 to 17. Fv4-IgG1 (Figure 8) with native human IgG1 showed only weak binding to rheumatoid factor, whereas existing Fc variants with increased FcRn binding, namely Fv4-YTE (Figure 9), Fv4 -LS (Figure 10), and Fv4-N434H (Figure 11) both showed significant increases in rheumatoid factor binding in some donors. On the other hand, all novel Fc region variants with increased FcRn binding, namely Fv4-F1847m (Figure 12), Fv4-F1848m (Figure 13), Fv4-F1886m (Figure 14), Fv4-F1889m (Figure 15 Figure), Fv4-F1927m (Figure 16), and Fv4-F1168m (Figure 17), show only weak rheumatoid factor binding, indicating that rheumatoid factor binding is significantly inhibited due to modification to increase FcRn binding. .

Figure 18 shows the average rheumatoid factor binding affinity in the sera of 30 RA patients for each variant. The six new variants all showed lower affinity than the three pre-existing variants (YTE, LS, and N434H), and they also showed lower affinity for rheumatoid factor than native human IgG1. As such, the Fc variants disclosed for the first time here may inhibit the concerns of pre-existing Fc variants when considering clinical development of therapeutic antibodies with improved affinity for FcRn for autoimmune diseases such as rheumatoid arthritis. risks associated with rheumatoid factor, so it can be used more safely than currently known Fc variants. [Example 7]

PK assessment of Fc variants with increased FcRn binding at acidic pH conditions in cynomolgus monkeys In Example 7, the effectiveness of improving plasma retention in Malay monkeys was evaluated using novel Fc region variant-containing antibodies provided herein that were confirmed to inhibit binding to rheumatoid factor.

(7-1) Production of novel antibodies containing Fc region variants The following anti-human IgE antibodies were produced: (a) OHB-IgG1, including OHBH-IgG1 (SEQ ID NO:56) as the heavy chain and OHBL-CK (SEQ ID NO:57) as the light chain; (b) OHB-LS, Including OHBH-LS (SEQ ID NO:58) as the heavy chain and OHBL-CK as the light chain; (c) OHB-N434A, including OHBH-N434A (SEQ ID NO:59) as the heavy chain and OHBL-CK as the light chain ; (d) OHB-F1847m, including OHBH-F1847m (SEQ ID NO:60) as the heavy chain and OHBL-CK as the light chain; (e) OHB-F1848m, including OHBH-F1848m (SEQ ID NO:61) as the heavy chain; chain and OHBL-CK as the light chain; (f) OHB-F1886m, including OHBH-F1886m (SEQ ID NO:62) as the heavy chain and OHBL-CK as the light chain; (g) OHB-F1889m, including OHBH-F1889m ( 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 analysis of novel antibodies containing Fc region variants To evaluate the in vivo kinetics of anti-human IgE antibodies in plasma following administration of anti-human IgE antibodies to Malay monkeys. The anti-human IgE antibody solution was administered intravenously once at 2 mg/kg. 5 minutes, (2 hours), 7 hours, 1 day, 2 days, 3 days, (4 days), 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and Blood was collected on day 56. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 5 min to obtain plasma. The separated plasma was stored frozen at -60°C or lower until measurement. 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-F1927m.

(7-3) Measurement of anti-human IgE antibody concentration in plasma using ELISA The concentration of anti-human IgE antibodies in the plasma of Malay monkeys was measured using ELISA. First, the anti-human IgG kappa chain antibody (antibody solution) was distributed in Nunc-ImmunoPlate, MaxiSorp (Nalge Nunc International), and allowed to stand at 4°C overnight to produce an anti-human IgG kappa chain antibody immobilized plate. Prepare calibration curve samples with plasma concentrations of 640, 320, 160, 80, 40, 20 or 10 ng/mL and measurement samples of Malay monkey plasma diluted 100 times or more. These calibration curve samples and plasma measurement samples were prepared so that Malay monkey IgE (a product prepared in-house) was added at a concentration of 1 μg/mL. Then, this sample was distributed to an anti-human IgG kappa chain antibody-immobilized plate and left to stand at room temperature for 2 hours. Then HRP-anti-human IgG gamma chain antibody (Southern Biotech) was dispensed and left to stand at room temperature for 1 hour. Then, TMB Chromogen Solution (Life Technologies) was used as the matrix to perform a color-forming reaction. After adding 1N sulfuric acid (Wako) to stop the reaction, the absorbance at 450 nm was measured with a microplate reader. The concentration of anti-human IgE antibodies in monkey plasma was calculated from the calibration curve using the analysis software SOFTmax PRO (Molecular Devices). The changes in anti-human IgE antibody concentration measured in monkey plasma are shown in Figure 19. Changes in anti-human IgE antibody concentrations were measured in monkey plasma, and elimination clearance was calculated using momentum analysis using Phoenix WinNonlin Ver. 6.2 (Pharsight Corporation). The calculated pharmacokinetic parameters are shown in Table 9. When calculating the change in anti-human IgE antibody concentration and the clearance rate in monkey plasma, samples from individuals who showed a positive reaction for antibodies in plasma against the administered sample were excluded.

[Table 9]

(7-4) Measurement of antibodies in plasma against administered samples using electrochemiluminescence method Antibodies against administered samples were measured in monkey plasma using electrochemiluminescence method. The administration sample for ruthenium calibration using SULFO-TAG NHS Ester (Meso Scale Discovery), the administration sample for biotinylation using EZ-Link Micro Sulfo-NHS-Biotinylation Kit (Pierce), and Malay were mixed in equal amounts. Monkey plasma measurement samples were left to stand at 4°C overnight. The samples were added to a MULTI-ARRAY 96-well Streptavidin Gold plate (Meso Scale Discovery), reacted at room temperature for 2 hours, and washed. Read Buffer T(x4) (Meso Scale Discovery) was then immediately allocated to the plate and measurements were performed using SECTOR Imager 2400 (Meso Scale Discovery).

Results Compared to the Fc region of native IgG1, all novel Fc variants were confirmed to show significantly improved plasma retention.

(7-5) Mouse PK analysis of Fc variants The following experiment was conducted to compare the Fc variant F1718 described in WO2013/046704 with the newly discovered Fc variant F1848m, as an Fc variant with increased FcRn binding at acidic pH.

A heavy chain sequence encoding the Fc region of the heavy chain (VH3-IgG1) of Fv4-IgG1 (anti-human IL-6 receptor antibody) introduced by amino acid modification was produced according to the method of Reference Example 1. Using these heavy chains, the following antibodies were produced according to the method of Reference Example 2: (a) Fv4-IgG1, including VH3-IgG1 as the heavy chain and VL3-CK as the light chain; and (b) Fv4-F1718, including VH3- F1718 (SEQ ID NO:65) as the heavy chain and VL3-CK as the light chain.

The tail vein of human FcRn gene transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice; Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010) was treated with 1 mg/kg The above anti-human IL-6 receptor antibody was administered once. 15 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days after administration of the anti-human IL-6 receptor antibody, and Blood was collected on day 28. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to obtain plasma. The separated plasma was frozen and stored at -20°C or lower until collection.

(7-6) The concentration of anti-human IL-6 receptor antibodies in plasma was measured by ELISA. The concentration of anti-human IL-6 receptor antibodies in mouse plasma was measured by ELISA. First, the anti-human IgG (gamma chain specificity) F(ab') ₂ fragment of the antibody (SIGMA) was allocated to Nunc-ImmunoPlate, MaxiSorp (Nalge nunc International), and left to stand at 4°C overnight to prepare an anti-human IgG immobilized plate. . Prepare calibration curve samples containing anti-human IL-6 receptor antibody plasma concentrations of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, or 0.0125 μg/mL, and mouse plasma measurement samples diluted 100-fold or more. Add 200 μL of 20 ng/mL soluble human IL-6 receptor to 100 μL of calibration curve sample or plasma measurement sample, and then let the mixed solution stand at room temperature for 1 hour. The mixed solution was then distributed into each well of the anti-human IgG immobilized plate, and the plate was allowed to stand at room temperature for 1 hour. Then, biotinylated anti-human IL-6R antibody (R&D) was added and reacted at room temperature for 1 hour. Then, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added, reacted at room temperature for 1 hour, and TMB One Component HRP Microwell Substrate (BioFX Laboratories) was used as the matrix to perform a color-forming reaction of the reaction solution. After stopping the reaction by adding 1 N sulfuric acid (Showa Chemical), the absorbance of the reaction solution in each well at 450 nm was measured on a microplate reader. The antibody concentration in mouse plasma was calculated from the absorbance value of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

The results are shown in Figure 20. F1718, an Fc variant described in WO2013/046704 that increases FcRn binding at acidic pH, did not show any effect in prolonging antibody PK, but showed the same plasma retention as natural IgG1.

F1718 has four mutations, namely N434Y/Y436V/Q438R/S440E introduced in the Fc region. In comparison, F1848m, first revealed here, also introduced four mutations, namely N434A/Y436V/Q438R/S440E. The only difference in the amino acid mutations introduced by these two types of Fc is that in F1718, the amino acid mutation introduced at position 434 according to the EU numbering method becomes Y (tyrosine), and in F1848m it is A (alanine). In Example (7-2), compared to native IgG1, F1848m showed improved plasma retention, while F1718 did not show any improvement in plasma retention. Therefore, without limitation, this suggests that it is ideal to introduce A (alanine) at position 434 as an amino acid mutation to improve plasma retention.

(7-7) Predicted immunogenicity scores of Fc variants The production of anti-drug antibodies (ADA) will affect the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes cause serious side effects; therefore, the production of ADA may limit the clinical utilization and drug efficacy of therapeutic antibodies. The immunogenicity of therapeutic antibodies is known to be affected by many factors. In particular, the importance of effector T cell epitopes carried by therapeutic antibodies has been reported many times.

In silico tools for predicting T cell epitopes have been developed, such as Epibase (Lonza), iTope/TCED (Antitope) and EpiMatrix (EpiVax). Using these computer simulation 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 Assessing the potential immunogenicity of therapeutic antibodies.

EpiMatrix was used to calculate the immunogenicity score of the Fc variants to be evaluated. EpiMatrix is a system used to predict the immunogenicity of a protein of interest. It cuts the amino acid sequence of the protein whose immunogenicity is to be predicted into 9 amino acids to automatically design the sequence of the peptide fragment, and then calculates them. Binding ability to eight major MHC class II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501) (Clin. Immunol . 131(2): 189-201 (2009)).

F1718 and F1756 (N434Y/Y436T/Q438R/S440E) are described in WO2013/046704 and contain the N434Y mutation. In contrast, the newly revealed F1848m and F1847m contain the N434A mutation.

The immunogenicity scores of these four variants, namely F1718, F1848m, F1756 and F1847m, calculated as above, are shown in the "EpiMatrix Score" column of Table 31. In addition, regarding the EpiMatrix score, the immunogenicity score corrected for the Tregitope content is displayed in the "tReg Adjusted Epx score" column. Tregitope is a peptide fragment sequence that mainly exists in naturally occurring antibody sequences and is considered to be a sequence that inhibits immunogenicity by activating regulatory T cells (Treg).

[Table 31]

According to these results, both "EpiMatrix score" and "tReg Adjusted Epx score" showed that the immunogenicity scores of N434A variants F1848m and F1847m were reduced compared to the N434Y variant. This indicates that it is ideal to introduce A (alanine) as an amino acid mutation at position 434 to obtain a lower immunogenicity score. [Example 8]

Production of humanized anti-human IL-8 antibodies (8-1) Production of humanized anti-human IL-8 antibody hWS-4 U.S. Patent No. 6,245,894 discloses humanized anti-IL-8 antibodies that bind to human IL-8 (hIL-8) and block its physiological functions. Modified humanized anti-IL-8 antibodies can be made by combining the heavy and light chain variable region sequences disclosed in US Pat. No. 6,245,894 with virtually any of the various known human antibody constant region sequences. Therefore, the human antibody constant region sequence of these modified antibodies is not particularly limited. The natural human IgG1 sequence or the natural human IgG4 sequence can be used as the heavy chain constant region, and the natural human Kappa sequence can be used as the light chain constant region sequence.

From among the humanized IL-8 antibodies disclosed in U.S. Patent No. 6,245,894, the coding sequence of hWS4H-IgG1 (SEQ ID NO:83), which combines the heavy chain variable region RVHg with the natural human anti-IgG1 sequence for the heavy chain The constant region was produced according to the method of Reference Example 1. The coding sequence of hWS4L-kOMT (SEQ ID NO:84), which combines the light chain variable region RVLa and the natural human Kappa sequence for the light chain constant region, was produced according to the method of Reference Example 1. An antibody combining the above heavy chain and light chain was produced and named humanized WS-4 antibody (hereinafter referred to as hWS-4).

(8-2) Production of humanized anti-human IL-8 antibody Hr9 New humanized antibodies were made using human consensus framework sequences that differed from the FR used in hWS-4.

Specifically, the mixed sequence of VH3-23 and VH3-64 was used as heavy chain FR1, the sequence found in VH3-15 and VH3-49 was used as FR2, and the sequence found in VH3-72 was used as FR3 (however, the sequences according to Kabat numbering were excluded. 82a), using the sequence found in JH1 as FR4. These sequences were linked to the CDR sequences of hWS-4 heavy chain to create Hr9-IgG1 (SEQ ID NO:85), a novel humanized antibody heavy chain.

Then 2 types of antibodies were made, namely: hWS-4, which has hWS4H-IgG1 as heavy chain and hWS4L-k0MT as light chain; and Hr9, which has Hr9-IgG1 as heavy chain and hWS4L-k0MT as light chain. In the context of disclosure C, when specifying the specific light chain, Hr9 is written as Hr9/hWS4L. This antibody was expressed using FreeStyle 293F cells (Invitrogen) according to the product protocol. The antibody was purified from the culture supernatant according to the method of Reference Example 2. The results were obtained at the antibody amounts shown in Table 11. Unexpectedly, the performance level of Hr9 was approximately 8 times that of hWS-4.

[Table 11]

(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 measured using BIACORE T200 (GE Healthcare) in the following manner.

Use a running buffer consisting of: 0.05% tween 20, 20 mM ACES, and 150 mM NaCl (pH 7.4). An appropriate amount of Protein A/G (PIERCE) was immobilized on Sensor chip CM4 (GE Healthcare) using the amine coupling method and the antibody of interest was captured. Human IL-8 and the antibodies captured on the sensor chip are then interacted by injecting a diluted human IL-8 solution with running buffer (as a reference solution). For running buffer, use the composition described above and also use this buffer to dilute the human IL-8. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 37°C. The KD (M) of human IL-8 for each antibody was calculated based on the kinetic parameters calculated from the sensorgram obtained by measurement, the binding rate constant kon (1/Ms) and the dissociation rate constant koff (1/s). Each parameter was calculated using BIACORE T200 Evaluation Software (GE Healthcare).

The results are shown in Table 12. It was confirmed that hWS-4 and Hr9 have equal binding affinity to human IL-8.

[Table 12]

In order to develop antibody medicines, the production level of antibody molecules is an important factor, and a high production level is generally desired. Of particular note is the selection of more appropriate human common framework derived sequences from the above tests to combine with the HVR sequence of hWS-4 and to produce Hr9 with improved production levels and maintained binding affinity for human IL-8. [Example 9]

Generation of antibodies with pH-dependent affinity for IL-8 (9-1) Production of Hr9-modified antibodies to impart pH dependence The purpose of the research is to impart pH-dependent IL-8 affinity to the Hr9 antibody obtained in Example 8.

Although not limited to a specific theory, antibodies with pH-dependent affinity for IL-8 may exhibit the following behavior in vivo. This antibody, when administered to a living organism, will bind strongly to IL-8 and block its function in an environment maintained at neutral pH (such as in plasma). Some of these IL-8/antibody complexes are taken up into cells through non-specific interactions with the cell membrane (pinocytosis) (hereinafter referred to as non-specific uptake). Under acidic pH conditions in endosomes, the binding affinity of the above-mentioned antibody to IL-8 becomes weak, so the antibody dissociates from IL-8. Antibodies dissociated from IL-8 can then return to the outside of the cell via FcRn. The above-mentioned antibodies returned to the outside of the cells (into the plasma) in this way can rebind to other IL-8 and block its function. Antibodies with pH-dependent affinity for IL-8 are thought to be able to bind to IL-8 multiple times via the mechanism described above.

On the contrary, when the antibody does not have the properties mentioned above, the antibody molecule can only neutralize the antigen once, but cannot neutralize the antigen multiple times. Generally, because IgG antibodies have two Fabs, a single antibody molecule can neutralize two molecules of IL-8. On the other hand, antibodies that can bind to IL-8 multiple times can bind to IL-8 any number of times as long as they remain in the body. For example, a single molecule of a pH-dependent IL-8-binding antibody, when taken up into cells 10 times after administration, can neutralize up to 20 molecules of IL-8 until it is eliminated. Therefore, antibodies that can bind IL-8 multiple times have the advantage of being able to neutralize several IL-8 molecules even in small amounts. From another point of view, antibodies that can bind IL-8 multiple times have the advantage of being able to maintain a state that can neutralize IL-8 for a longer period of time than when the same amount of antibodies without this property is administered. From another point of view, 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 the same amount of antibodies without this property when administered.

In order to achieve these advantages, amino acid modifications, mainly histidine, were introduced into the variable regions of Hr9-IgG1 and WS4L-k0MT to produce antibodies that can bind to IL-8 multiple times. Specifically, the variants shown in Table 13 were produced according to the method of Reference Examples 1 and 2.

Labels such as "Y97H" in Table 13 show the mutation introduction position according to Kabat numbering, the amino acid before mutation and the amino acid after mutation. Specifically, when labeled "Y97H", it indicates that the amino acid residue at position 97 of the Kabat numbering system is substituted from Y (tyrosine) to H (histidine). When a plurality of amino acids are substituted and introduced in combination, it is described as "N50H/L54H", for example.

[Table 13]

(9-2) pH-dependent IL-8 affinity The human IL-8 binding affinity of the antibody produced in Example 9-1 was measured using BIACORE T200 (GE Healthcare) according to the following method. Use the following 2 running buffers: (1) 0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

An appropriate amount of Protein A/G (PIERCE) was immobilized on Sensor chip CM4 (GE Healthcare) by amine coupling method and the antibody of interest was captured. Human IL-8 and the antibodies captured on the sensor chip are then interacted by injecting a diluted human IL-8 solution with running buffer (as a reference solution). For the running buffer, use any of the above solutions, and dilute human IL-8 using each buffer. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 37°C. The KD (M) of human IL-8 for each antibody was calculated based on the kinetic parameters calculated from the sensorgram obtained by measurement, the binding rate constant kon (1/Ms) and the dissociation rate constant koff (1/s). Each parameter was calculated using BIACORE T200 Evaluation Software (GE Healthcare).

The results are shown in Table 14-1. First, compared with Hr9, Hr9/L16 with L54H modification in the light chain slightly increases the binding affinity of human IL-8 at neutral pH (pH 7.4), but decreases the binding affinity of human IL-8 at acidic pH (pH 5.8). . on the other hand. Anti-IL-8 antibodies (H89/WS4L, H89/L12 and H89/L16) prepared by combining various light chains and H89 containing Y97H modification on the heavy chain all showed reduced binding affinity to human IL-8 at acidic pH and reduced human IL-8 binding affinity at neutral pH.

[Table 14-1]

(9-3) Preparation and evaluation of antibodies modified to impart pH dependence The promising modifications found in 9-2 were evaluated in combination with new amino acid mutations, and the following combinations were found.

[Table 14-2]

This variant was produced according to the methods of Reference Examples 1 and 2, and the binding affinity to human IL-8 was evaluated using a method similar to that of Example 9-2.

The results are also shown in Table 14. The binding affinity of H89/L63 with H89-IgG1 (SEQ ID NO:86) as the heavy chain and L63-k0MT (SEQ ID NO:87) as the light chain is equivalent to that of human IL-8 at neutral pH (pH 7.4) Hr9, and has reduced binding affinity to human IL-8 at acidic pH (pH 5.8). Specifically, the koff (dissociation rate constant) and KD (dissociation constant) of H89/L63 at pH 5.8 are both higher than that of Hr9. This means that H89/L63 has the property of easily releasing human IL-8 under the acidic pH conditions of endosomes.

Surprisingly, it was found that H89/L118, which has H89-IgG1 as the heavy chain and L118-k0MT (SEQ ID NO:88) as the light chain, has enhanced human IL-8 binding under neutral pH conditions compared to Hr9 Affinity (KD), but compared to Hr9, the binding affinity (KD) of human IL-8 under acidic pH conditions is weaker. It is not particularly limited. Generally, if an antibody that can bind to an antigen multiple times is used as a pharmaceutical product, the pH-dependent antigen-binding antibody should have strong binding affinity (small KD) so that it can neutralize strongly under neutral pH conditions. This antigen (such as in plasma). On the other hand, the antibody should have a large dissociation rate constant (koff) and/or weak binding affinity (large KD) so that the antigen can be rapidly released under acidic pH conditions (such as in endosomes) . Compared with Hr9, H89/L118 has obtained these favorable properties under both neutral pH and acidic pH conditions.

Therefore, useful amino acid modifications have been identified for Hr9, such as Y97H for its heavy chain and N50H/L54H/Q89K for its light chain. Although not limited thereto, it is shown that a pH-dependent IL-8-binding antibody that is advantageous as a medicine can be produced by introducing a single amino acid modification or a combination of multiple amino acid modifications selected from these modifications.

Although not limited to a specific theory, it is believed that an important factor in using pH-dependent antigen-binding antibodies as medicines is whether the administered antibodies can release the antigens in the endosome. In this regard, a sufficiently weak binding (large dissociation constant (KD)) or a sufficiently fast dissociation rate (large dissociation rate constant (koff)) under acidic pH conditions is considered to be important. Therefore, the following experiment was performed to check whether the obtained KD or koff of H89/L118 is sufficient to dissociate this antigen in endosomes in vivo using BIACORE. [Example 10]

Production of high-affinity antibodies for mouse PK assay The method used to confirm the effect of the antibody on the elimination rate of human IL-8 in mice is not particularly limited. For example, this method involves administering the antibody to mice after mixing it with human IL-8, and then comparing the rate of elimination of human IL-8 from the mouse plasma.

Here, the reference antibody used for mouse PK analysis should have strong enough binding affinity under both neutral pH and acidic pH conditions. A search was then performed for modifications that would confer high affinity to Hr9, creating H998/L63 with H998-IgG1 (SEQ ID NO:89) as the heavy chain and L63-k0MT as the light chain.

Human IL-8 binding affinity was evaluated using H998/L63 in a similar manner to Example 9-2. The sensor pattern obtained is shown in Figure 21.

Unexpectedly, H998/L63 showed a slow dissociation rate under both neutral pH and acidic pH conditions, and had stronger IL-8 binding affinity than Hr9. However, it is known that due to the mechanical limitations of BIACORE, analytical values such as dissociation rate constant (koff) and dissociation constant (KD) cannot be calculated correctly when protein-protein interactions have low dissociation rates. Because the correct analytical value cannot be obtained for H998/L63, its analytical value is not shown here. However, the experimental results confirmed that H998/L63 has very strong binding affinity at both neutral pH and acidic pH, and is suitable as a comparison antibody for mouse PK analysis. [Example 11]

Mouse PK assay using pH-dependent IL-8 binding antibody H89/L118 (11-1) Mouse PK assay using H89/L118 H89/L118 produced in Example 9 and H998/L63 produced in Example 10 were used to evaluate the elimination rate of human IL-8 in vivo.

The pharmacokinetics of human IL-8 were evaluated after simultaneous administration of human IL-8 and anti-human IL-8 antibody in mice (C57BL/6J, Charles river). A mixed solution of human IL-8 and anti-human IL-8 antibody (10 μg/mL and 200 μg/mL each) was administered into the tail vein in a single dose of 10 mL/kg. At this time, there is a sufficient excess of anti-human IL-8 antibody relative to human IL-8, so it is believed that almost all human IL-8 has been bound to the antibody. Blood was collected at 5 minutes, 2 hours, 4 hours, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 min to obtain plasma. The separated plasma was stored frozen at -20°C or lower until measurement.

(11-2) Measurement of human IL-8 concentration in plasma The concentration of human IL-8 in mouse plasma was determined by electrochemiluminescence method. First, the anti-human IL-8 antibody (prepared in-house) including the mouse IgG constant region was dispensed into a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and left to stand at room temperature for 1 hour. Then use PBS-Tween solution containing 5% BSA (w/v) to block for 2 hours at room temperature to prepare an anti-human IL-8 antibody immobilized plate. Prepare calibration curve samples containing human IL-8 with plasma concentrations of 275, 91.7, 30.6, 10.2, 3.40, 1.13, or 0.377 ng/mL, and mouse plasma measurement samples diluted 25 times or more. The sample and hWS-4 were mixed and allowed to react overnight at 37°C. Then 50 μL of this mixed solution was distributed into each well of the anti-human IL-8 antibody immobilized plate, and the solution was stirred at room temperature for 1 hour. Adjust the final concentration of hWS-4 to 25 μg/mL. Then react with biotin mouse anti-human Igκ light chain (BD Pharmingen) for 1 hour at room temperature, then react with SULFO-TAG-labeled streptavidin (Meso Scale Discovery) for 1 hour at room temperature, and distribute Read Buffer T (x1 ) (Meso Scale Discovery), immediately perform measurements with SECTOR Imager 2400 (Meso Scale Discovery). The concentration of human IL-8 was calculated based on the response of the calibration curve using the analysis software SOFT Max PRO (Molecular Devices).

Data on human IL-8 concentration in plasma are shown in Figure 22, and human IL-8 clearance (CL) values from mouse plasma are shown in Table 15.

[Table 15]

As shown in Figure 22, compared with simultaneous administration of human IL-8 and H998/L63, human IL-8 and H89/L118 were eliminated unexpectedly faster from the plasma of mice than when human IL-8 and H998/L63 were administered simultaneously. The CL value, which also quantitatively represents the elimination rate of human IL-8 from mouse plasma, indicates that the elimination rate of human IL-8 in H89/L118 is approximately 19 times higher than that in H998/L63.

Without being bound by a specific theory, the following can be surmised from the available data. Most of the human IL-8 administered simultaneously with the antibody is bound to the antibody in plasma and exists as a complex. Human IL-8 has been bound to H998/L63. Due to the strong affinity of this antibody, it will still exist in the bound state of the antibody even in the endosome under acidic pH conditions. Afterwards, H998/L63 is still bound to human IL-8 and can return to the plasma via FcRn; therefore, when this occurs, human IL-8 will also return to the plasma at the same time. Therefore, most of the human IL-8 that enters the cells will return to the plasma. That is, the rate of human IL-8 elimination from plasma was significantly reduced when H998/L63 was administered simultaneously. On the other hand, as mentioned above, human IL-8 that enters cells becomes a complex with H89/L118 (pH-dependent IL-8-binding antibody), and may be dissociated from this antibody under acidic pH conditions in endosomes. Human IL-8 dissociated from the antibody may be degraded after being transported to lysosomes. Therefore, pH-dependent IL-8-binding antibodies can significantly accelerate the elimination of human IL-8, compared with IL-8-binding antibodies such as H998/L63, which have strong binding affinity at both acidic pH and neutral pH.

(11-3) Mouse PK assay with increasing doses of H89/L118 Then, experiments to confirm the effects of dose changes of H89/L118 were performed as follows. The pharmacokinetics of human IL-8 were evaluated after simultaneous administration of human IL-8 and H89/L118 (2 mg/kg or 8 mg/kg) to mice (C57BL/6J, Charles river). A mixed solution of human IL-8 (2.5 μg/mL) and anti-human IL-8 antibody (200 μg/mL or 800 μg/mL) was administered into the tail vein in a single dose of 10 mL/kg. At this point, since there is a sufficient excess of anti-human IL-8 antibody compared to human IL-8, it is believed that almost all of the human IL-8 has bound to the antibody. Blood was collected at 5 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 min to obtain plasma. The separated plasma was stored frozen at -20°C or lower until measurement.

The concentration of human IL-8 in mouse plasma was measured in a method similar to Example 11-2. The results of human IL-8 concentration in plasma are shown in Figure 23, and the values of human IL-8 clearance (CL) from mouse plasma are shown in Table 16.

[Table 16]

As a result, it was confirmed that the group administered with 8 mg/kg of antibody had an elimination rate of human IL-8 that was approximately 2 times slower than the group administered with 2 mg/kg of H89/L118.

The inventor below will record the possible factors that may bring about the above results based on scientific background, but the content of disclosure C is not limited to the following discussion.

Among the antibodies returned to the plasma via FcRn from the endosome, it is preferable that the proportion of antibodies that bind to human IL-8 is low. Focusing on human IL-8 present in vivo, a high proportion of free rather than bound antibody was expected. When human IL-8 is administered together with an antibody that has no pH-dependent IL-8-affinity, most (nearly 100%) of the human IL-8 is thought to exist as an antibody complex in endosomes, with small amounts (nearly 0%) is considered free. On the other hand, when administered with a pH-dependent IL-8 binding antibody (eg, H89/L118), a certain proportion of human IL-8 should exist in the free form in the body. Hypothetically, the free proportion can be understood as follows: [Proportion of free human IL-8 in endosomes (%)] = [Concentration of free human IL-8 in endosomes] / [Concentration of free human IL-8 in endosomes] total human IL-8 concentration] x 100.

The ratio of free human IL-8 in the endosome understood from the above formula is ideally higher, for example, 20% is better than 0%, 40% is better than 20%, 60% is better than 40%, and 80% is better than 60 %, and 100% are better than 80%.

Therefore, there is a correlation between the above-mentioned proportion of free human IL-8 in endosomes and the binding affinity (KD) and/or dissociation rate constant (koff) for human IL-8 at acidic pH. That is, the weaker the binding affinity and/or the greater the dissociation rate for human IL-8 at acidic pH, the higher the proportion of free human IL-8 in endosomes. However, in the case of pH-dependent IL-8-binding antibodies that can bring the proportion of free human IL-8 close to 100% in endosomes, further weakening the binding affinity and/or increasing the dissociation rate at acidic pH may not necessarily be effective. Increase the proportion of free human IL-8. It is readily understood that even if, for example, the proportion of free human IL-8 improves from 99.9% to 99.99%, the extent of this improvement may not be significant.

According to the general chemical equilibrium theory, when anti-IL-8 antibody and human IL-8 coexist, and their binding reaction and dissociation reaction have reached equilibrium, the proportion of free human IL-8 will be clearly determined by the following three parameters: Antibody concentration, antigen concentration, and dissociation constant (KD). Here, when the antibody concentration is high, when the antigen concentration is high, or when the dissociation constant (KD) is small, complexes are easily formed, and the proportion of free human IL-8 decreases. On the other hand, when the antibody concentration is low, when the antigen concentration is low, or when the dissociation constant (KD) is large, it is difficult to form a complex, and the proportion of free human IL-8 increases.

In this experiment, when H89/L118 was administered at a dose of 8 mg/kg, the rate of elimination of human IL-8 was slower than when the antibody was administered at a dose of 2 mg/kg. This therefore suggests that in endosomes, the proportion of free human IL-8 is lower when the antibody is administered at 8 mg/kg compared to when the antibody is administered at 2 mg/kg. The reason for the decrease may be that increasing the antibody dose in endosomes to 4 times the antibody concentration promotes the formation of IL-8-antibody complexes in endosomes. That is, in the group in which the antibody dose was increased, the proportion of free human IL-8 in endosomes decreased, and therefore the elimination rate of human IL-8 decreased. This also implies that when the antibody is administered at a dose of 8 mg/kg, the dissociation constant (KD) of H89/L118 under acidic pH conditions is not sufficient to achieve almost 100% free human IL-8. More specifically, if it is an antibody with a larger dissociation constant (KD) (weaker binding) under acidic pH conditions, almost 100% free IL-8 can be achieved even when the antibody is dosed at 8 mg/kg. In this state, the elimination rate of human IL-8 is equivalent to that when the antibody is administered at a dose of 2 mg/kg.

Based on the above, in order to confirm whether the pH-dependent IL-8 binding antibody of interest can achieve a ratio of almost 100% free human IL-8 in vivo, without particular limitation, we can verify whether there is room for increasing the elimination of this antigen in vivo degree of effect. For example, a method is used to compare the elimination rate of human IL-8 using a novel pH-dependent IL-8-binding antibody, which has weaker binding affinity at acidic pH and H89/L118 compared to H89/L118. /or have an increased dissociation rate at acidic pH. If the above-mentioned novel pH-dependent IL-8-binding antibody shows the same elimination rate of human IL-8 as H89/L118, this implies that the binding affinity and/or dissociation rate of H89/L118 is sufficient to achieve at acidic pH. The ratio of endosomal levels to almost 100% free human IL-8. On the other hand, when the above novel pH-dependent IL-8 binding antibody shows a higher elimination rate of human IL-8, it implies that there is room for improvement in the binding affinity and/or dissociation rate of H89/L118 at acidic pH. [Example 12]

Production and evaluation of pH-dependent IL-8 binding antibody H553/L118 (12-1) Production of antibody H553/L118 with pH-dependent IL-8 binding ability The inventors' aim here was to produce antibodies that have weaker human IL-8 binding affinity and/or greater off-rate than H89/L118 under acidic pH conditions.

Following a method similar to Example 9, using H89/L118 as a base, amino acid modifications mainly involving histidine were introduced to produce modified antibodies shown in Table 17. In addition, the binding affinity of human IL-8 to these antibodies was measured in a method similar to that of Example 9-2.

Some results are shown in Table 17. Antibody H553/L118 including H553-IgG1 (SEQ ID NO:90) as the heavy chain and L118-k0MT as the light chain, and antibody H553/L118 including H496-IgG1 (SEQ ID NO:101) as the heavy chain and L118-k0MT as the light chain. Antibody H496/L118 showed a more increased pH dependence than H89/L118.

[Table 17]

In the obtained H553/L118, two amino acid modifications, namely Y55H and R57P, were introduced into the heavy chain of H89/L118. On the other hand, H496/L118, in which only R57P is introduced into the heavy chain of H89/L118, has enhanced binding affinity for human IL-8 at neutral pH compared with H89/L118, but at acidic pH, human IL-8 8 Binding affinity is almost unchanged. More specifically, the R57P modification introduced into H89/L118 is a modification that only increases the binding affinity of human IL-8 at neutral pH but does not change the binding affinity at acidic pH. In addition, H553/L118, which is produced by introducing Y55H into the heavy chain of H496/L118, has maintained or slightly improved binding affinity at neutral pH. However, compared to H89/L118, H553/L118 has higher binding affinity at acidic pH. The binding affinity is reduced. That is, when the combination of two amino acid modifications, namely Y55H and R57P, is introduced into H89/L118, the binding affinity that decreases at acidic pH can be further improved while the binding affinity at neutral pH can be maintained or slightly improved.

(12-2) Mouse PK assay using H553/L118 By a method similar to Example 11-2, H553/L118 was used to evaluate the elimination rate of human IL-8 in mice. The resulting data on human IL-8 concentration in plasma are shown in Figure 24, and the values of human IL-8 clearance (CL) from mouse plasma are shown in Table 18.

[Table 18]

As a result, when comparing the data from mice administered 2 mg/kg antibody, no major differences were observed between H553/L118 and H89/L118; however, when comparing the data from mice administered 8 mg/kg antibody, It was confirmed that H553/L118 accelerated the elimination of human IL-8 by 2.5 times or more compared to H89/L118. From another point of view, the elimination rate of human IL-8 showed no difference in H553/L118 between 2 mg/kg and 8 mg/kg, and no decrease in the antigen elimination rate due to an increase in the antibody dose of H89/L118 was observed. .

Without being particularly limited, the reasons for obtaining such results can be discussed as follows. H533/L118 showed equivalent elimination rates of human IL-8 when this antibody was administered at 2 mg/kg and 8 mg/kg. It was shown that the proportion of free IL-8 in endosomes can reach a level close to 100%, because IL-8 binding by H553/L118 at acidic pH is still weak enough even when administered at 8 mg/kg. In other words, this suggests that although the human IL-8 elimination effect of H89/L118 is maximal at a dose of 2 mg/kg, its effect may be weakened at a high dose of approximately 8 mg/kg. On the other hand, H553/L118 can still achieve the maximum effect of eliminating human IL-8 even at a high dose of 8 mg/kg.

(12-3) Stability evaluation using H553/L118 In mice, it was shown that H553/L118 is an antibody that can significantly accelerate the elimination of human IL-8 compared with H89/L118. However, in order for this antibody to maintain its inhibitory effect on human IL-8 for a long period of time in vivo, the neutralization of IL-8 by the antibody must be maintained stably while the administered antibody is present in vivo (for example, in plasma). Stability of activity) IL-8 neutralizing activity is also important. Therefore, the following method was used to evaluate the stability of these antibodies in mouse plasma.

Mouse plasma was collected from the blood of C57BL/6J (Charles River) according to methods known in the art, and 200 μL of 200 mM PBS (Sigma, P4417) was added to 800 μL of mouse plasma to reach 1 mL. In addition, sodium azide (sodium azide) was added to a final concentration of 0.1% as an antibacterial agent. Each antibody (Hr9, H89/L118, and H553/L118) was then added to the above mouse plasma to a final concentration of 0.2 mg/mL. At this point, a portion of the sample is collected as a starting sample. The remaining samples were stored at 40°C. After 1 and 2 weeks of storage, a part of each sample was collected and used as a 1-week storage sample and a 2-week storage sample. All samples were frozen at -80°C and stored until each analysis was performed.

Their human IL-8 neutralizing activity was then evaluated using anti-IL-8 antibodies contained in mouse plasma, as follows: CXCR1 and CXCR2 are known receptors for human IL-8. PathHunter (registered trademark) CHO-K1 CXCR2 Beta-Arrestin cell line (DiscoveRx Co., Cat.# 93-0202C2) expresses human CXCR2 and is an artificial cell line that transmits signals in human IL-8- to emit chemical electricity. Causes luminescence. Although not particularly limited, such cells can be used to evaluate the human IL-8 neutralizing activity possessed by anti-human IL-8 antibodies. When human IL-8 is added to cell culture fluid, a certain amount of chemiluminescence appears in a manner that is dependent on the concentration of human IL-8 added. When human IL-8 and anti-human IL-8 antibodies are added to the culture medium together, when the anti-human IL-8 antibody binds to human IL-8, it can block human IL-8 signal transmission. Therefore, the chemiluminescence caused by the added human IL-8 can be inhibited by the anti-human IL-8 antibody, and the chemiluminescence will be weaker than that without the added antibody, or there will be no chemiluminescence at all. Therefore, as the neutralizing activity of human IL-8 carried by the antibody increases, the degree of chemiluminescence becomes weaker; and as the neutralizing activity of human IL-8 carried by the antibody weakens, the degree of chemiluminescence increases.

This is also true for antibodies that have been added to mouse plasma and stored for a certain period of time. If the neutralizing activity of the antibody has not changed due to storage in mouse plasma, the degree of the above-mentioned chemiluminescence before and after storage will not change. should be changed. On the other hand, if the neutralizing activity of the antibody is reduced due to preservation in mouse plasma, the extent of chemiluminescence using the preserved antibody will be increased compared to before preservation.

Next, the above-mentioned cell lines were used to examine whether the antibodies preserved in mouse plasma maintained neutralizing activity. First, the cell lines were suspended in AssayComplete(tm) Cell Plating 0 Reagent, and then seeded in a 384-well plate at 5,000 cells/well. One day after starting the cell culture, the following experiment was performed to determine the concentration of human IL-8 to be added. Serially diluted human IL-8 solutions with final concentrations ranging from 45 nM (400 ng/mL) to 0.098 nM (0.1 ng/mL) were added to the cell culture medium. Then add detection reagents according to the product's experimental procedures, and use a chemical electroluminescence detector to detect the relative chemical electroluminescence level. 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. Here, the human IL-8 concentration was set to 2 nM.

Then, the neutralizing activity of the antibodies contained in the mouse plasma to which the anti-human IL-8 antibody was added was evaluated. Human IL-8 at a concentration determined as above and the aforementioned mouse plasma containing anti-human IL-8 antibodies were added to the cell culture. Determine the amount of mouse plasma to be added to contain a gradient concentration of anti-human IL-8 antibody ranging from 2 μg/mL (13.3 nM) to 0.016 μg/mL (0.1 nM). Then add detection reagents according to the product experimental procedures, and use a chemical electroluminescence detector to detect the relative chemical electroluminescence level.

Here, the relative value of the relative chemiluminescence level for each antibody concentration is defined as follows: the average relative chemiluminescence level when no human IL-8 and antibody are added to the well is 0%, and when only human IL-8 is added, the average relative chemiluminescence level is 0%. The average relative chemiluminescence level without antibody in the wells was 100%.

The results of the human IL-8 inhibition assay using human CXCR2-expressing cells are shown in Figure 25, Figure 25A shows the results from the starting sample (mouse plasma treated without preservative), Figure 25B shows results preserved at 40 Results for samples stored at 40°C for 1 week. Figure 25C shows the results for samples stored at 40°C for 2 weeks.

Results Regarding Hr9 and H89/L118, no difference in the neutralizing activity of human IL-8 was observed before and after storage in mouse plasma. On the other hand, H553/L118 showed a decrease in human IL-8 neutralizing activity after 2 weeks of storage. Therefore, the human IL-8 neutralizing activity of H553/L118 in mouse plasma is easier to decrease than that of Hr9 and H89/L118, which means that H553/L118 is an unstable antibody in terms of IL-8 neutralizing activity. [Example 13]

Using in silico systems to produce antibodies with reduced predicted immunogenicity scores (13-1) Predicted immunogenicity scores of various IL-8 binding antibodies The production of anti-drug antibodies (ADA) will affect the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes cause serious side effects; therefore, the clinical utilization and drug efficacy of therapeutic antibodies may be limited by the production of ADA. The immunogenicity of therapeutic antibodies is known to be affected by many factors, and there have been many reports describing the importance of effector T cell epitopes in therapeutic antibodies.

Computer simulation tools for predicting T cell epitopes have been developed, such as Epibase (Lonza), iTope/TCED (Antitope) and EpiMatrix (EpiVax). Using these computer simulation tools, T cell epitopes at each amino acid sequence can be predicted (Walle et al., Expert Opin. Biol. Ther. 7(3):405-418 (2007)) and can be evaluated Potential immunogenicity of therapeutic antibodies.

Here EpiMatrix is used to calculate the immunogenicity score of each anti-IL-8 antibody. EpiMatrix is a system used to predict the immunogenicity of proteins of interest. It cuts the amino acid sequence of the protein to be predicted to be immunogenic by 9 amino acids to automatically design the sequence of the peptide fragment and then calculates their relationship with the 8 amino acids. Binding ability of major MHC class II allele genes (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301 and DRB1*1501) (De Groot et al., Clin . Immunol. 131(2):189-201 (2009)).

The immunogenicity scores of the heavy and light chains of each anti-IL-8 antibody calculated in the above manner are shown in the "EpiMatrix Score" column of Table 19. In addition, regarding the EpiMatrix score, the immunogenicity score corrected for the Tregitope content is displayed in the "tReg Adjusted Epx score" column. Tregitope is a peptide fragment sequence that mainly appears in natural antibody sequences and is considered to inhibit immunogenicity by activating regulatory T cells (Tregs).

In addition, regarding the score, the total score for the heavy chain and the light chain is shown in the "Total" column.

[Table 19]

In accordance with these results, the "EpiMatrix Score" and "tReg Adjusted Epx Score" show the immunogenicity scores of H89/L118, H496/L118 and H553/L118 compared to hWS-4, a known humanized anti-human IL-8 antibody for reduction.

Furthermore, using EpiMatrix, the frequency of ADA production of predicted antibody molecules can be compared as a whole by considering the heavy chain and light chain fractions of the actual frequency of ADA production caused by various commercially available antibodies. The results of such analysis are shown in Figure 26. Due to system limitations, Figure 26 uses "WS4" to label hWS-4, "HR9" to label Hr9, "H89L118" to label H89/L118, "H496L118" to label H496/L118, and "H553L118" to label H553/L118.

As shown in Figure 26, the frequency of ADA production in humans caused by various commercially available antibodies is known to be 45% for Campath (Alemtuzumab), 27% for Rituxan (Rituximab), and 14% for Zenapax (Daclizumab). On the other hand, the ADA production frequency predicted from the amino acid sequence is 10.42% for the known humanized anti-human IL-8 antibody hWS-4, but the newly identified H89/L118 (5.52%) and H496 The frequency of /L118 (4.67%) or H553/L118 (3.45%) was significantly lower compared to hWS-4.

(13-2) Making modified antibodies with lower predicted immunogenicity scores As mentioned above, compared with hWS-4, the immunogenicity scores of H89/L118, H496/L118, and H553/L118 are lower; but from Table 19, it can be seen that the immunogenicity scores of the heavy chain are higher than those of the light chain, suggesting that There is still room for improvement of the heavy chain amino acid sequence, especially from an immunogenicity point of view. A search was then performed against the heavy chain variable region of H496 to identify amino acid modifications that would reduce the immunogenicity fraction. As a result of diligent search, 3 variants were found, namely H496v1, in which alanine at position 52c was replaced by aspartic acid according to Kabat numbering, and H496v2, in which glutamic acid at position 81 was replaced by Kabat numbering. Threonine, H496v3, in which serine at position 82b is replaced by aspartic acid according to Kabat numbering. Furthermore, H1004 containing all three modifications was produced.

The results of the immunogenicity scores calculated in a similar manner to Example 13-1 are shown in Table 20.

[Table 20]

Three heavy chains, namely H496v1, H496v2 and H496v3, all contain a single modification and show a smaller immunogenicity fraction than H496. And H1004, containing a combination of 3 modifications, achieved a significant improvement in the immunogenicity score.

Here, in addition to L118, L395 was also identified as a suitable light chain for combination with H1004. Therefore, when calculating the immunogenicity score, both the L118 combination and the L395 combination are used. As shown in Table 20, H1004/L118 and H1004/L395, which combine heavy and light chains, also showed very low immunogenicity scores.

The frequency of ADA produced by these combinations is then predicted using a method similar to Example 13-1. The results are shown in Figure 27. In Figure 27, "V1" is used to mark H496v1/L118, "V2" is used to mark H496v2/L118, "V3" is used to mark H496v3/L118, "H1004L118" is used to mark H1004/L118, and "H1004L395" is used to mark H1004/L395.

Unexpectedly, H1004/L118 and H1004/L395, which had significantly lower immunogenicity scores, also showed an improvement in the predicted value of ADA production frequency, with a predicted value of 0%.

(13-3) Measure the IL-8 binding affinity of H1004/L395 H1004/L395 was produced, which is an antibody including H1004-IgG1m (SEQ ID NO:91) as the heavy chain and L395-kOMT (SEQ ID NO:82) as the light chain. The binding affinity of H1004/L395 to human IL-8 was measured using BIACORE T200 (GE Healthcare) according to the following method.

The following 2 running buffers were used and measurements were performed at their respective temperatures: (1) 0.05% tween20, 40 mM ACES, 150 mM NaCl, pH 7.4, 40°C; and (2) 0.05% tween20, 40 mM ACES, 150 mM NaCl, pH 5.8, 37°C.

An appropriate amount of Protein A/G (PIERCE) was immobilized on Sensor chip CM4 (GE Healthcare) using the amine coupling method, and the antibody of interest was captured. Human IL-8 and the antibodies captured on the sensor chip are then interacted by injecting a diluted human IL-8 solution or running buffer (as a reference solution). For the running buffer, use one of the two buffers above, and dilute human IL-8 using each buffer. To regenerate the sensor chip, use 25 mM NaOH with 10 mM glycine-HCl (pH 1.5). The KD (M) of human IL-8 for each antibody was calculated based on the kinetic parameters calculated from the sensorgram obtained by measurement, the binding rate constant kon (1/Ms) and the dissociation rate constant koff (1/s). BIACORE T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

The measurement results are shown in Table 21. Compared with H89/L118, H1004/L395 has a lower immunogenicity score, has the same KD for human IL-8 at neutral pH, but has a larger KD and koff at acidic pH; showing that it is easily excreted from the endosome. Properties of IL-8 dissociation.

[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 Through the evaluation in Example 13, H1004/L395 was obtained, which has pH-dependent IL-8 binding ability and a lower immunogenicity score. Elaborate investigations were then performed to generate variants with these beneficial properties and stability in mouse plasma.

Based on H1004/L395, the following modified antibodies were produced by introducing various modifications.

[Table 21-2]

[Table 21-3]

A total of 36 types of antibodies were produced using a combination of the above 18 types of heavy chains and 2 types of light chains. Various evaluations were performed for these antibodies, as shown below.

The human IL-8 binding affinity under neutral and acidic pH conditions was measured in a similar manner to Example 13-3. Among the results obtained, KD at pH 7.4 and KD and koff at pH 5.8 are shown in Table 22.

The stability of this antibody in binding to IL-8 when stored in PBS was then evaluated according to the method shown below.

Each antibody was dialyzed against DPBS (Sigma-Aldrich) overnight, and then the antibody concentration was adjusted to 0.1 mg/mL. At this point, collect some of this antibody sample as a starting sample. The remaining samples were stored at 50°C for one week and then collected as samples for thermal acceleration testing.

BIACORE measurement of IL-8 binding affinity was then performed using the starting sample and the sample from the heat acceleration test in the following manner.

Levels of human IL-8 binding to modified antibodies were analyzed using BIACORE T200 (GE Healthcare). Measurements were performed at 40°C using 0.05% tween20, 40 mM ACES, and 150 mM NaCl at pH 7.4 as running buffer.

An appropriate amount of Protein A/G (PIERCE) was immobilized on Sensor chip CM4 (GE Healthcare) using the amine coupling method to capture the antibody of interest. Human IL-8 and the antibodies captured on the sensor chip are then interacted by injecting a diluted human IL-8 solution or running buffer (as a reference solution). Running buffer was also used to dilute human IL-8. To regenerate the sensor chip, use 25 mM NaOH with 10 mM glycine-HCl (pH 1.5). Measured human IL-8 binding levels and the amount of capture antibody at that binding level were extracted using BIACORE T200 Evaluation Software (GE Healthcare).

The amount of human IL-8 bound per 1000 RU of captured antibody was calculated for the starting sample and for the sample in the heat-accelerated test. Furthermore, the human IL-8 binding level ratio of the starting sample relative to the sample of the heat accelerated test was calculated.

The results of the human IL-8 binding level ratio of the starting sample relative to the sample of the heat accelerated test are also shown in Table 22.

[Table 22]

Using the above test, H1009/L395 was obtained, which is an antibody containing H1009-IgG1m (SEQ ID NO:92) as the heavy chain and L395-kOMT as the light chain.

As shown in Table 22, compared to H89/L118, H1009/L395 has slightly increased human IL-8 binding affinity at neutral pH, but has reduced binding affinity at acidic pH, i.e., the pH dependence has been further strengthened. When exposed to severe conditions, such as 50°C in PBS, H1009/L395 has slightly improved IL-8 binding stability compared to H89/L118.

Therefore, H1009/L395 was selected as an antibody whose neutralizing activity could be stably maintained in mouse plasma while still maintaining pH-dependent IL-8 binding ability.

(14-2) Stability evaluation of H1009/L395 Then, in a manner similar to Example 12-3, it was evaluated whether the IL-8 neutralizing activity of H1009/L395 was stably maintained in mouse plasma. Here, H1009/L395-F1886s, which will be described in detail in Example 19 later, is used. This antibody has the same variable region as H1009/L395, and its constant region has modifications that enhance FcRn binding under acidic pH conditions and reduce FcγR binding compared to natural human IgG1. The variable region of H1009/L395, especially the region around the HVR, is responsible for human IL-8 binding and the IL-8 neutralizing activity of the antibody. It is believed that introducing modifications into the constant region will not affect these properties.

Stability assessment in mouse plasma was performed as follows. Add 150 μL of 200 mM phosphate buffer (pH 6.7) to 585 μL of mouse plasma. Sodium azide was then added as an antibacterial agent to 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 is collected as a starting sample. Store the remaining samples at 40°C. After 1 and 2 weeks of storage, a part of each sample was collected and used as a 1-week storage sample and a 2-week storage sample. All samples were frozen at -80°C and stored until each analysis was performed.

Human IL-8 neutralizing activity was measured using human CXCR2-expressing cells in a manner similar to Example 12-3. However, at this time, the human IL-8 concentration used to confirm the neutralizing activity of the anti-human IL-8 antibody was 1.2 nM.

The results of human IL-8 inhibition assay using human CXCR2-expressing cells and the above antibodies are shown in Figure 28A, Figure 28A shows the starting sample (without preservation in mouse plasma), and Figure 28B shows the results for 40°C storage Results for 1 week samples, Figure 28C shows results for samples stored at 40°C for 2 weeks.

As a result, surprisingly, even when mouse plasma was stored at 40°C for 2 weeks, the human IL-8 neutralizing activity of H1009/L395-F1886s was still maintained, and the IL-8 neutralizing activity was more stable than that of H553/L118. maintain.

(14-3) Mouse PK assay using H1009/L395 The rate of elimination of human IL-8 by H1009/H395 in mice was evaluated as follows. H1009/L395, H553/L118 and H998/L63 were used as antibodies. The methods shown in Example 11 were used to administer drugs to mice, collect blood, and measure the concentration of human IL-8 in mouse plasma.

The human IL-8 concentration results in plasma are shown in Figure 29, and the human IL-8 clearance (CL) values from mouse plasma are shown in Table 23.

[Table 23]

Results When the dosage of H1009/L395 was 2 mg/kg, the elimination rate of human IL-8 in mice was equivalent to that of H553/L118, indicating that H1009/L395 reached almost 100% free IL-8 in endosomes. Clearance (CL) values, which quantitatively represent the rate of elimination of human IL-8 from mouse plasma, were shown to be approximately 30-fold higher than H998/L63.

Without being particularly limited, the effect of increasing the elimination rate of human IL-8 can be understood as follows. Generally, in a living body, the concentration of the antigen is maintained at an almost certain level, and the production rate and elimination rate of the antigen are also maintained at an almost certain value. When antibodies are administered under such conditions, even if the rate of antigen production is not affected, the rate of antigen elimination may be altered due to the formation of complexes between the antigen and the antibody. Generally, since the elimination rate of the antigen is greater than the elimination rate of the antibody, in this case, the elimination rate of the antigen that has formed a complex with the antibody is reduced. When the antigen elimination rate decreases, the antigen concentration in the plasma increases, but the degree of increase in this case can also be defined by the ratio of the elimination rate of the antigen alone relative to the elimination rate of the antigen in complexes. That is, if the elimination rate when a complex is formed is reduced to one-tenth compared to the elimination rate when the antigen exists alone, the concentration of the antigen in the plasma of the organism to which the antibody is administered may increase to About 10 times as much. Here, the clearance rate (CL) can be used as the elimination rate. More specifically, the increase in antigen concentration (antigen accumulation) that occurs after antibody is administered to an organism can be defined as the antigen CL before and after antibody administration under each condition.

Here, there is an approximately 30-fold difference in the CL of human IL-8 when H998/L63 and H1009/L395 are administered, suggesting that when these antibodies are administered to humans, the concentration of human IL-8 in plasma increases. There is about 30 times difference. Furthermore, a 30-fold difference in the concentration of human IL-8 in plasma means that the amount of antibody required to completely block the biological activity of human IL-8 also differs approximately 30-fold under each condition. That is, compared to H998/L63, H1009/L395 can block the biological activity of IL-8 in plasma at about 1/30 of the amount, and the amount of this antibody is very small. And when H1009/L395 and H998/L63 are administered to humans individually at the same dose, H1009/L395 can block the biological activity of IL-8 with greater intensity for a longer period of time. In order to block the biological activity of IL-8 for a long time, the neutralizing activity of IL-8 needs to be stably maintained. As in Example 14, experiments using mouse plasma have demonstrated that H1009/L395 can maintain its human IL-8 neutralizing activity for a long time. H1009/L395, which has such remarkable properties, is also shown to be an antibody with superior effects from the viewpoint of neutralizing IL-8 in vivo. [Example 15]

Assessment of extracellular matrix binding using pH-dependent IL-8 binding antibody H1009/L395 The 30-fold superior effect of H1009/L395 in eliminating human IL-8 shown in Example 14 is an unexpected effect. It is known that when pH-dependent antigen-binding antibodies are administered, the rate of antigen elimination depends on the rate of uptake of the antibody-antigen complex into the cell. That is, if the rate of uptake of pH-dependent antigen-binding antibodies into cells increases, the antigen-eliminating effect of pH-dependent antibodies will increase when an antigen-antibody complex is formed compared to when the complex is not formed. Known methods for increasing the rate of uptake of antibodies into cells include: imparting FcRn-binding ability to antibodies under neutral pH conditions (WO 2011/122011), and methods for increasing the binding ability of antibodies to FcγR (WO 2013/047752) , and a method for promoting the formation of a complex including a multivalent antibody and a multivalent antigen (WO 2013/081143).

But the above technology is not used in the constant region of H1009/L395. Furthermore, although IL-8 is known to form homodymes, human IL-8 bound by H1009/L395 was found to exist in the form of a monomer because H1009/L395 recognizes homodymes of human IL-8. The body forms the surface. Therefore, antibodies do not form multivalent complexes.

More specifically, although the above technology was not used in H1009/L395, H1009/L395 still showed up to 30 times the elimination effect of human IL-8.

The inventors then conducted the following discussion as possible factors that may bring about the above-mentioned properties of pH-dependent IL-8 binding antibodies represented by H1009/L395. However, the following is only the inventor's possible speculation based on the technical background, and the content of disclosure C is not limited to the following discussion.

Human IL-8 is a protein with a high isoelectric point (pI), and the theoretical isoelectric point calculated according to known methods is about 10. That is, under neutral pH conditions, human IL-8 is a protein with a charge biased toward the positive side. pH-dependent IL-8-binding antibodies represented by H1009/L395 are also proteins with a positive charge bias, and the theoretical isoelectric point of H1009/L395 is about 9. That is, the isoelectric point of the complex formed by combining H1009/L395 (which has a high isoelectric point and is originally rich in positive electricity) with human IL-8 (which has a high isoelectric point) will be higher than that of H1009/L395 alone.

As in Example 3, increasing the isoelectric point of the antibody, including increasing the number of positive charges and/or reducing the number of negative charges on the antibody, is believed to increase the non-specific uptake of the antibody-antigen complex into cells. The isoelectric point of the complex formed by anti-IL-8 antibody and human IL-8 (which has a high isoelectric point) is higher than that of anti-IL-8 antibody alone, and this complex can enter more easily cells.

As mentioned previously, affinity for the extracellular matrix is also a factor that may influence uptake into cells. We then examined whether there was a difference in extracellular matrix binding when the antibody alone was complexed with human IL-8-antibody.

The amount of antibody bound to the extracellular matrix was evaluated using the ECL (electroluminescence) method. The extracellular matrix (BD Matrigel basement membrane matrix/manufactured by BD) was diluted to 2 mg/mL using TBS (Takara, T903). The diluted extracellular matrix was dispensed into a MULTI-ARRAY 96-well plate, high-binding, bare plate (MSD manufactured by Meso Scale Discovery) at 5 μL per well, and immobilized at 4°C overnight. Then block using 20 mM ACES buffer (pH 7.4) containing 150 mM NaCl, 0.05% Tween20, 0.5% BSA, 0.01% NaN ₃ .

Antibodies to be evaluated were prepared as follows. The antibody sample added separately was diluted to 9 μg/mL with buffer 1 (20 mM ACES buffer, containing 150 mM NaCl, 0.05% Tween20, and 0.01% NaN ₃ , pH 7.4), and then buffer 2 (20 mM ACES buffer, containing 150 mM NaCl, 0.05% Tween20, 0.1% BSA, and 0.01% NaN ₃ , pH 7.4) was prepared by diluting it to a final concentration of 3 μg/mL.

On the other hand, the antibody sample to be added as a complex with human IL-8 was prepared in the following manner: human IL-8 with a molar concentration 10 times that of the antibody was added to the antibody sample, and then each antibody was diluted with buffer-1 , so that the antibody concentration was 9 μg/mL respectively, and then further diluted using buffer-2 to a final antibody concentration of 3 μg/mL. At this point, the human IL-8 concentration is approximately 0.6 μg/mL. It was shaken at room temperature for 1 hour to allow for complex formation.

Then add the solution of the antibody alone or the antibody in the form of a complex to the plate from which the blocking solution has been removed, and shake at room temperature for 1 hour. Then, after removing the solution of the antibody alone or the complex solution, add buffer-1 containing 0.25% Glutaraldehyde. Then, the plate was allowed to stand for 10 minutes, and then washed with DPBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween20. The antibody system for ECL detection was prepared by sulfo-labeling goat anti-human IgG (gamma) (manufactured by Zymed Laboratories) using Sulfo-Tag NHS Ester (manufactured by MSD). The antibody for ECL detection was diluted to 1 μg/mL with buffer-2, added to the plate, and shaken in the dark at room temperature for 1 hour. The antibody for ECL detection was removed, a solution of MSD Read Buffer T (4x) (manufactured by MSD) diluted 2-fold with ultrapure water was added, and the luminescence amount was measured with SECTOR Imager 2400 (manufactured by MSD).

The results are shown in Figure 30. Interestingly, all anti-IL-8 antibodies such as H1009/L395 showed almost no binding to the extracellular matrix when the antibody was used alone (-IL8), but when in complex with human IL-8 ((+hIL8 )), will bind to the extracellular matrix.

As mentioned above, no one has yet elucidated the properties of anti-IL-8 antibodies that possess affinity for the extracellular matrix by binding to human IL-8. Also, without limitation, combining such properties with a pH-dependent IL-8 binding antibody can effectively increase the rate of IL-8 elimination. [Example 16]

Mouse PK assay using non-FcRn-binding antibodies The following method was used to confirm whether a complex of human IL-8 and a pH-dependent IL-8-binding antibody is formed, and whether uptake of the complex into cells is increased in mice.

First, an antibody variant was produced, which included the variable region of H1009/L395 and the Fc region lacking binding affinity to various Fc receptors. Specifically, as a modification to delete the binding ability to human FcRn under acidic pH conditions, the isoleucine at position 253 of heavy chain H1009-IgG1 was replaced with alanine and serine at position 254 according to the EU numbering method. is aspartic acid. Furthermore, as modifications to delete binding to mouse FcγR(s), leucine at position 235 was replaced with arginine, glycine at position 236 was replaced with arginine, and serine at position 239 was replaced with ionine. acid. H1009-F1942m (SEQ ID NO:93) was produced as a heavy chain containing 4 of these modifications. Furthermore, H1009/L395-F1942m was produced, which contains H1009-F1942m as a heavy chain and L395-k0MT as a light chain.

Because antibodies with this Fc region lack FcRn binding affinity under acidic pH conditions, they will not be transferred from endosomes to plasma. Therefore, such antibodies will be eliminated from plasma faster in vivo than antibodies containing the native Fc region. In this case, when the antibody containing the native Fc region is taken up into the cell, only the part that is not rescued by FcRn will be degraded after being transferred to lysosomes. However, if the antibody contains an Fc that does not have FcRn binding affinity, area, all antibodies taken into cells will be degraded in lysosomes. More specifically, for antibodies that include such modified Fc regions, the rate of elimination of the administered antibody from the plasma may be equivalent to the rate of entry into cells. The rate of intracellular uptake of an antibody whose FcRn binding affinity has been deleted can also be confirmed by measuring the rate of elimination of this antibody from the plasma.

We then tested whether intracellular uptake of the complex formed by H1009/L395-F1942m and human IL-8 was increased compared to uptake of H1009/L395-F1942m alone. Specifically, it was tested whether the rate of elimination of the antibody from plasma changes when the antibody is administered alone and when the antibody is administered in complex with human IL-8.

For when anti-human IL-8 antibody was administered alone to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice; Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)), and the biokinetics of anti-human IL-8 antibodies were evaluated when human IL-8 and anti-human IL-8 antibodies were administered simultaneously to human FcRn gene transgenic mice. The anti-human IL-8 antibody solution (200 μg/mL) and the mixed solution of human IL-8 (10 μg/mL) and anti-human IL-8 antibody (200 μg/mL) were separately added to the tail vein at 10 mL/mL. kg is administered once. In this case, since the anti-human IL-8 antibody is present in sufficient excess for human IL-8, it is believed that nearly all human IL-8 is bound to this antibody. Blood was collected at 5 minutes, 2 hours, 7 hours, 1 day, and 2 days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 min to obtain plasma. The separated plasma was stored frozen at -20°C or lower until measurement.

Anti-human IL-8 antibody concentration in mouse plasma was measured by electrochemiluminescence method. First, anti-human Kappa light chain goat IgG Biotin ( IBL) was reacted at room temperature for 1 hour to produce an anti-human antibody immobilized plate. Prepare samples for the calibration curve, containing anti-human IL-8 antibody concentrations in plasma at 3.20, 1.60, 0.800, 0.400, 0.200, 0.100 and 0.0500 μg/mL, and prepare samples for mouse plasma measurement, which are diluted 100 times or higher. Each sample and human IL-8 were mixed, then 50 μL per well was dispensed into the anti-human antibody immobilized plate, and then stirred at room temperature for 1 hour. Human IL-8 was adjusted to a final concentration of 333 ng/mL.

Then, anti-human IL-8 antibody containing mouse IgG constant region (prepared in-house) was added to the plate and allowed to react at room temperature for 1 hour. Anti-mouse IgG (BECKMAN COULTER) calibrated with ruthenium-SULFO-TAG NHS Ester (Meso Scale Discovery) was added to the plate and allowed to react for 1 hour. Measurements were then taken using SECTOR Imager 2400 (Meso Scale Discovery) immediately after the Read Buffer T(x1) (Meso Scale Discovery) was allocated to the plate. Anti-human IL-8 antibody concentration was calculated based on the response in the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

The results of the antibody concentration in mouse plasma are shown in Figure 31, and the antibody clearance rates under each condition are shown in Table 24.

[Table 24]

Compared with the intracellular uptake rate of H1009/L395-F1942m, the intracellular uptake rate of the complex of H1009/L395-F1942m and human IL-8 was shown to be increased by at least 2.2 times. It is marked as "at least 2.2 times" here for the following reasons: one is to include the possibility that the actual value is 5 times, 10 times or 30 times. Because the rate of elimination of human IL-8 is quite rapid compared to the rate of elimination of H1009/L395-F1942m from mouse plasma, the proportion of H1009/L395-F1942m bound to human IL-8 in the plasma decreases rapidly after administration. More specifically, even if human IL-8 is administered at the same time, not all H1009/L395-F1942m in the plasma exists in the human IL-8-bound form. In fact, most of them have been in the free form about 7 hours after administration. exist. Since the uptake rate is evaluated under such conditions, even compared to the uptake rate of H1009/L395-F1942m, the intracellular uptake rate of the complex of H1009/L395-F1942m and human IL-8 is actually increased by 5 times, 10 times or 30 times. times, but the results are only partially reflected in the experimental system; therefore, the effect presented may be 2.2 times, etc. Therefore, from the results obtained, although the intracellular uptake rate of the complex of H1009/L395 and IL-8 is increased compared to the intracellular uptake rate of H1009/L395 in vivo, this effect is not limited to a 2.2-fold increase.

It is not particularly limited, but the following inferences can be drawn from the findings so far. When H1009/L395, a pH-dependent IL-8 binding antibody, forms a complex with human IL-8, the complex has a higher isoelectric point and is more positively charged than the antibody alone. At the same time, the affinity of the complex to the extracellular matrix is increased compared to the affinity of the antibody alone. For example, increasing the isoelectric point and increasing extracellular matrix binding are considered factors that promote antibody uptake into living cells. From mouse experiments, the intracellular uptake of the complex of H1009/L395 and human IL-8 increased by 2.2 times or more compared to the uptake rate of H1009/L395. From the above, theoretical explanations as well as in vitro properties and in vivo phenomena consistently support the following hypothesis: H1009/L395 forms a complex with human IL-8 to promote the uptake of the complex into cells and leads to a significant increase in the elimination of human IL-8.

So far, some antibodies against IL-8 have been reported, but there are no reports on increasing the binding affinity to the extracellular matrix and increasing the uptake of the complex into cells after forming a complex with IL-8.

Furthermore, based on the observation that when the antibody forms a complex with IL-8, the intracellular uptake of the anti-IL-8 antibody increases, it is believed that the anti-IL-8 antibody that has formed a complex with IL-8 in the plasma will quickly enter the cell. , while free antibodies that have not formed a complex with IL-8 tend to remain in the plasma without entering cells. In this case, when the anti-IL-8 antibody is pH-dependent, the anti-IL-8 antibody that has entered the cell will release IL-8 molecules in the cell and return to the outside of the cell, and then it can bind to another IL-8 8 molecules; therefore, the increased intracellular uptake after the formation of the complex will have a stronger elimination effect of IL-8. That is, selecting an anti-IL-8 antibody with increased binding to extracellular matrix or an anti-IL-8 antibody with increased uptake into cells may also be another embodiment of Disclosure C. [Example 17]

Prediction of immunogenicity of pH-dependent IL-8 binding antibody H1009/L395 using computer simulation system Then, predict the immunogenicity score of H1009/L395 and the frequency of ADA production according to a method similar to Example 13-1. The results are shown in Table 25 and Figure 32. In Figure 32, H1009/L395 is marked as "H1009L395".

[Table 25]

The results in Table 25 show that H1009/L395 and H1004/L395 have low immunogenicity scores to the same extent. In addition, in Figure 32, the predicted ADA production frequency result for H1009/L395 is 0%, and the result for H1004/L395 is similar.

Therefore, the predicted immunogenicity of H1009/L395 is significantly reduced compared to the known anti-human IL-8 antibody hWS-4. Therefore, H1009/L395 is considered to have very low immunogenicity in humans and can stably maintain anti-IL-8 neutralizing activity for a long time. [Example 18]

Malay monkey PK assay using H89/L118 variants with enhanced FcRn binding in acidic pH conditions As mentioned in the previous examples, among the antibodies with natural IgG1 as the constant region, the pH-dependent IL-8 binding antibody H1009/L395 is an antibody with superior properties. Such antibodies can also be used as antibodies containing amino acid substitutions in the constant region, as shown in Example 5, including an Fc region that enhances FcRn binding at acidic pH. Therefore, H89/L118 was used to confirm that the Fc region that enhances FcRn binding at acidic pH can also be used as a pH-dependent IL-8 binding antibody.

(18-1) H89/L118 Fc region modified antibody produced at acidic pH with enhanced FcRn binding Various modifications for improving FcRn binding described in Example 5-1 were introduced into the Fc region of H89/L118. Specifically, by introducing modifications used in F1847m, F1848m, F1886m, F1889m, F1927m, and F1168m into the Fc region of H89-IgG1 to create the following variants: (a) H89/L118-IgG1, including H89-IgG1m (SEQ ID NO:94) as the heavy chain and L118-KOMT as the light chain; (b) H89/L118-F1168m includes H89-F1168m (SEQ ID NO:95) as the heavy chain and L118-KOMT as the light chain; (c) ) H89/L118-F1847m, including H89-F1847m (SEQ ID NO:96) as the heavy chain and L118-K0MT as the light chain; (d) H89/L118-F1848m, including H89-F1848m (SEQ ID NO:97) as the light chain; Heavy chain and L118-K0MT as light chain; (e) H89/L118-F1886m, including H89-F1886m (SEQ ID NO:98) as heavy chain and L118-K0MT as light chain; (f) H89/L118-F1889m, including H89-F1889m (SEQ ID NO:99) as the heavy chain and L118-KOMT as the light chain; and (g) H89/L118-F1927m, including H89-F1927m (SEQ ID NO:100) as the heavy chain and L118-KOMT as a light chain. The Malay monkey PK assay using these antibodies was performed according to the following formula.

(18-2) Novel Malay monkey PK assay for antibodies containing Fc region variants The biokinetics of anti-human IL-8 antibodies were evaluated after administration of anti-human IL-8 antibodies to Malay monkeys. The anti-human IL-8 antibody solution was administered intravenously once at 2 mg/kg. Blood was collected at 5 minutes, 4 hours, 1 day, 2 days, 3 days, 7 days, 10 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 15,000 rpm and 4°C for 10 min to obtain plasma. The separated plasma was stored frozen at -60°C or lower until measurement.

The concentration of anti-human IL-8 antibody in the plasma of Malay monkeys was measured by electrochemiluminescence method. Anti-hKappa Capture Ab (Antibody Solutions) was first dispensed into a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and stirred at room temperature for 1 hour. Then use PBS-Tween solution containing 5% BSA (w/v) to block for 2 hours at room temperature to prepare anti-human antibody immobilized plates. Prepare samples for the calibration curve, which contain anti-human IL-8 antibody concentrations in plasma of 40.0, 13.3, 4.44, 1.48, 0.494, 0.165 and 0.0549 μg/mL, and prepare samples for Malay monkey plasma measurement, Dilute 500 times or more. Dispense 50 μL of the solution into each well of the anti-human antibody immobilized plate, and stir the solution at room temperature for 1 hour. Then, anti-hKappa Reporter Ab, Biotin conjugate (Antibody Solutions) was added to the aforementioned plate and allowed to react at room temperature for 1 hour. SULFO-TAG labeled streptavidin (Meso Scale Discovery) was further added and allowed to react at room temperature for 1 hour. Read Buffer T (x1) (Meso Scale Discovery) was distributed to the plate and SECTOR Imager 2400 (Meso Scale Discovery) was used. ) to measure immediately. Anti-human IL-8 antibody concentration was calculated based on the response in the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

The half-life (t1/2) and clearance (CL) results obtained for each antibody are shown in Table 26, and the changes in antibody concentration in the plasma of Malay monkeys are shown in Figure 33.

[Table 26]

The above results confirmed that all Fc region variants showed longer plasma retention than antibodies with the native IgG1 Fc region. In particular, H89/L118-F1886m has the most ideal hemodynamics. [Example 19]

Fc region with low binding ability to FcγRs It is known that the Fc region of natural human IgG1 binds to Fcγ receptors (hereinafter referred to as FcγR) in various cells of the immune system and exhibits effector functions on target cells, such as ADCC and ADCP.

On the other hand, IL-8 is a soluble interleukin, and the use of anti-IL-8 antibodies as medicines is mainly expected to show pharmacological effects by neutralizing the function of IL-8 at locations where excess IL-8 exists. The location where IL-8 is excessively present is not particularly limited, and may be an inflammatory site, for example. It is generally known that various immune cells accumulate and activate at such inflamed sites. It is not always beneficial to transmit unintended activation signals to these cells via Fc receptors and induce activities such as ADCC and ADCP in the unintended cells. Therefore, it is not particularly limited. From the viewpoint of safety, it is preferable that the anti-IL-8 antibody administered in vivo has low affinity for FcγR.

(19-1) Production of modified antibodies with lower binding to FcγR In order to reduce the binding ability to various human and Malay monkey FcγRs, further amino acid modifications were introduced into the Fc region of H1009/L395-F1886m. Specifically, H1009-F1886s (SEQ ID NO:81) was produced by performing the following substitutions on the H1009-F1886m heavy chain: according to the EU numbering method, L at position 235 was replaced with R, G at position 236 was replaced with R, S at position 239 is replaced by K. Similarly, make the following substitutions for H1009-F1886m to make H1009-F1974m (SEQ ID NO:80): According to the EU numbering method, replace the L at position 235 with R and replace the G at position 236 with R. According to the EU numbering method, The region from position 327 to position 331 was replaced with the native human IgG4 sequence. H1009/L395-F1886s and H1009/L395-F1974m were manufactured as antibodies with these heavy chains and L395-k0MT as the light chain.

(19-2) Confirmation of affinity for various human FcγRs Then, the affinity of H1009/L395-F1886s or H1009/L395-F1974m to soluble FcγRIa or FcγRIIIa in humans or Malay monkeys was confirmed according to the following method.

Binding of soluble forms of FcγRIa or FcγRIIIa was analyzed using BIACORE T200 (GE Healthcare) against H1009/L395-F1886s or H1009/L395-F1974m in humans or Malay monkeys. Soluble FcγRIa and FcγRIIIa in both humans and Malay monkeys were prepared as His-tagged molecules according to methods known to those skilled in the art. An appropriate amount of rProtein L (BioVision) was immobilized on the sensor chip CM4 (GE Healthcare) using the amine coupling method, and the antibody of interest was captured. Soluble FcγRIa or FcγRIIIa is then injected together with running buffer (as a reference solution) to allow it to interact with the antibodies captured on the sensor chip. HBS-EP+ (GE Healthcare) was used as running buffer and HBS-EP+ was also used to dilute soluble FcγRIa or FcγRIIIa. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 20°C.

The results are shown in Figure 34. Here, the markers of human FcγRIa, human FcγRIIIa, Malay monkey FcγRIa and Malay monkey FcγRIIIa are hFcγRIa, hFcγRIIIa, cynoFcγRIa and cynoFcγRIIIa in order. H1009/L395-F1886m was shown to bind to all FcγRs, but H1009/L395-F1886s and H1009/L395-F1974m were confirmed not to bind to any FcγR.

(19-3) Mouse IL-8 elimination assay for Fc variants Then, for H1009/L395-F1886s and H1009/L395-F1974m, the following experiments were performed to confirm the elimination rate of human IL-8 and the retention of the antibody in mouse plasma. Three doses of H1009/L395-F1886s were used here for evaluation, namely 2 mg/kg, 5 mg/kg, and 10 mg/kg, so the impact of increasing antibody doses on H1009/L395-F1886s can also be evaluated.

Human FcRn gene transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice; Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) were simultaneously administered with human IL-8 and anti-human IL-8 antibodies to evaluate the biokinetics of human IL-8. A single dose of 10 mL/kg of a mixed solution of human IL-8 (10 μg/mL) and anti-human IL-8 antibody (200 μg/mL, 500 μg/mL or 1000 μg/mL) was administered via the tail vein. In this case, since the anti-human IL-8 antibody is present in sufficient excess relative to human IL-8, almost all of the human IL-8 is believed to be bound to this antibody. Blood was collected at 5 minutes, 2 hours, 4 hours, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 min to obtain plasma. The separated plasma was stored frozen at -20°C or lower until measurement.

The concentration of human IL-8 in mouse plasma was measured in a manner similar to Example 11. The human IL-8 concentration results in plasma are shown in Figure 35, and the human IL-8 clearance (CL) values from mouse plasma are shown in Table 27.

First, when comparing the groups dosed at 2 mg/kg, H1009/L395 containing the Fc region of natural IgG1 showed the same human IL-8 elimination effect as H1009/L395-F1886s containing the modified Fc region.

Then when the dose of H1009/L395-F1886s antibody was changed, although there was a slight difference in the concentration of IL-8 in plasma 1 day after administration, the clearance rate of human IL-8 at doses of 2 mg/kg and 10 mg/kg was not significant. difference. This strongly suggests that antibodies including the variable region of H1009/L395 show sufficient IL-8 ablation even when the antibodies are administered at high doses.

[Table 27]

(19-4) Fc variant Malay monkey PK analysis method The plasma retention of the antibody in Malay monkeys was then verified using H1009/L395-F1886s or H1009/L395-F1974m as follows.

The biokinetics of anti-human IL-8 antibodies were evaluated in Malay monkeys when anti-human IL-8 antibodies were administered alone and when human IL-8 and anti-human IL-8 antibodies were administered simultaneously. Give anti-human IL-8 antibody solution (2 mg/mL) or a mixed solution of human IL-8 (100 μg/kg) and anti-human IL-8 antibody (2 mg/kg) intravenously as a single dose of 1 mL/kg. Mainland investment. Blood was collected at 5 minutes, 4 hours, 1 day, 2 days, 3 days, 7 days, 10 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 15,000 rpm and 4°C for 10 min to obtain plasma. The separated plasma was stored frozen at -60°C or lower until measurement.

The concentration of anti-human IL-8 antibody in the plasma of Malay monkeys was measured according to the method of Example 18. The results of the anti-human IL-8 antibody concentration in plasma are shown in Figure 36, and the values of the anti-human IL-8 antibody half-life (t _1/2 ) and clearance (CL) from Malay monkey plasma are shown in Table 28.

First, compared to Hr9 and H89/L118 with the Fc region of native human IgG1, H1009/L395-F1886s with enhanced Fc region showed significantly longer plasma retention.

And when H1009/L395-F1886s and human IL-8 were administered simultaneously, the concentration change in plasma was equivalent to that of the antibody alone. Without being particularly limited, the following discussion can be derived from the above findings. As mentioned above, the intracellular uptake of the complex of H1009/L395 and human IL-8 was shown to be increased compared to H1009/L395 alone. Generally, it is believed that high molecular weight proteins enter cells in a non-specific or receptor-dependent manner and are then transferred to lysosomes for degradation by various degradative enzymes present in lysosomes. Therefore, if the rate of protein uptake into cells is increased, plasma retention of the protein may also worsen. However, if the antibody has the property of returning to plasma via FcRn in endosomes, as long as there is sufficient FcRn rescue function, plasma retention will not be affected even if the intracellular uptake rate is accelerated. Here, even if H1009/L395-F1886s and human IL-8 are administered simultaneously to Malay monkeys, plasma retention is not affected. This suggests the possibility that even if the rate of uptake of antibodies against H1009/L395-F1886s into cells is increased, FcRn may be sufficient to rescue it and return it to the plasma.

Yet another Fc variant, H1009/L395-F1974m, also showed equivalent plasma retention relative to H1009/L395-F1886s. These Fc variants have introduced different modifications as described above that reduce the binding ability of various FcγRs, but have not been shown to have an impact on the plasma retention of the antibody itself. From the above, both H1009/L395-F1886s and H1009/L395-F1974m showed significantly prolonged plasma retention in Malay monkeys, and compared with antibodies with natural IgG1 Fc region, they are extremely satisfactory.

[Table 28]

As demonstrated in the above examples, H1009/L395 became the first to significantly increase the elimination rate of human IL-8 in vivo through its properties including pH-dependent IL-8 binding ability and formation of complexes with IL-8 for rapid uptake into cells. of antibodies. In addition, the IL-8 binding affinity of this antibody under neutral pH conditions is also increased compared to the known hWS-4 antibody. This antibody can neutralize human IL-8 more strongly under neutral pH conditions, such as plasma. 8. In addition, it is an antibody with excellent stability under plasma conditions, and its IL-8 neutralizing activity does not decrease after in vivo administration. In addition, H1009/L395, which is constructed based on Hr9, has a significantly improved production level compared to hWS-4. From the perspective of manufacturing level, it is an antibody suitable for manufacturing. Furthermore, in the in silico immunogenicity prediction, this antibody showed a very low score for immunogenicity, which is a significantly lower score compared to known hWS-4 antibodies and several other commercially available antibodies. It can be expected that H1009/L395 is not likely to produce ADA in humans and can be used safely for a long time. Therefore, compared with known anti-human IL-8 antibodies, H1009/L395 shows improvement in various aspects and is very useful as a pharmaceutical.

As mentioned above, H1009/L395 with the Fc region of native IgG is sufficiently useful; however, variants of H1009/L395 including a functionally improved Fc region may also be suitably used as antibodies with enhanced uses. Specifically, it can increase FcRn binding in acidic pH conditions to prolong plasma retention and maintain effects over the long term. Variants including the introduction of modifications in the Fc region to reduce the binding ability to FcγR can also be used as highly safe therapeutic antibodies to avoid unwanted immune cell activation and cytotoxic activity when administered to organisms. As such Fc variants, the use of F1886s or F1974m utilized here is particularly advantageous, but is not limited to such Fc variants; therapeutics including other modified Fc regions may be used as long as the Fc variant has similar functions. Antibodies serve as embodiments of Disclosure C.

Therefore, the C-revealing antibodies including H1009/L395-F1886s and H1009/L395-F1974m generated by the careful research of the present invention can maintain the following conditions: the biological activity of human IL-8 is strongly inhibited safely and long-term. Here, it is understood that known anti-IL-8 antibodies cannot achieve levels, and it is revealed that the antibody of C can be expected to be used as a high-quality finished anti-IL-8 antibody medicine. [Example 20]

Anti-factor IXa/factor X bispecific antibody The humanized anti-Factor IXa/Factor X bispecific antibody disclosed in WO2012/067176 binds to human Factor IXa and Factor X and induces blood co-aggregation activity. Utilize the humanized anti-Factor IXa/Factor :331)/Common L chain (SEQ ID NO:332)) In this embodiment, F8M includes 2 different H chains and 2 common L chains. F8M is manufactured according to the method described in the examples of WO2012/067176.

(20-1) Production of anti-factor IXa/factor X bispecific antibodies Based on F8M, the following three antibodies were produced according to the method of Reference Example 2 as anti-Factor IXa/Factor 323) with F8M-F1847mv2 (SEQ ID NO:324) as the heavy chain and F8ML (SEQ ID NO:325) as the light chain; (b) F8M-F1868mv, a conventional antibody, including F8M-F1868mv1 (SEQ ID NO :326) with 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, including F8M-F1927mv1 (SEQ ID NO:328) with F8M-F1927mv2 (SEQ ID NO:329) as the heavy chain and F8ML (SEQ ID NO:325) as the light chain.

The heavy chain sequence includes the same Fc variant sequence mentioned in Example 5 regarding increased FcRn binding and reduced rheumatoid factor binding, as follows:

[Table 29]

(20-2) Pharmacokinetic study of monoclonal antibodies, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv in Malay monkeys The pharmacokinetics of monoclonal antibodies, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were evaluated separately in male Malay monkeys following intravenous administration of single bolus doses of 0.6 mg/kg. The 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 are 29.3 days, 54.5 days, and 35.0 days, respectively. A PK study of F8M using Malay monkeys was conducted on different days at a dose of 6 mg/kg, and the results showed a half-life of 19.4 days. It was found that the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv are longer than those of F8M. This suggests that the half-life of the anti-Factor IXa/X bispecific antibody may be extended by the same modification of the Fc region sequence as described in Example 5.

[Table 30] [Example 21]

Assessment of IgE clearance from plasma using Fab variants with increased pI In order to increase the clearance of human IgE, in this example a pH-dependent antigen-binding antibody was used to evaluate pi-increasing substitutions of the Fab portion of the antibody. The method of adding amino acid substitutions to the antibody variable region to increase the pI is not particularly limited, but can be implemented using methods disclosed in WO2007/114319 or WO2009/041643. Amino acid substitutions introduced into the variable region are preferably ones that will decrease the number of negatively charged amino acids (such as aspartic acid and glutamic acid) and increase the number of positively charged amino acids (such as arginine and lysine) By. In addition, amino acid substitutions can be introduced at any position in the variable region of the antibody. Without particular limitation, the site for introducing amino acid substitution is preferably a position where the amino acid side chain may be exposed on the surface of the antibody molecule.

(21-1) Produce antibodies with increased isoelectric point values by modifying amino acids in the variable region The antibodies to be tested are summarized in Table 32 and Table 33.

Heavy chain Ab1H003 (also known as H003, SEQ ID NO:144) was obtained by introducing the pI-increasing substitution H32R into Ab1H (SEQ ID NO:38). Other heavy chain variants were also prepared by introducing each of the substitutions presented in Table 32 into Ab1H according to the method shown in Reference Example 1. All heavy chain variants were represented with Ab1L (SEQ ID NO:39) as the light chain. The pH-dependent binding status of this antibody is summarized in Table 5 (Ab1).

Likewise, we also evaluate pi-increasing substitutions in the light chain.

Light chain Ab1 L001T (also known as L001, SEQ ID NO:164) was prepared by introducing the pI-increasing substitution G16K into Ab1L. Other light chain variants can also be implemented by introducing the substitutions shown in Table 33 into Ab1L according to the method shown in Reference Example 1. All light chain variants were expressed with Ab1H as the heavy chain.

[Table 32]

[Table 33]

(21-2) Human FcγRIIb binding assay was performed using BIACORE using variants with increased pI. For produced antibodies containing Fc region variants, soluble human FcγRIIb and antigen were performed using binding assay using BIACORE T200 (GE Healthcare). -Binding between antibody complexes. Soluble human FcγRIIb (NCBI accession NM_004001.3) was prepared 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) using the His capture kit (GE Healthcare) by the amine coupling method to capture human FcγRIIb. The antibody-antigen complex is then injected with running buffer (as a reference solution) and interacts with human FcγRIIb captured on the sensor chip. Use 20 mM N-(2-acetamide)-2-aminoethanesulfonic acid, 150 mM NaCl, _1.2 mM CaCl, and 0.05% (w/v) Tween 20, pH 7.4, as running buffer, also with Each buffer dilutes soluble human FcγRIIb. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 25°C. The analysis was performed based on the calculated binding (RU) from the sensorgram obtained from the measurements and the relative value when the binding amount of Ab1H/Ab1L (raw Ab1) was defined as 1.00. For calculation of parameters, BIACORE T100 Evaluation Software (GE Healthcare) was used.

The SPR analysis results are summarized in Tables 32 and 33. Some variants showed improved binding to human FcγRIIb immobilized on BIACORE sensor chips.

Antibodies prepared by introducing a modification that increases the pI into the variable region of an antibody have a variable region that is more positively charged than before the modification was introduced. Therefore, it can be considered that the Coulomb interaction between the variable region (positive charge) and the surface of the sensing chip (negative charge) is strengthened by the modification by increasing pI. Furthermore, it is expected that such an effect will also occur on the surface of negatively charged cell membranes; therefore, it is expected that it will also show an effect of accelerating uptake into cells in vivo.

Here, this variant binds to hFcγRIIb about 1.2 times or more than the original Ab1 binds to hFcγRIIb, which is thought to be due to the strong charge effect of binding the antibody to hFcγRIIb on the sensor chip.

Among the heavy chain variants with increased pI, there were antibodies showing higher binding to hFcγRIIb alone or in combination with substitutions of Q13K, G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G or Q105R (according to Kabat numbering). Single amino acid substitutions or combinations of these substitutions in the heavy chain are hypothesized to have a strong charge effect on hFcγRIIb bound to the sensor chip. Therefore, one or more positions that are expected to accelerate the uptake into cells in vivo by introducing pI-increasing modifications to the antibody heavy chain variable region may include, for example, positions 13, 15, 64, and 64 according to Kabat numbering. 77, 82a, 82b, 85 and 105. The amino acid substitution introduced into such a position may be aspartic acid, glycine, serine, arginine or lysine, preferably arginine or lysine.

In pI-increased light chain variants bearing G16K, Q24R, A25R, S26R, E27R, Q37R, G41R, Q42K, S52K, S52R, S56K, S56R, S65R, T69R, T74K, S76R, S77R, Q79K, alone or in combination Substituted antibodies (according to Kabat numbering) showed higher binding to human FcγRIIb. Single amino acid substitutions or combinations of such substitutions in the light chain are hypothesized to have a strong charge effect on human FcγRIIb bound to the sensor chip. Therefore, one or more positions that are expected to accelerate uptake into cells in vivo by introducing pI-increasing modifications to the antibody light chain variable region may include, for example, positions 16, 24, 25, according to Kabat numbering. 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77, and 79. The amino acid substitution introduced into such a position may be arginine or lysine.

(21-3) pI-increased cellular uptake of antibodies containing Fab region variants To evaluate the rate of intracellular uptake into hFcγRIIb-expressing cell lines using manufactured antibodies containing Fab region variants, an assay similar to (4-5) above was performed, except that the amount of uptaken antigen was expressed relative to Ab1H/Ab1L (original Ab1 ) value (let it be 1.00) is represented by the relative value.

Quantitative results of cellular uptake are summarized in Tables 32 and 33. Strong fluorescence from this antigen in cells was observed in various Fc variants. Here, when the fluorescence intensity of the antigen taken up into the cells of this variant is about 1.5 times or more compared to the fluorescence intensity of the original Ab1, it is considered that the antigen enters the cell and has a strong charge effect.

Among the heavy chain variants with increased pI, antibodies with P41R, G44R, T77R, D82aN, D82aG, D82aS, S82bR or E85G substitutions (according to Kabat numbering) alone or in combination showed stronger antigen uptake into cells. Single amino acid substitution or combination substitution in the heavy chain is presumed to have a strong charge effect on the uptake of antigen-antibody complexes into cells. It is therefore expected that the introduction of pI-increasing modifications into the variable region of the antibody heavy chain will result in faster or greater uptake of the antigen-antibody complex into the cell at one or more positions, which may include, for example, positions 41, 44 according to Kabat numbering. , 77, 82a, 82b or 85. The amino acid substitution introduced into such a position may be aspartic acid, glycine, serine, arginine or lysine, preferably arginine or lysine.

Light chain variants with increased pI, alone or in combination, G16K, Q24R, A25R, A25K, S26R, S26K, E27R, E27Q, E27K, Q37R, G41R, Q42K, S52K, S52R, S56R, S65R, T69R, T74K, S76R, Antibodies with S77R or Q79K substitutions (according to Kabat numbering) show stronger antigen uptake into cells. Single amino acid substitutions or combinations of such substitutions in light chains are hypothesized to have a strong charge effect on the uptake of antigen-antibody complexes into cells. Variants with 4 or more amino acid substitutions tend to show stronger charge effects than variants with fewer amino acid substitutions. It is therefore expected that the introduction of pI-increasing modifications into the antibody light chain variable region will result in faster or greater uptake of the antigen-antibody complex into the cell at one or more positions, which may include, for example, positions 16, 24 according to Kabat numbering. , 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77 or 79. The amino acid substitution introduced into such a position may be glutamine, arginine or lysine, preferably arginine or lysine.

Although not limited to a particular theory, this result can be explained as follows: the antigen and antibody added to the cell culture medium form an antigen-antibody complex in the culture medium. This antigen-antibody complex binds to human FcγRIIb expressed on the cell membrane via the antibody Fc region, and is taken up into cells in a receptor-dependent manner. The antibody used in this experiment binds to the antigen in a pH-dependent manner; therefore, the antibody can dissociate from the antigen in intracellular endosomes (acidic pH conditions). Because the dissociated antigen will be transported to lysosomes and accumulated, it will fluoresce within the cell. Therefore, strong fluorescence intensity within cells is thought to indicate that uptake of antigen-antibody complexes into cells occurs faster or more.

(21-4) Evaluation of clearance rate of human IgE in mouse co-injection model Several anti-IgE antibodies with pH-dependent antigen binding (original Ab1, Ab1H/Ab1L013, Ab1H/Ab1L014, Ab1H/Ab1L007) were tested in a mouse coinjection model to evaluate their ability to accelerate clearance of IgE from plasma. In the coinjection model, C57BL6J mice (Jackson Laboratories) were administered IgE premixed with anti-IgE antibody as a single intravenous injection. All groups received 0.2 mg/kg IgE and 1.0 mg/kg anti-IgE antibodies. Total IgE plasma concentration was determined by anti-IgE ELISA. First, anti-human IgE (selected clone 107, MABTECH) was dispensed into a microwell plate (Nalge nunc International) and left to stand at room temperature for 2 hours or at 4°C overnight to prepare an anti-human IgE antibody immobilized plate. Samples against the standard curve and samples are mixed with an excess of anti-IgE antibodies (made in-house) to form a homogeneous structure of immune complexes. These samples were added to the anti-human IgE antibody-immobilized plate and allowed to remain at 4°C overnight. These samples were then reacted sequentially with human GPC3 nuclear protein (manufactured in-house), biotinylated anti-GPC3 antibody (manufactured in-house), and streptavidin Poly HRP80 conjugate (Stereospecific Detection Technologies) for 1 hour. Then SuperSignal (registered trademark) ELISA Pico chemiluminescent substrate (Thermo Fisher Scientific) was added. Chemiluminescence was read with SpectraMax M2 (Molecular Devices). Human IgE concentrations were calculated using SOFTmax PRO (Molecular Devices). Figure 37 depicts IgE plasma concentration time curve in C57BL6J mice.

Following administration of Fab variants with increased pI for pH-dependent antigen binding, total plasma IgE concentrations were lower than those of the original Ab1. These results show that this antigen-antibody immune complex with a high pI variant of pH-dependent antigen binding will bind more strongly to plasma membrane receptors such as FcγR, increasing cellular uptake of the antigen-antibody immune complex. This antigen taken into cells can be effectively released from the antibody in the endosome, accelerating the elimination of IgE. In vitro experiments showed weak potency in Ab1H/Ab1L007-treated mice with higher IgE concentrations than other antibodies containing Fab variants with increased pI. These results also imply that for the purpose of estimating the antigen clearance rate in plasma in vivo, the sensitivity of the in-vitro system using the above-mentioned fluorescence intensity of InCell Analyzer 6000 will be higher than the sensitivity of the above-mentioned in-vitro BIACORE system. [Example 22]

Assessment of C5 clearance from plasma using pI-increased Fab variants To enhance the clearance of human IgE, a pH-dependent antigen-binding antibody was used in this example to evaluate pi-increasing substitutions in the Fab portion of the antibody.

(22-1) Preparation of C5 [Performance and Purification of Recombinant Human C5] Recombinant human C5 (NCBI GenBank accession number: NP_001726.2, SEQ ID NO:207) was transiently expressed using FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). The adjusted culture medium expressing human C5 was diluted with an equal volume of milliQ water, then applied to a Q-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden), and then eluted with a NaCl gradient. The human C5-containing fractions were pooled, and the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4 respectively. The obtained sample was applied to an SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. The human C5-containing fractions were combined and applied to a CHT ceramic hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 eluate was applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). The human C5-containing fractions were combined and stored at -150°C. In-house prepared recombinant human C5 or plasma derived from human C5 (CALBIOCHEM, Cat#204888) was used in this study.

Recombinant Malay monkey C5 (NCBI GenBank Accession Number: XP_005580972, SEQ ID NO:208) was expressed and purified in exactly the same manner as the human part.

(22-2) Preparation of synthetic calcium library (calcium libary) A gene library of antibody heavy chain variable regions was used as a synthetic human heavy chain library consisting of 10 heavy chain libraries. The germ line frameworks VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59, VH4-61 were selected based on their frequency in the human B cell inventory and the biophysical properties of the V gene family. , VH4-b, VH5-51, and VH6-1 are libraries. The synthetic human heavy chain repertoire contains diversity in antibody binding sites that mimics the human B cell antibody repertoire.

Design a gene library of antibody light chain variable regions with calcium-binding motifs and diversity in positions that may contribute to antigen recognition, as compared to a human B-cell antibody library. The design of a gene library of antibody light chain variable regions exhibiting properties for calcium-dependent binding to antigens is described in WO 2012/073992.

Insert the combination of the heavy chain variable region library and the light chain variable region library into the phagemid vector and construct the phage library, refer to (de Heard et al., Meth. Mol. Biol. 178:87-100 (2002) )). The trypsin cleavage site is introduced into the linker region between Fab and pill protein in the phagemid vector. A modified M13KO7 helper phage with a trypsin cleavage site between the N2 and CT domains of gene III was used to prepare phage for Fab presentation.

(22-3) Isolated calcium-dependent anti-C5 antibody The phage display library was diluted in TBS supplemented with BSA and CaCl ₂ to a final concentration of 4% and 1.2 mM respectively. As a panning method, the commonly known magnetic bead selection method was used according to the general experimental procedures (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 Proteomics 2 (2):61-69 (2003). Use NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin) for magnetic beads. Human C5 (CALBIOCHEM , Cat #204888) labeled with EZ-Link NHS-PEG4-Biotin (PIERCE, Cat No. 21329).

To initiate a round of phage selection, the phage display library was incubated with biotinylated human C5 (312.5 nM) for 60 minutes at room temperature. Magnetic beads are then used to capture phage exhibiting Fab-binding variants.

After incubating with the magnetic beads at room temperature for 15 minutes, wash the magnetic beads three times with 1 mL TBS containing 1.2 mM CaCl ₂ and 0.1% Tween20, and then wash the magnetic beads twice with 1 mL TBS _containing 1.2 mM CaCl 2. Phage were eluted by suspending the beads in TBS containing 1 mg/mL trypsin for 15 minutes. The eluted phage were infected with ER2738 and rescued with helper phage. The rescued phages were precipitated with polyethylene glycol, resuspended in TBS supplemented with BSA and CaCl ₂ at a final concentration of 4% and 1.2 mM, and used for the next round of panning.

After the first round of panning, phage were selected in a calcium-dependent manner, with this antibody binding more strongly to C5 in the presence of calcium ions. Panning in rounds 2 and 3 was performed in the same manner as in round 1, except for the following differences: 50 nM (round 2) or 12.5 nM (round 3) biotinylated antigen was used, and the final Elute with 0.1 mL of elution buffer (50mM MES, 2mM EDTA, 150mM NaCl, pH5.5) and contact with 1 μL of 100 mg/mL trypsin to select for calcium dependence. After selection, the selected phage clones are converted into IgG format.

The binding ability of converted IgG antibodies to human C5 was evaluated under 2 different conditions: binding and dissociation at 1.2 mM CaCl ₂ -pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl ₂ ), and binding at 1.2 mM CaCl ₂ -pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl ₂ ) and dissociated in 3 μM CaCl ₂ -pH 5.8 (20 mM MES, 150 mM NaCl, 3 μM CaCl ₂ ) at 30°C using the Octet RED384 system (Pall Life Sciences). A total of 25 pH-calcium-dependent antigen-binding colonies were isolated. The induction pattern of these antibodies is shown in Figure 38.

(22-4) Identification of anti-C5 bispecific antibodies From the clones isolated in Example B-3, 9 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 using methods known to those skilled in the art to improve the properties of the antibody, such as physicochemical properties. The CFP0016 variant, CFP0016H019, was used for further analysis instead of CFP0016. The amino acid sequences of the VH and VL regions of these nine antibodies are reported in Table 34. In this table, names enclosed in parentheses represent abbreviated names.

[Table 34]

Full-length genes encoding the nucleotide sequences of antibody heavy and light chains are synthesized and prepared according to general methods known to those skilled in the art. Heavy chain and light chain expression vector systems are prepared by inserting the obtained plasmid fragments into vectors for expression in mammalian cells. The obtained expression vectors are sequenced according to general methods known to those skilled in the art. For antibody expression, the prepared plasmids were temporarily transfected into FreeStyle293-F cell line (Thermo Fisher Scientific). Purification of expressed antibodies from the conditioned culture medium was performed using rProtein A Sepharose Fast Flow (GE Healthcare) in a general manner known to those skilled in the art.

(22-5) Generation and characterization of pH-dependent anti-C5 bispecific antibodies Bispecific antibodies that recognize two different epitopes of C5 are produced using a combination of CFP0020 and CFP0018. The bispecific antibody system uses general methods known to those skilled in the art to prepare an IgG format with two different clones of the Fab at each binding site of the antibody. In this bispecific IgG antibody, the two heavy chains include different heavy chain constant regions (G1dP1, SEQ ID NO: 227 and G1dN1, SEQ ID NO: 228) to efficiently form the two heavy chains. Yuan Ti. Anti-C5 bispecific antibodies that include the binding sites of anti-C5 MAb "X" and anti-C5 MAb "Y" are represented by "X//Y".

By introducing some amino acid substitutions into the heavy chain and light chain CDRs using general methods known to those skilled in the art, we obtained the light chain consensus variant of 20//18, named 'optimized 20 //18' (consisting of 2 heavy chains: CFP0020H0261-G1dP1, SEQ ID NO:229 and CFP0018H0012-G1dN1, SEQ ID NO:230, and a common light chain: CFP0020L233-k0, SEQ ID NO:231).

Kinetic parameters of optimal 20//18 against recombinant human C5 were evaluated using a BIACORE T200 instrument (GE Healthcare) at 37°C under 2 different conditions (e.g. (A) binding and dissociation at pH 7.4, (B) binding at pH 7.4 , dissociates at pH 5.8). Protein A/G (Pierce, Cat No. #21186) or anti-human IgG (Fc) antibody (in human antibody capture kit; GE Healthcare, Cat No. BR-1008-39) was immobilized using amine coupling. Series S CM4 (GE Healthcare, Cat No. BR-1005-34). Anti-C5 antibodies are captured on immobilized molecules, and human C5 is then injected. The running buffer used was ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20). Kinetic parameters for both pH conditions were determined by fitting sensorgrams to a 1:1 binding-RI (without substantial effect adjustment) model using BIACORE T200 evaluation software, version 2.0 (GE Healthcare). The kinetic parameters, namely the association rate (ka), dissociation rate (kd) and binding affinity (KD) at pH 7.4, and the dissociation rate (kd) determined only by calculating the dissociation phase at each pH condition, are recorded in Table 35 . Optimized at 20//18, the dissociation rate of human C5 at pH 5.8 is faster than the dissociation rate at pH 7.4.

[Table 35]

(22-6) Modify the amino acids in the variable region to produce antibodies with increased isoelectric point values The antibodies tested are summarized in Tables 36 and 37.

The heavy chain, CFP0020H0261-001-G1dP1 (also known as 20H001, SEQ ID NO:232), was prepared by introducing P41R/G44R with increased pI in place of CFP0020H0261-G1dP1 (SEQ ID NO:229). Likewise, the heavy chain, CFP0018H0012-002-G1dN1 (also known as 18H002, SEQ ID NO:251), was prepared by introducing T77R/E85R with increased pI substitution into CFP0018H0012-G1dN1 (SEQ ID NO:230). Other heavy chain variants were also prepared by introducing each of the substitutions shown in Table 36 into CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1 according to the method shown in Reference Example 1. The CFP0020H0261-G1dP1 variant was expressed together with two heavy chain variants of the CFP0018H0012-G1dN1 variant and CFP0020L233-k0 (SEQ ID NO:231) as the light chain to obtain a bispecific antibody.

Likewise, we also evaluate substitutions that increase the pi of the light chain. The light chain, CFP0020L233-001-k0 (also known as 20L233-001, SEQ ID NO:271) was prepared by introducing the pI-increasing substitution G16K into CFP0020L233-k0. Other light chain variants were also prepared by introducing each of the substitutions shown in Table 37 into CFP0020L233-k0 according to the method shown in Reference Example 1. All light chain variants and CFP0020H0261-G1dP1 as heavy chain were expressed together with CFP0018H0012-G1dN1 to obtain bispecific antibodies.

[Table 36]

[Table 37]

(22-7) Binding analysis of human FcγRIIb using BIACORE using variants with increased pI Regarding the produced antibodies containing Fc region variants, binding analysis of soluble hFcγRIIb and antigen-antibody complexes was performed using BIACORE T200 (GE Healthcare). Soluble hFcyRIIb is formed as a His-tagged molecule according to methods known in the art. An appropriate amount of anti-His antibody was immobilized on the sensor chip CM5 (GE Healthcare) using the His capture kit (GE Healthcare) using the amine coupling method to capture hFcγRIIb. The antibody-antigen complex is then injected with running buffer (as a reference solution), allowing it to interact with hFcγRIIb captured on the sensor chip. Use 20 mM N-(2-acetamide)-2-aminomethanesulfonic acid, 150 mM NaCl, _1.2 mM CaCl, and 0.05% (w/v) Tween 20, pH 7.4 as running buffer and use each Buffer to dilute soluble hFcγRIIb. To regenerate the sensor chip, use 10 mM glycine-HCl at pH 1.5. All measurements were performed at 25°C, and the analysis was performed based on calculated binding (RU) from the measured sensorgrams, showing the binding amount defined as 1.00 for CFP0020H0261-G1dP1/ CFP0018H0012-G1dN1/ CFP0020L233-k0 (original Ab2) Relative value. To calculate parameters, BIACORE T100 evaluation software (GE Healthcare) was used.

The SPR analysis results are summarized in Tables 36 and 37. Some variants showed improved binding to hFcγRIIb immobilized on the BIACORE sensor chip. When this variant binds hFcγRIIb about 1.2-fold or more compared to the original Ab2 binding to hFcγRIIb, the antibody is considered to have a strong charge effect on the binding of hFcγRIIb on the sensor chip.

Among the heavy chain variants with increased pI, antibodies with L63R, F63R, L82K or S82bR substitutions (according to Kabat numbering) showed higher binding to hFcγRIIb. Single amino acid substitutions or combinations of such substitutions in the heavy chain are hypothesized to have a strong charge effect on the binding of hFcγRIIb on the sensor chip. It is therefore expected that introduction of pi-increasing modifications into the antibody heavy chain variable region will result in faster or greater uptake into cells at one or more positions in vivo, which may include, for example, positions 63, 82 or 82b according to Kabat numbering. The amino acid substitution introduced into such a position may be arginine or lysine.

Antibodies with G16K, Q27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (according to Kabat numbering) showed higher binding to hFcγRIIb in light chain variants with increased pI. Single amino acid substitutions in the light chain or combinations of such substitutions are thought to have a strong charge effect on the binding of human FcγRIIb on the sensor chip. Therefore, one or more positions that are expected to result in faster or greater in vivo uptake into cells by introducing pI-increasing modifications into the variable region of the antibody light chain may include, for example, positions 16, 27, 41, according to Kabat numbering. 52, 56, 65, 69, 74, 76, 77 or 79 position. The amino acid substitution introduced into such a position may be arginine or lysine. Variants with 4 or more amino acid substitutions tend to show stronger charge effects than variants with fewer amino acid substitutions.

(22-8) pI-increased cellular uptake of antibodies containing Fab region variants To assess the rate of intracellular uptake into hFcγRIIb-expressing cell lines using manufactured antibodies containing Fab region variants, the following analysis was performed.

MDCK (Madin-Darby canine kidney) cell lines that continuously express hFcγRIIb were produced according to known methods. These cells are used to assess intracellular uptake of antigen-antibody complexes. Specifically, Alexa555 (Life Technologies) was used to label human C5 according to established experimental procedures, and an antigen-antibody complex was formed in a culture medium with an antibody concentration of 10 mg/mL and an antigen concentration of 10 mg/mL. The culture medium containing the antigen-antibody complex was added to the culture plate of the above-mentioned MDCK cells that continuously expressed hFcγRIIb, incubated for 1 hour, and then the fluorescence intensity of the antigen absorbed into the cells was quantified using InCell Analyzer 6000 (GE healthcare). The amount of antigen ingested is expressed relative to the original Ab2 value, which was taken as 1.00.

Quantitative results of cellular uptake are shown in Tables 36 and 37. Strong fluorescence from this antigen in cells was observed in some heavy and light chain variants. Here, when the fluorescence intensity of the cells in which the antigen is taken up into the variant is about 1.5 times or more compared to the fluorescence intensity of the original Ab2, it is considered that the antigen is taken up into the cells and there is a strong charge effect.

Among the heavy chain variants with increased pI, those with G8R, L18R, Q39K, P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN, S82bR, T83R, A85R or E85G substitutions (according to Kabat numbering) Antibodies show strong uptake of antigen into cells. Single amino acid substitutions or combinations of such substitutions in the heavy chain are hypothesized to have a strong charge effect on the uptake of antigen-antibody complexes into cells. It is therefore expected that by introducing modifications that increase the pI into the variable region of the antibody heavy chain, one or more positions that result in faster or greater uptake of the antigen-antibody complex into the cell may include, for example, positions 8, 18 according to Kabat numbering. , 39, 41, 44, 63, 64, 77, 82, 82a, 82b, 83 or 85. The amino acid substitution introduced into such a position may be aspartic acid, glycine, serine, arginine or lysine and is preferably arginine or lysine.

Among the pI-increased light chain variants, antibodies with G16K, Q27R, S27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (according to Kabat numbering) showed stronger antigen uptake Enter the cell. Single amino acid substitutions or combinations of such substitutions in light chains are hypothesized to have a strong charge effect on the uptake of antigen-antibody complexes into cells. Variants with 4 or more amino acid substitutions tend to have a stronger charge effect than variants with fewer amino acid substitutions. As in Example 21, (21-3), the combination of 42K and 76R substitution is effective in the IgE antibody system. In the case of the C5 antibody, the amino acid at position 42 of the Kabat numbering is already lysine, so one can observe the charge effect of 42K/76R by a single substitution of 76R. The fact that the variant with 76R substitution has a strong charge effect shows that regardless of the antigen, the C5 antibody also shows a strong charge effect of the 42K/76R combination. It is therefore expected that introduction of modifications that increase the pI into the variable region of the antibody light chain will result in faster or greater uptake of the antigen-antibody complex into the cell at one or more positions, which may include, for example, positions 16, 27 according to Kabat numbering. , 41, 52, 56, 65, 69, 74, 76, 77 or 79. The amino acid substitution introduced into such a position may be arginine or lysine.

(22-9) Evaluate the clearance rate of C5 in mouse co-injection model Several anti-C5 bispecific antibodies (original Ab2, 20L233-005, 20L233-006, and 20L233-009) were tested in a mouse coinjection model to assess the ability to accelerate clearance of C5 from plasma. In the coinjection model, C5 premixed with an anti-C5 bispecific antibody was administered as a single intravenous injection to C57BL6J mice (Jackson Laboratories). All groups received 0.1 mg/kg C5 and 1.0 mg/kg anti-C5 bispecific antibody. Total C5 plasma concentration was determined by anti-C5 ELISA. First, anti-human C5 mouse IgG was dispensed onto ECL plates and incubated at 5°C overnight to prepare anti-human C5 mouse IgG immobilized plates. Mix the sample for the standard curve and the sample with anti-human C5 rabbit IgG. Add these samples to the anti-human C5 mouse IgG immobilized plate and leave it at room temperature for 1 hour. These samples were then reacted with anti-rabbit IgG (Jackson Immuno Research) conjugated to HRP. After the plate was incubated for 1 hour at room temperature, conjugated thio-labeled anti-HRP antibody was added. The ECL signal was read with Sector Imager 2400 (Meso Scale discovery). Human C5 concentration was calculated from the ECL signal in the standard curve using SOFTmax PRO (Molecular Devices). Figure 39 depicts C5 plasma concentration time curve in C57BL6J mice.

All of the pi-increased substituted bispecific antibodies tested in this study demonstrated rapid C5 clearance from plasma compared to the original Ab2. Therefore, it is suggested that the T74K/S77R, S76R/Q79K and Q37R amino acid substitutions in the light chain will also accelerate the elimination of C5-antibody immune complexes in vivo. In addition, the elimination of C5 in 20L233-005 and 20L233-006 was faster than that in 20L233-009, which was consistent with in vitro angiography and BIACORE analysis. These results suggest that even in vivo, in vitro systems using the InCell Analyzer 6000 to measure fluorescence intensity, or in the in vitro BIACORE system described above at locations that appear unlikely to contribute to antigen clearance from plasma, more sensitive in vivo The system finds contributors. These results also suggest that in order to estimate the clearance rate of antigens cleared from plasma in vivo, the sensitivity of the in-vitro system using the fluorescence intensity of the above-mentioned InCell Analyzer 6000 may be higher than that of the above-mentioned in-vitro BIACORE system. [Example 23]

Assessment of IgE clearance in plasma using pI-increased Fc variants To enhance clearance of human IgE or human C5, pH-dependent antibodies were used to evaluate pi-increasing substitutions in the Fc portion of the antibodies. The method of adding amino acid substitutions to the antibody constant region to increase the pI is not particularly limited. For example, it can be implemented according to the method described in WO2014/145159.

(23-1) Production 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 Ab1H. Other heavy chain variants were also prepared by introducing each of the substitutions shown in Table 38 into Ab1H according to the method shown in Reference Example 1. All heavy chain variants are represented, as well as Ab1L as the light chain.

[Table 38]

(23-2) Human FcγRIIb binding assay using BIACORE using antibodies containing Fc region variants with increased pI In order to evaluate the charge effect of the formed antigen-antibody complex on FcRγRIIb binding using the antibodies described in Table 38, FcRγRIIb binding analysis was performed in a manner similar to that described in Example 21 and (21-2). The analysis results are shown in Table 38. Here, when the variant binds to hFcγRIIb about 1.2 times or more than the original Ab1 binds to hFcγRIIb, it can be considered that the antibody binds to hFcγRIIb on the sensor chip to have a strong charge effect.

Among the variants with a single amino acid substitution and an increased pI relative to the original Ab1, some variants such as P1398m, P1466m, P1482m, P1512m, P1513m and P1514m formed antigen-antibody complexes with the highest binding to hFcγRIIb. D413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K or G402K single amino acid substitution is speculated to have a strong charge effect on the binding of hFcγRIIb on the sensor chip. Therefore, one or more positions that are expected to result in accelerated uptake into cells by introducing pI-increasing modifications into the antibody constant region or Fc region may include, for example, positions 311, 343, 384, 401, 402, or 413 according to Kabat numbering. According to EU numbering law. The amino acid substitution introduced into such a position may be arginine or lysine.

(23-3) pI-increased cellular uptake of antibodies containing Fc region variants In order to evaluate the intracellular uptake of the antigen-antibody complex formed by the antibodies described in Table 38, a cytography analysis method similar to that described in Example 21 and (21-3) was performed. The analysis results are shown in Table 38. Here, compared to the fluorescence intensity of the original Ab1, the variant in which the antigen enters the cells has a fluorescence intensity of about 1.5 times or more, which is considered to have a strong charge effect on the antigen entering the cells.

Among the variants with single amino acid substitutions with increased pI relative to the original Ab1, some variants such as P1398m, P1466m, P1469m, P1470m, P1481m, P1482m, P1483m, P1512m, P1513m and P1653m showed stronger antigen uptake into cells. Single amino acid substitutions at D413K, Q311R, N315K, N384R, Q342K, P343R, P343K, D401R, D401K or D413R are presumed to have a strong charge effect on the uptake of antigen-antibody complexes into cells. Therefore, positions expected to result in faster or more uptake of the antigen-antibody complex into cells by introducing pI-increasing modifications into the constant region or Fc region of the antibody may include, for example, positions 311, 315, 342, and 342 according to EU numbering. 343, 384, 401 or 413. The amino acid substitution introduced into such a position may be arginine or lysine.

(23-4) Evaluating the clearance rate of human IgE in a mouse co-injection model Several anti-IgE antibodies with pH-dependent antigen binding (original Ab1, P1466m, P1469m, P1470m, P1480m, P1482m, P1512m, P1653m) were tested in a mouse coinjection model to evaluate their ability to accelerate the clearance of IgE from plasma. The analysis method was carried out in a manner similar to Example 21, (21-4). Figure 40 depicts plasma concentration time curves in C57BL6J mice.

After administration of high pI variants (only single amino acid substitutions) with pH-dependent antigen binding, total plasma IgE concentrations were lower than the original concentrations except for P1480m. P1480m, both in vitro studies showed weak efficacy and failed to accelerate the elimination of IgE. In mice treated with high pI variants without pH-dependent antigen binding, total plasma IgE concentrations were significantly higher than those with pH-dependent antigens. Binding high pI variants (data not shown). These results show that cellular uptake of antigen-antibody immune complexes is increased by introducing modifications that increase pi. Antigens that are taken up into cells and complexed with pH-dependent antigen-binding antibodies can be efficiently released from the antibodies in endosomes, thereby accelerating the elimination of IgE. These results suggest that even though the substitution positions examined by the in vitro BIACORE system described above do not appear to contribute to the clearance of antigens from plasma in vivo, contributors can still be found using more sensitive in vivo systems. These results also suggest that in order to estimate the clearance rate of antigens cleared from plasma in vivo, the sensitivity of measuring fluorescence intensity using the above-mentioned InCell Analyzer 6000 in vitro system may be higher than that in the above-mentioned in vitro BIACORE system.

Reference Example 1 Expression vector for constructing amino acid-substituted IgG antibodies Mutants were prepared using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) using the method described in the accompanying instruction manual. The plasmid fragment containing this mutant is inserted into the animal cell expression vector to construct the desired H chain and L chain expression vector. The nucleotide sequence of the obtained expression vector is determined according to methods known in this technical field.

Reference Example 2 Expression and Purification of IgG Antibodies Antibodies were expressed using the following method. The HEK293H cell line (Invitrogen) derived from human embryonic kidney cancer cells was suspended in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). Cells were seeded at 10 mL per dish at a density of 5 to 6 x ¹⁰ cells/mL for adherent cells (10 cm in diameter; CORNING) and cultured in a _CO2 incubator (37°C, 5% _CO2 ) for 1 sky. The medium was then aspirated to remove and 6.9 mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plastids were introduced into cells by lipofection method. The obtained culture supernatant was collected, centrifuged (approximately 2,000 g, 5 minutes, room temperature) to remove cells, and filtered through a 0.22-μm filter MILLEX (registered trademark)-GV (Millipore) to obtain the supernatant. Antibodies were purified from the culture supernatants obtained using rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) using methods known in the art. To determine the concentration of the purified antibody, the absorbance at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated from the measured value using the absorbance coefficient according to the method described in Pace et al., Protein Science 4:2411-2423 (1995).

Reference Example 3 Preparation of soluble human IL-6 receptor Recombinant soluble human IL-6 receptor as an antigen was prepared according to the following method. Methods known in this technical field are used to construct a CHO cell line that continuously expresses soluble human IL-6 receptor. The expressed soluble human IL-6 receptor consists of the amino acid sequence of amino acids No. 1 to 357 from the N-terminus. As reported by Mullberg et al., J. Immunol. 152:4958-4968 (1994). Soluble human IL-6 receptor was expressed by culturing this CHO cell line. Soluble human IL-6 receptor was purified from the culture supernatant of the obtained CHO cell line in two steps: Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. Use the fraction that eluted the main peak in the final step as the final purified product.

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Figure 1 shows changes in human IL-6 receptor concentration in the plasma of human FcRn transgenic mice administered human IL-6 receptor-binding antibodies, which bind to human IL-6 receptors in a pH-dependent manner. IL-6 receptor, and its constant region is natural IgG1 (Low_pI-IgG1), or an antibody is administered, and the isoelectric point value of the variable region of the antibody is increased (High_pI-IgG1). Figure 2 shows changes in human IL-6 receptor concentration in plasma of human FcRn gene transgenic mice that were individually administered with human IL-6 receptor-binding antibodies. The antibodies were pH-dependent. Binds to the human IL-6 receptor in a sexual manner and confers binding to FcRn under neutral pH conditions (Low_pI-F939); and administers an antibody that increases the isoelectric point value of the variable region of the antibody (Middle_pI-F939 , High_pI-F939). Figure 3 shows changes in human IL-6 receptor concentration in plasma of human FcRn gene transgenic mice that were individually administered with human IL-6 receptor-binding antibodies. The antibodies were pH-dependent. Binds to the human IL-6 receptor in a sexual manner, and its FcγR binding increases under neutral pH conditions (Low_pI-F1180), and the antibody is administered, and the isoelectric point value of the variable region of the antibody increases (Middle_pI-F1180 , High_pI-F1180). Figure 4 shows that the concentration of human IL-6 receptor in the plasma of human FcRn gene transgenic mice changes, and the concentration of soluble human IL-6 receptor in the plasma remains at a steady state. The human FcRn gene transgenic mice individually A human IL-6 receptor-binding antibody was administered, which binds to the human IL-6 receptor in a pH-dependent manner, and its constant region is natural IgG1 (Low_pI-IgG1); and an antibody was administered, which Comprising an Fc region variant, wherein the Fc region in the antibody has increased FcRn binding under neutral pH conditions (Low_pI-F11); and administering the antibody at an isoelectric point value of the variable region of the antibody Increase (High_pI-IgG1, High_pI-F11). Figure 5 shows the extracellular matrix binding degree of each of three types of antibodies with different isoelectric point values (Low_pI-IgG1, Middle_pI-IgG1 and High_pI-IgG1) that bind to human IL-6 receptor in a pH-dependent manner. And the extracellular matrix binding degree of two types of antibodies with different isoelectric point values (Low_pI(NPH)-IgG1 and High_pI(NPH)-IgG1) that do not bind to human IL-6 receptor in a pH-dependent manner. Within the context of Disclosure A described herein, "NPH" means independent of pH. Figure 6 shows the relative range of soluble human FcγRIIb binding (measured with BIACORE (registered trademark)) of an antibody that includes an Fc region variant whose isoelectric point has been modified by modifying the constant region of the Ab1H-P600 antibody. The antibody binds to IgE in a pH-dependent manner. Ab1H-P600 is set to 1.00. Figure 7 shows the relative rate of uptake of an antibody into cells of an hFcγRIIb-expressing cell line. The antibody includes an Fc region variant whose isoelectric point has been modified by modifying one amino acid in the constant region of the Ab1H-P600 antibody. increase in residues. Ab1H-P600 is set to 1.00. Figure 8 shows the degree of binding of Fv4-IgG1 with the Fc region of native human IgG1 to rheumatoid factors in the serum of each RA patient. Figure 9 shows the extent of Fv4-YTE binding of Fc region variants with increased FcRn binding to rheumatic factors in the sera of various RA patients. Figure 10 shows the extent of binding of Fv4-LS with Fc region variants with increased FcRn binding to rheumatic factors in the sera of various RA patients. Figure 11 shows the extent of binding of the Fc region variant Fv4-N434H with increased FcRn binding to rheumatoid factors in the sera of various RA patients. Figure 12 shows the extent of binding of Fv4-F1847m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the sera of various RA patients. Figure 13 shows the extent of binding of Fv4-F1848m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the sera of various RA patients. Figure 14 shows the extent of binding of the Fc region variant Fv4-F1886m with increased FcRn binding to rheumatoid factors in the sera of various RA patients. Figure 15 shows the extent of binding of Fv4-F1889m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the sera of various RA patients. Figure 16 shows the extent of binding of Fv4-F1927m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the sera of various RA patients. Figure 17 shows the extent of binding of the Fc region variant Fv4-F1168m with increased FcRn binding to rheumatoid factors in the sera of various RA patients. Figure 18 shows the average binding of Fv4-IgG1 to rheumatoid factors in the serum of each RA patient. Fv4-IgG1 has the Fc region of natural human IgG1, and each of these antibodies includes novel Fc region variants. This Fc Regions have Fc region variants that increase binding for each FcRn. Figure 19 shows the concentration changes of anti-human IgE antibodies in the plasma of Malay monkeys, which were administered OHB-IgG1, which is an anti-human IgE antibody and has the Fc region of natural human IgG1. Each of these antibodies has a novel Fc region. Variants, each Fc region has an Fc region variant with increased binding to FcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m). Figure 20 shows the concentration changes of anti-human IL-6 receptor antibodies in the plasma of human FcRn gene transgenic mice, which were administered Fv4-IgG1, which is an anti-human IL-6 receptor antibody and has natural human IgG1. Fc region, or administration of Fv4-F1718, which increases FcRn binding under acidic pH conditions. Figure 21 shows the sensogram of IL-8 on the binding of H998/L63 and Hr9 measured by Biacore at pH 7.4 and pH 5.8. Figure 22 shows changes in the concentration of human IL-8 in the plasma of mice when H998/L63 or H89/L118 was administered as a mixture with human IL-8 at 2 mg/kg. Figure 23 shows the changes in human IL-8 concentration in the plasma of mice when H89/L118 was administered at 2 mg/kg or 8 mg/kg and human IL-8 as a mixture. Figure 24 shows changes in the concentration of human IL-8 in the plasma of mice when H89/L118 or H553/L118 was administered as a mixture with human IL-8 at 2 mg/kg or 8 mg/kg. Figure 25A shows the relative value changes in the concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H553/L118 before storage in plasma. Figure 25B shows the relative value changes in the concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H553/L118 after being stored in plasma for 1 week. Figure 25C shows the relative value changes in the concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H553/L118 after being stored in plasma for 2 weeks. Figure 26 shows the predicted frequency of ADA predicted by EpiMatrix for each anti-IL-8 antibody (hWS4, Hr9, H89/L118, H496/L118 or H553/L118) and for other pre-existing therapeutic antibodies. Predicted frequency of ADA occurrence. Figure 27 shows the predicted frequency of ADA for each anti-IL-8 antibody (H496/L118, H496v1/L118, H496v2/L118, H496v3/L118, H1004/L118 or H1004/L395) predicted by EpiMatrix, and Predicted frequency of ADA against other pre-existing therapeutic antibodies. Figure 28A shows the relative value changes in the concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H1009/L395-F1886 before storage in plasma. Figure 28B shows the relative value changes in the concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H1009/L395-F1886 after being stored in plasma for 1 week. Figure 28C shows the relative value changes in the concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H1009/L395-F1886 after being stored in plasma for 2 weeks. Figure 29 shows changes in the concentration of human IL-8 in the plasma of mice when H1009/L395, H553/L118 and H998/L63 are administered as a mixture with human IL-8. Figure 30 shows the extent of extracellular matrix binding when Hr9, H89/L118 or H1009/L395 are added to the extracellular matrix alone and as a mixture with human IL-8. Figure 31 shows that when the antibody containing the variable region of H1009/L395 and the Fc region that does not bind to FcRn (F1942m) is administered alone or in a mixture with human IL-8 to human FcRn gene transgenic mice, the Changes in antibody concentration in mouse plasma. Figure 32 shows the predicted frequency of ADA predicted by EpiMatrix against H1009/L395 and H1004/L395, and the predicted frequency of ADA against other pre-existing therapeutic antibodies. Figure 33 shows the concentration changes of each anti-human IL-8 antibody in the plasma of Malay monkeys, which were administered H89/L118-IgG1, which has the variable region of H89/L118 and the Fc region of natural human IgG1, and each antibody has For Fc region variants with increased FcRn binding (H89/L118-F1168m, H89/L118-F1847m, H89/L118-F1848m, H89/L118-F1886m, H89/L118-F1889m and H89/L118-F1927m). Figure 34 shows the binding of an antibody that has the variable region of H1009/L395 and whose Fc region is a variant (F1886m, F1886s or F1974m) directed against each FcγR. Figure 35 shows changes in human IL-8 concentration in mouse plasma when human FcRn gene transgenic mice were administered a mixture of anti-IL-8 antibody and human IL-8. In this case, the anti-IL-8 antibody is H1009/L395-IgG1 (2 mg/kg), including the variable region of H1009/L395 and the Fc region of native human IgG1, or H1009/L395-F1886s (2, 5 or 10 mg/kg), including the variable region and modified Fc region of H1009/L395. Figure 36 shows the changes in antibody concentration in the plasma of Malay monkeys administered Hr9-IgG1 or H89/L118-IgG1, both of which include the Fc region of native human IgG1, or administered H1009/L395-F1886s or H1009/L395-F1974m, both include modified Fc regions. Figure 37 shows IgE plasma concentration time profiles for some anti-IgE antibodies in C57BL6J mice with respect to antibody variable region modifications. Figure 38 (Figures 38A-38D) shows the Octet sensorgrams of selected 25 [25] pH-dependent and/or calcium-dependent antigen-binding selections. Figure 38B is a continuation of Figure 38A. Figure 38C is a continuation of Figure 38B. Figure 38D is a continuation of Figure 38C. Figure 39 shows C5 plasma concentration time profiles for some anti-C5 bispecific antibodies in C57BL6J mice with respect to antibody variable region modifications. Figure 40 shows IgE plasma concentration time profiles for some anti-IgE antibodies in C57BL6J mice with respect to antibody constant region modification.

Claims (1)

An isolated anti-IL-8 antibody as described in the instructions and drawings.

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