TW202214688A - 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 uses thereof

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

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 the treatment of, eg, IL-8-related disorders.

Antibodies are highly stable in plasma and have few side effects, so they have been valued as pharmaceuticals. Among them, most of the therapeutic antibodies of IgG type have been marketed, and most of them 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 techniques have been developed in terms of techniques applicable to second-generation therapeutic antibodies, including techniques for enhancing effector function, antigen-binding capacity, pharmacokinetics or stability, and reducing the risk of immunogenicity. Technology (Kim et al., Mol. Cells. 20(1):17-29 (2005)). The amount of therapeutic antibodies administered is usually very high, so the development of therapeutic antibodies has encountered difficulties such as preparation of subcutaneous administration formulations and high manufacturing costs. Methods for improving the pharmacokinetics, pharmacodynamics, and antigen-binding properties of therapeutic antibodies provide avenues that can be used to reduce the dosage and manufacturing costs associated with therapeutic antibodies.

Substitution of amino acid residues in the constant region 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)). The technique of affinity maturation provides a method for enhancing 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 antigen-binding activity due to amino acid introduction mutations in the CDRs and/or framework regions of antibody variable domains. Improving the antigen-binding properties of the antibody may improve the biological activity of the antibody in vitro or reduce the dose, and may further improve in vivo (in vivo) efficacy (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 that 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 various known methods (see, eg, Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471 (2005)). Furthermore, if the antigen can be covalently bonded and the affinity is infinite, one antigen molecule can theoretically be neutralized by one antibody molecule (two antigens can be neutralized when the antibody is bivalent). 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 bivalent). Recently, it has been reported that an antibody that binds to an antigen in a pH-dependent manner (hereinafter also referred to as "pH-dependent antibodies" or "pH-dependent binding antibodies"), which enable a single 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 in plasma under neutral conditions and dissociate from antigen under acidic conditions within the endosome of cells. After dissociation from the antigen, the antibody is recirculated in the plasma by FcRn to bind and neutralize another antigen molecule again, so one pH-dependent antibody can repeatedly bind and neutralize multiple antigen molecules.

It has recently been reported that antibody recovery properties can be achieved by focusing on the difference in calcium (Ca) ion concentrations between plasma and endosomes, and using antibodies that exhibit calcium-dependent antigen-antibody interactions (hereinafter also referred to as "calcium ions") 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 have a long retention time in plasma. Binding between IgG antibodies and FcRn is strong at acidic pH conditions (eg pH 5.8), but almost no binding at neutral pH conditions (eg pH 7.4). IgG antibodies are carried into cells nonspecifically and bind to FcRn in the acidic pH conditions of the endosome, thereby returning to the cell surface. This IgG is then dissociated from FcRn at neutral pH 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; administration of this antibody thus enables elimination of antigen from plasma (WO2011/122011). According to this report, pH-dependent antibodies modified to increase binding to FcRn at neutral pH conditions (eg, pH 7.4) can further accelerate antigen elimination compared to pH-dependent antibodies comprising the Fc region of native IgG antibodies ( WO2011/122011).

When a mutation is introduced into the Fc region of an IgG antibody to eliminate its binding to FcRn under acidic pH conditions, it is no longer recovered from the endosome to the plasma, which will significantly compromise the retention of the antibody in the plasma. In connection with this, it has been reported that a method of increasing 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 at acidic pH conditions can improve the efficiency of recovery from endosomes to plasma, thereby improving plasma retention. For example modified 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 longer antibody half-lives compared to 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 at acidic pH conditions, there have also been reports that the administration of therapeutic antibodies Binding to anti-drug antibodies (hereinafter also referred to as "pre-existing ADA") such as rheumatoid factor is increased in pre-existing patients (WO2013/046722, WO2013/046704). WO2013/046704 reports that Fc region variants containing specific mutations (represented by EU numbering, as a two-residue modification of Q438R/S440E) have increased binding to FcRn under acidic pH conditions, and compared to unmodified native Fc, Significantly reduced binding was shown for rheumatoid factor. However, WO2013/046704 does not specifically demonstrate that this Fc region variant has better plasma retention than the antibody with the native Fc region.

Therefore, a safe and more favorable Fc region variant with further improved plasma retention, and which has not shown binding to pre-existing ADA, is desired.

Antibody-dependent cytotoxicity (hereinafter referred to as "ADCC"), complement-dependent cytotoxicity (hereinafter referred to as "CDC"), antibody-dependent cellular phagocytosis (ADCP) by phagocytosis of target cells mediated by IgG antibodies, according to Reporter functions as effector of IgG antibodies. In order for IgG antibodies to mediate ADCC activity or ADCP activity, the Fc region of IgG antibodies must bind to antibody receptors present on the surface of effector cells such as killer cells, natural killer cells, or activated macrophages (here Also referred to as "Fcy receptor", "FcgR", "Fc gamma receptor" or "FcyR" within the scope of the disclosure. In humans, FcyRIa, FcyRIIa, FcyRIIb, FcyRIIIa, and FcyRIIIb isoforms have been reported as FcyR family proteins, and their respective allotypes have also been reported (Jefferis et al., Immunol. Lett. 82:57 -65 (Non-Patent Document 13)). Achieving the balance of the antibody's respective affinity for an activating receptor including FcyRIa, FcyRIIa, FcyRIIIa or FcyRIIIb and an inhibitory receptor including FcyRIIb is important in optimizing antibody effector function.

Numerous techniques have been reported to increase or improve the activity of therapeutic antibodies against antigens. For example, the activity of an antibody to bind to activating FcγRs plays an important role in the cytotoxicity of antibodies, so antibodies that target membrane-type antigens and increase cytotoxicity due to enhanced binding of activating FcγR(s) have been developed. See eg 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. USA 95:652-656 (1998); Clynes et al., Nat. Med. 6:443-446 (2000)). Similarly, the binding activity of inhibitory FcγR (in humans, FcγRIIb) plays an important role in immunosuppressive activity and agonist activity. Therefore, studies have been conducted on antibodies targeting membrane antigens with increased inhibitory FcγR binding activity. . (Li et al., Proc. Nat. Acad. Sci. USA. 109(27):10966-10971 (2012)). In addition, the influence of FcγR binding of an antibody that binds to a water-soluble antigen was examined mainly from the viewpoint of side effects (Scappaticci et al., J. Natl. Cancer Inst. 99(16):1232-1239 (2007)). For example, when an antibody with increased FcyRIIb binding is used as a drug, a reduced risk of developing anti-drug antibodies can be expected (Desai et al., J. Immunol. 178(10):6217-6226 (2007)).

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

Plasma retention of soluble antigens is very brief compared to antibodies with FcRn-mediated recovery mechanisms, and thus soluble antigens can be exhibited by binding to antibodies with such recovery mechanisms (eg, antibodies without pH/Ca concentration dependence) Increased plasma retention and plasma concentrations. 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. Increased retention and/or plasma concentrations. In this case, in addition to the above-exemplified methods for antibody modification to accelerate antigen elimination, such as the use of multiple pH/Ca concentration-dependent antibodies to form multivalent immune complexes with multiple antigens, it has also been reported that increased Binding method for FcRn, FcγR(s), complement receptors (WO2013/081143).

Even if the Fc region is not modified, 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 variable region of the antibody to increase or decrease the antibody's isoelectric point. Controlling the half-life of antibodies in blood regardless of the type of target antigen or antibody, without substantially reducing the antigen-binding activity of the antibody (WO2007/114319: Technology of amino acid substitution mainly at FR; WO2009/041643: Amine substitution mainly at CDR base acid technology). These documents show that the plasma half-life of an antibody can be prolonged by decreasing the isoelectric point of the antibody, and conversely, the plasma half-life of an antibody can be shortened by increasing the isoelectric point of the antibody.

Regarding the modification of the charge of amino acid residues in the constant region of antibodies, it has been reported that the entry of antigens into cells can be facilitated by modifying the charge of specific amino acid residues, especially the charge of specific amino acid residues in the CH3 domain, In order to increase the isoelectric point value of the antibody, it has also been recorded 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 antibody constant region (mainly the CH1 domain) to reduce the isoelectric point value can prolong the half-life of the antibody in plasma, and combined amino acid residue mutations can increase FcRn. Binding can enhance binding to FcRn and prolong the plasma half-life of the antibody (WO2012/016227).

It is not clear, however, whether combining modification techniques designed to increase or decrease the isoelectric point of the antibody with other modification techniques to increase or decrease binding to FcRn or FcγR(s) would enhance plasma retention or increase the antibody's plasma retention. The effect of eliminating antigens from plasma.

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

ECM is reported to be involved in the in vivo kinetics of proteins administered in vivo. Blood levels of VEGF-Trap molecules, which are fusion proteins of the VEGF receptor and Fc, have been examined by subcutaneous administration (Holash et al., Proc. Natl. Acad. Sci., 99(17):11393-11398 (2002) ). Subcutaneously administered VEGF-Trap molecules with a high electrical point have low plasma concentrations and therefore low bioavailability. Modified VEGF-Trap molecules with reduced isoelectric point values using amino acid substitutions have higher plasma concentrations and improved bioavailability. In addition, the change of bioavailability is related to the binding strength to ECM. It can be seen that the bioavailability of VEGF-Trap molecules administered subcutaneously depends on the binding strength to ECM at the subcutaneous site.

WO2012/093704 reports that antibody binding to ECM has an inverse relationship with plasma retention, so that antibody molecules that are not bound to ECM have better plasma retention than antibodies that bind to ECM.

As above, techniques to reduce extracellular matrix binding in order to improve protein bioavailability and plasma retention in vivo have been reported. In contrast, 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 "chemokines" is a general term for a family of proteins with a molecular weight of 8-12 kDa and containing four cysteines that form intermolecular disulfide bonds. Chemokines are classified into CC chemokines, CXC chemokines, C chemokines, and CA3C chemokines according to the cysteine arrangement. IL-8 is classified in the CXC chemokine, also known as CXCL8.

IL-8 exists in the form of monomeric and homodimeric in solution. The IL-8 unit body comprises an antiparallel beta sheet 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 crosslinks in between. The IL-8 homodimers 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 homodimers.

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 by inflammatory cytokines (Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014)).

Chemokines are generally undetectable or only weakly detectable in normal tissue, but are strongly detected in inflamed sites and are involved in inducing inflammation by promoting leukocyte involvement at inflamed tissue sites. IL-8 is a pro-inflammatory chemokine known to activate neutrophils, promote expression of cell adhesion molecules, and enhance neutrophil adhesion to vascular endothelial cells. IL-8 also has neutrophil chemotaxis ability. IL-8 produced in injured tissue can promote the chemotaxis of neutrophils adhered to vascular endothelial cells into tissues, and induce inflammation together with neutrophil infiltration. . IL-8 is also known to be a potent angiogenic factor for endothelial cells and is involved in promoting tumor angiogenesis.

Inflammatory disorders associated with elevated IL-8 levels (eg, excess), including inflammatory disorders of the skin, eg, inflammatory keratinization (eg, psoriasis), atopic dermatitis, contact dermatitis; chronic inflammatory disorders, 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 disease, such as glomerulonephritis; inflammatory respiratory disease, such as bronchitis and asthma; inflammatory chronic vascular disease, such as atherosclerosis; multiple sclerosis Symptoms, oral ulcers, vocal corditis, and related inflammation due to artificial organs and/or artificial blood vessels. Elevated levels of IL-8 (eg, excess) are also associated with malignancies such as ovarian, lung, prostate, stomach, breast, melanoma, head and neck, and kidney cancers; sepsis due to infection; cystic fibrosis; and pulmonary fibrosis. (See, eg, Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014) (not, hereby incorporated by reference in its entirety).

For several of these diseases, human anti-IL-8 antibodies with high affinity have been developed as pharmaceutical compositions (Desai et al., J. Immunol. 178(10):6217-6226 (2007)) , but not yet listed. Currently, only one pharmaceutical composition comprising IL-8 antibody is available, which is a murine anti-IL-8 antibody, which is used as a topical drug for psoriasis. New anti-IL-8 antibodies for the treatment of disease are expected.

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

In a non-exclusive aspect, the non-limiting purpose of the embodiments of disclosure B is to provide safe and more favorable Fc region variants with longer half-lives and reduced binding to pre-existing anti-drug antibodies (ADAs) .

In a non-exclusive aspect, it is a non-limiting purpose of the embodiments disclosed in C to provide anti-IL-8 antibodies that have pH-dependent binding affinity for IL-8. Additional embodiments pertain to anti-IL-8 antibodies that, when administered to an individual, have a faster effect of eliminating IL-8 than a reference antibody. In another embodiment, line C is disclosed for anti-IL-8 antibodies capable of stably maintaining their IL-8 neutralizing activity when administered to an individual. In some embodiments, the anti-IL-8 antibody exhibits lower immunogenicity. In other embodiments, disclosure C relates to methods of making and using the above-described anti-IL-8 antibodies. Another alternative non-limiting objective of the disclosure of C is to provide novel anti-IL-8s that can be included in pharmaceutical compositions.

In a non-exclusive aspect, within the scope of Disclosure A provided herein, the inventors unexpectedly discovered that ion concentration-dependent antibodies (including antibodies of ion concentration-dependent antigen-binding domains ("antigen-binding activity dependent on ion concentration conditions") The ability of an 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 found that ion concentration-dependent antibodies with increased isoelectric point values can further increase extracellular matrix binding of the antibodies. Therefore, without being bound by a particular theory, the inventors have discovered that the elimination of antigens from plasma can be increased by increasing the binding of the antibody to the extracellular matrix.

In a non-exclusive aspect, within the scope of Disclosure B provided herein, the inventors conducted a study on a safe and advantageous Fc region variant that does not bind to anti-drug antibodies (pre-existing ADA) and may more improve plasma retention. precise research. As a result, the inventors unexpectedly found that the substitution of amino acid substitution at position 434 to Ala(A) according to EU numbering method and the mutation of 2 specific residues (represented by Q438R/S440E according to EU numbering method) were used as a combination of amino acid residue mutations A variant of the Fc region is ideal for maintaining significantly reduced binding to rheumatoid factors while simultaneously achieving plasma retention of the antibody.

In a non-exclusive aspect, within the scope of Disclosure C provided herein, the inventors made some pH-dependent anti-IL-8 antibodies (anti-IL-8 that binds to IL-8 in a pH-dependent manner. Antibody). From the results of various verifications, the inventors identified that the pH-dependent anti-IL-8 antibody has a more rapid IL-8 elimination effect than the reference antibody when administered to an individual. In some embodiments, the present disclosure 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 succeeded in obtaining an anti-IL-8 antibody comprising an Fc region whose FcRn binding affinity at acidic pH was increased compared to that of the native Fc region. In an alternative aspect, the inventors succeeded in obtaining an anti-IL-8 antibody comprising an Fc region with reduced binding affinity for the Fc region to pre-existing ADA compared to the native Fc region for pre-existing ADA . In an alternative aspect, the inventors succeeded in obtaining an Fc region anti-IL-8 antibody with an increased plasma half-life of the Fc region compared to that of the native Fc region. In an alternative aspect, the inventors succeeded in obtaining a pH-dependent anti-IL-8 antibody comprising an Fc region with binding affinity for effector receptors compared to that of the native Fc region for effector receptors Affinity decreases. In a different aspect, the inventors identified nucleic acids encoding the aforementioned anti-IL-8 antibodies. In another aspect, the inventors obtained a host containing the above nucleic acid. In another aspect, the inventors develop a method for producing the above-mentioned anti-IL-8 antibody, comprising the step of culturing the above-mentioned host. In another aspect, the inventors developed a method of promoting the elimination of IL-8 relative to a reference antibody in an individual comprising the step of administering to the individual an anti-IL-8 antibody as described above.

In one embodiment, Disclosure A is related to but 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 whose antigen-binding activity is higher under conditions of high ion concentration than under conditions of low ion concentration. [4] The antibody according to any one of [1] to [3], wherein the ion concentration is a hydrogen ion concentration (pH) or a calcium ion concentration. [5] The antibody of [4], wherein, with respect to the antigen, the ratio of the KD of the acidic pH range to the neutral pH range, that is, KD (acid pH range)/KD (neutral pH range), is 2 or more high. [6] The antibody of any one of [1] to [5], wherein in the antigen-binding domain, at least one amino acid residue is substituted with histidine or at least one histidine is inserted. [7] The antibody according to any one of [1] to [6], which promotes elimination of the antigen from plasma compared to before the antibody is modified. [8] The antibody of any one of [1] to [7], wherein the extracellular matrix-binding activity of the antibody is enhanced compared to that before the antibody is modified. [9] The antibody according to any one of [1] to [8], wherein the amino acid residue modification is an amino acid residue substitution. [10] The 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 into an uncharged amino acid residue, (b) substituting a negatively charged amino acid residue for a positively charged amino acid residue, and (c) Substituting uncharged amino acid residues into positively charged amino acid residues. [11] The antibody of 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 item [11], wherein the variable region comprises a complementarity determining region (CDR) and/or a framework region (FR). [13] The antibody of [12], wherein the variable region comprises a heavy chain variable region and/or a light chain variable region, and CDRs or FRs are selected from the group consisting of the following according to the Kabat numbering system One of the positions has at least 1 amino acid residue modified: (a) 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44 of 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 CDRs of the heavy chain variable region; (c) 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 bits; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 of the CDRs of the light chain variable region. [14] The antibody of [13], wherein at least 1 amino acid residue in one of the positions selected from the group consisting of the following in the CDRs and/or FRs is modified: (a) 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105 bits; (b) positions 31, 61, 62, 63, 64, 65, and 97 of the CDRs 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 CDRs of the light chain variable region. [15] The antibody of any one of [11] to [14], wherein in the constant region at least 1 amino acid residue is modified at a position selected from the group consisting of: According to EU Numbering Act 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 bits. [16] The antibody of [15], wherein in the constant region at least 1 amino acid residue is modified at a position selected from the group consisting of: 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 bits. [17] The antibody of [16], wherein at least 1 amino acid residue in a position selected from the group consisting of the following in the constant region is modified: According to EU Numbering Act 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413. [18] The antibody of any one of [1] to [17], wherein the constant region has Fc gamma receptor (FcyR) binding activity, and the FcγR binding activity at neutral pH conditions is higher than that of native IgG The reference antibody of the constant region is enhanced. [19] The antibody of [18], wherein the FcγR is FcγRIIb. [20] The antibody of any one of [1] to [17], wherein the constant region has binding activity to one or more activating FcγRs selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa have binding activity to FcγRIIb, and the FcγRIIb binding activity is maintained or enhanced and the binding activity to the activating FcγR is reduced compared to a reference antibody that is different from IgG whose constant region is native only. [21] The antibody according to any one of [1] to [20], wherein the constant region has FcRn-binding activity and is at neutral pH compared to a reference antibody that is different from IgG in which only the constant region is native Conditions (eg, pH 7.4) enhance the FcRn binding activity of the constant region. [22] The antibody of any one of [1] to [21], wherein the antibody is a polyspecific antibody that binds to at least two antigens. [23] The antibody of any one of [1] to [22], wherein the antibody is an IgG antibody. [24] A pharmaceutical composition comprising the antibody of any one of [1] to [23]. [25] The pharmaceutical composition according to item [24], which is used for promoting elimination of an antigen from plasma. [26] The pharmaceutical composition according to item [24] or [25], which is used for enhancing the binding of an antibody to an extracellular matrix. [27] A nucleic acid encoding the antibody of any one of [1] to [23]. [28] A vector comprising the nucleic acid of [27]. [29] A host cell comprising the vector of [28]. [30] A method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, comprising the steps of: Host cells were cultured as in [29] and antibodies were collected from the cell culture. [30A] A method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, comprising the steps of: At least one amino acid residue possibly exposed on the surface of the antibody is modified to increase the isoelectric point (pi). [30B] The method for producing an antibody according to [30A], wherein at least one amino acid residue is modified at one position in the CDR or FR according to the Kabat numbering method 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, 84, 85, 86, 105, 108, 110, and 112; (b) the position of the heavy chain variable region 31, 61, 62, 63, 64, 65, and 97 in the CDR; (c) 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) 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDRs of the light chain variable region bit; or (II) A position in the constant region selected from the group consisting of: According to EU Numbering Act 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 bits. [31] The method for producing an antibody according to [30A] or [30B], wherein the amino acid residue modification is selected from the group consisting of: (a) Substituting negatively charged amino acid residues into uncharged amino acid residues; (b) substituting negatively charged amino acid residues into positively charged amino acid residues; (c) replacing uncharged amino acid residues into positively charged amino acid residues; (d) Substitution or insertion of histidine in CDRs or FRs. [32] The method for producing an antibody according to any one of [30], [30A] or [30B], more optionally comprising any one or more of the following steps: Compared to the reference antibody, (a) selection of antibodies that promote elimination of antigens from plasma; (b) selecting antibodies with enhanced binding activity to the extracellular matrix; (c) select antibodies with enhanced FcγR binding activity at neutral pH conditions (eg pH 7.4); (d) select antibodies with enhanced FcγRIIb binding activity at neutral pH conditions (eg pH 7.4); (e) selecting an antibody having maintained or enhanced FcγRIIb binding activity and reduced binding activity to one or more activating FcγRs, preferably selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc , FcγRIIIa, FcγRIIIb and FcγRIIa; (f) selecting antibodies with enhanced FcRn binding activity at neutral pH conditions (eg pH 7.4); (g) select antibodies with increased isoelectric point (pI); (h) confirm the isoelectric point (pI) of the collected antibodies, and then select the antibody with an increased isoelectric point (pI); and (i) Selection of antibodies whose antigen-binding activity changes or increases according to ion concentration conditions.

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

According to various embodiments, disclosure A includes part or all of one or more of the descriptions in [1] to [30], [30A], [30B], [31], [32], and [A1] to [A11] A combination of elements, provided that such 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 comprising an antigen binding domain that facilitates elimination of an antigen from plasma compared to the antibody prior to modification, comprising: (a) Modification of at least 1 amino acid residue that may be exposed on the surface of the antibody, which is: (1) a position in a CDR or FR according to Kabat numbering 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, 84, 85, 86, 105, 108, 110, and 112; (b) positions 31, 61, 62, 63, 64, 65, and 97 in the CDRs of the heavy chain variable region; (c) the light 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, and (d) the light Positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDRs of the chain variable region; or (II) a position in the constant region according to EU numbering selected from the group consisting of: 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 bits; (b) modifying the antigen-binding domain such that the obtained antigen-binding activity varies 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 comprise one or more of the following steps: Compared with the antibody before modification, (e) selection of antibodies that promote elimination of antigens from plasma; (f) selecting antibodies with enhanced binding activity to the extracellular matrix; (g) select antibodies with enhanced FcγR binding activity at neutral pH conditions (eg pH 7.4); (h) select antibodies with enhanced FcγRIIb binding activity at neutral pH conditions (eg pH 7.4); (i) selecting an antibody that has maintained or enhanced FcγRIIb binding activity and reduced binding activity to one or more activating FcγRs, preferably selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc , FcγRIIIa, FcγRIIIb and FcγRIIa; (j) selecting antibodies with enhanced FcRn binding activity at neutral pH conditions (eg pH 7.4); (k) select antibodies with increased isoelectric point (pI); (l) Confirm the isoelectric point (pI) of the collected antibodies, and then select the antibody with an increased isoelectric point (pI); and (m) Selection of antibodies whose antigen-binding activity changes or increases according to ion concentration conditions.

Another embodiment of Disclosure A relates to, for example and without limitation: [D1] A method for producing a modified antibody, the half-life of the modified antibody in plasma is prolonged or shortened compared to that before modification, comprising the following steps: (a) modifying the nucleic acid encoding the antibody prior to modification to alter the charge of at least 1 amino acid residue in a position selected from the group consisting of: According to EU Numbering Act 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 bits; (b) culturing host cells to express the nucleic acid; and (c) collecting the antibody from the host cell culture. [D2] A method for prolonging or shortening the half-life of an antibody in plasma, comprising the steps of: Modifies at least 1 amino acid residue at a position selected from the group consisting of: According to EU Numbering Act 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 bits.

In one embodiment, it is disclosed that B relates to, but is not limited to: [33] an Fc region variant comprising an FcRn binding domain comprising: (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 comprises: 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 comprises: 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 comprises: According to EU numbering, Leu is at position 428; and/or Val or Thr is at position 436. [37] The Fc region variant of any one of [33] to [36], wherein the FcRn binding domain comprises a combination of amino acid substitutions selected from the group consisting of: N434A/Q438R/S440E; N434A /Q438R/S440D; N434A/Q438K/S440E; N434A/Q438K/S440D; N434A/Y436T/Q438R/S440E; N434A/ Y436T/Q438R/S440D; N434A/Y436T/Q438K/S440E; N434A/Y436T/Q438K/S440D; N434A /Y436V/Q438R/S440E; N434A/Y436V/Q438R/S440D; N434A/Y436V/Q438K/ S440E; N434A/Y436V/Q438K/S440D; N434A/R435H/F436T/Q438R/S440E; N434A/ R435H/F436T/Q438R/S440D ; N434A/R435H/F436T/Q438K/S440E; N434A/R435H/ F436T/Q438K/S440D; N434A/R435H/F436V/Q438R/S440E; N434A/R435H/F436V/ Q438R/S440D; N434A/R435H/F436V/Q438K/S440E ; N434A/R435H/F436V/Q438K/ S440D; M428L/N434A/Q438R/S440E; M428L/N434A/Q438R/S440D; M428L/N434A/ Q438K/S440E; M428L/N434A/Q438K/S440D; M428L/N434A/Y436T/Q438R /S440E; M428L/N434A/Y436T/Q438R/S440D; M428L/N434A/Y436T/Q438K/S440E; M428L/ N434A/Y436T/Q438K/S440D; M428L/N434A/Y436V/Q438R/S440E; M428L/N434A/ Y436V/Q438R /S440D; M428L/N434A/Y436V/Q438K/S440E; M428L/N434A/Y436V/Q438K/S440D; L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and L235R/G 236R/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 comprises 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; M428L/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 EU numbering. [39] The Fc region variant of any one of [33] to [38], wherein the FcRn-binding activity under acidic pH conditions (eg, pH 5.8) is enhanced compared to the Fc region of native IgG. [40] The Fc region variant of any one of [33] to [39], wherein, Compared to the Fc region of native IgG, the binding activity for anti-drug antibodies (ADA) at neutral pH was 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 to the Fc region of native IgG, plasma clearance (CL) is decreased, plasma retention time is increased, or 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 comprising the following combination of amino acid substitutions: N434Y/Y436V/Q438R/S440E, according to EU numbering method. [44] An antibody comprising the Fc region variant of any one of [33] to [43]. [45] The antibody of [44], wherein the antibody is an IgG antibody. [46] A pharmaceutical composition comprising the antibody of [44] or [45]; [47] The pharmaceutical composition according to [46], which is used to increase the retention of antibodies in plasma. [48] A nucleic acid encoding an Fc region variant as in any one of [33] to [43] or an antibody as in [44] or [45]. [49] A vector comprising the nucleic acid of [48]. [50] A host cell comprising the vector of [49]. [51] A method of making an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant, comprising the steps of: Host cells as in [50] are cultured, and then the Fc region variant or antibody containing the variant is collected from the cell culture. [52] The method for producing an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant according to [51], further optionally comprising any one or more of the following steps: (a) selection of Fc region variants with enhanced FcRn binding activity under acidic pH conditions compared to the Fc region of native IgG; (b) selection of Fc region variants that do not significantly enhance the binding activity of anti-drug antibodies (ADA) at neutral pH compared to the Fc region of native IgG; (c) selecting Fc region variants with increased plasma retention compared to the Fc region of native IgG; and (d) selecting 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 making an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant, comprising the steps of: Substitute amino acids such that the resulting Fc region variant or antibody containing the variant comprises, according to EU numbering, Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440 bit.

In one embodiment, B is disclosed with respect to, for example and without limitation: [B1] Use of the Fc region variant according to any one of [33] to [43] or the antibody according to [44] or [45] for the manufacture of a medicament for increasing plasma retention. [B2] Use of the Fc region variant according to any one of [33] to [43] or the antibody according to [44] or [45] to produce an Fc region compared to native IgG at neutral pH Condition drugs that do not significantly enhance the binding activity of anti-drug antibodies (ADA). [B3] Use of an Fc region variant as in any one of [33] to [43] or an antibody as in [44] or [45] for increasing retention in plasma. [B4] Use of an Fc region variant according to any one of [33] to [43] or an antibody according to [44] or [45], for comparison with the Fc region of native IgG at neutral pH The conditions did not significantly increase the binding activity of anti-drug antibodies (ADA). [B5] An Fc region variant or an antibody containing the variant obtained by the method for producing an antibody according to any one of [51], [52] and [53].

According to various embodiments, disclosure B includes a combination of part or all of one or more of the elements recited in [33] to [53] and [B1] to [B5], provided that such combination is not technically compatible with Common technical knowledge in this technical field conflicts. For example, in some embodiments, it is disclosed that B includes an Fc region variant comprising an FcRn binding domain that 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 at position 434; Arg or Lys at position 438; and Glu or Asp at position 440; (c) according to EU numbering, Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr, or Phe at position 436; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp , or Gln at bit 440; (d) according to EU numbering, Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr, or Phe at position 436; Arg or Lys at position 438; and Glu or Asp at position 440; (e) according to EU numbering, Leu at position 428; Ala at position 434; Val or Thr at position 436; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440; or (f) according to EU numbering, Leu at position 428; Ala at position 434; Val or Thr at position 436; Arg or Lys at position 438; and Glu or Asp at position 440.

In one embodiment, C is disclosed with respect to, for example and without limitation: [54] An isolated anti-IL-8 antibody that binds to human IL-8, comprises at least 1 amino acid substitution at any of the following (a) to (f) positions, and binds in a pH-dependent manner On IL-8: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO:67; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO:68; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO:69; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO:70; (e) HVR-L2, including the amino acid sequence of SEQ ID NO:71; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO:72. [55] The anti-IL-8 antibody of [54], comprising: 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 Arginine at position 3, and amino acid substitution with tyrosine at position 3 of the amino acid sequence of SEQ ID NO:69. [56] The anti-IL-8 antibody of [54] or [55], further comprising alanine at the sixth position 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 the 8th position. [57] The anti-IL-8 antibody according to any one of [54] to [56], comprising aspartic acid at position 1 of the amino acid sequence of SEQ ID NO:71, The amino acid substitution of leucine at position 5 of the amino acid sequence of SEQ ID NO: 71 and glutamic acid at position 1 of the amino acid sequence of SEQ ID NO: 72. [58] The anti-IL-8 antibody of any one of [54] to [57], comprising (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 67, (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 73, and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 73 : The amino acid sequence of 74. [59] The anti-IL-8 antibody of any one of [54] to [58], comprising (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO:70, (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO:75, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. [60] The anti-IL-8 antibody according to any one of [54] to [59], comprising 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 of any one of [54] to [60], comprising an Fc region having at least one property selected from the following (a) to (f): (a) increased binding affinity for FcRn under acidic pH conditions relative to the binding affinity of the native Fc region for FcRn; (b) reduced binding affinity for pre-existing ADA relative to the binding affinity of the native Fc region for pre-existing ADA; (c) increased plasma half-life relative to the plasma half-life of the native Fc region; (d) decreased plasma clearance of the Fc region relative to the plasma clearance 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 comprises amino acid substitutions at one or more positions selected from the group consisting of: 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440 bits. [63] The anti-IL-8 antibody of [62], comprising an Fc region comprising one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, A327G, A330S , P331S, M428L, N434A, Y436T, Q438R and S440E. [64] The anti-IL-8 antibody of [63], wherein the Fc region comprises the following amino acid substitutions: L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E. [65] The anti-IL-8 antibody of [63], wherein the Fc region comprises the following amino acid substitutions: L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E. [66] An anti-IL-8 antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO:81, and a light chain comprising the amino acid sequence of SEQ ID NO:82. [67] An anti-IL-8 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:80, and a light chain comprising the amino acid sequence of SEQ ID NO:82. [68] An isolated nucleic acid encoding the anti-IL-8 antibody of any one of [54] to [67]. [69] A vector comprising the nucleic acid of [68]. [70] A host cell comprising the vector of [69]. [71] A method of producing an anti-IL-8 antibody, comprising the step of culturing a host as in [70]. [72] The method for producing an anti-IL-8 antibody according to [71], comprising the step of isolating the antibody from the culture supernatant. [73] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of [54] to [67], and a pharmaceutically acceptable carrier. [74] The use of the anti-IL-8 antibody according to any one of [54] to [67], in a pharmaceutical composition. [75] The use of the anti-IL-8 antibody according to any one of [54] to [67] in the treatment of a disorder in which excess IL-8 is present. [76] The use of the anti-IL-8 antibody according to any one of [54] to [67] in the manufacture of a pharmaceutical composition for a condition in which excess IL-8 is present. [77] A method of treating a patient suffering from a disorder of excess IL-8, comprising the steps of: administering to the individual the anti-IL-8 antibody of any one of [54] to [67]. [78] A method of promoting elimination of IL-8 from an individual, comprising the steps of: The anti-IL-8 antibody of any one of [54] to [67] is administered to the individual. [79] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of [54] to [67], which antibody binds to IL-8 and binds to an extracellular matrix. [80] A method of making an anti-IL-8 antibody, the antibody comprising: a variable region with pH-dependent IL-8 binding activity, comprising the steps of: (a) assessing the binding of anti-IL-8 antibody to extracellular matrix, (b) select anti-IL-8 antibodies that bind strongly to the extracellular matrix, (c) culturing a host comprising a vector encoding nucleic acid for the antibody, and (d) Isolation of the antibody from the culture solution.

In an alternative embodiment, it is disclosed that C relates to: [C1] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for the manufacture of a pharmaceutical combination for inhibiting the accumulation of biologically active IL-8 thing. [C2] 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. [C3] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for the manufacture of a pharmaceutical composition for inhibiting angiogenesis. [C4] Use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for inhibiting angiogenesis. [C5] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for the manufacture of a pharmaceutical composition for inhibiting the promotion of neutrophil migration . [C6] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for inhibiting the promotion of neutrophil migration. [C7] Use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for inhibiting the accumulation of biologically active IL-8. [C8] A method of inhibiting accumulation of biologically active IL-8, comprising administering to an individual the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31]. [C9] A pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, comprising an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] . [C10] 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 steps of: An anti-IL-8 antibody such as any one of [54] to [67] and [C26] to [C31] is administered to the individual. [C12] A pharmaceutical composition for inhibiting angiogenesis, comprising the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31]. [C13] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for inhibiting the promotion of neutrophil migration. [C14] A method for inhibiting neutrophil migration promotion in an individual, comprising administering to the individual an anti-IL of any one of [54] to [67] and [C26] to [C31] -8 steps for antibodies. [C15] A pharmaceutical composition for inhibiting neutrophil migration promotion in an individual, comprising 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], for use in the treatment of a condition in which there is an excess of IL-8. [C17] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for the manufacture of a medicine for treating a condition in which IL-8 is excessive combination. [C18] 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 there is an excess of IL-8. [C19] A method of treating a condition in which an excess of IL-8 exists in an individual, comprising the steps of: administering to the individual an anti-IL of any one of [54] to [67] and [C26] to [C31] -8 antibody. [C20] A pharmaceutical composition for treating a condition in which an excess of IL-8 exists in an individual, comprising 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], for use in promoting elimination of IL-8. [C22] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for the treatment of a pharmaceutical composition for promoting elimination of IL-8. [C23] Use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for promoting the elimination of IL-8. [C24] A method of promoting elimination of IL-8 in an individual, comprising the steps of: 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, in order to promote 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 comprising amino acid substitutions at one or more positions selected from the group consisting of: 235 according to EU numbering, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440 bits. [C27] The anti-IL-8 antibody of [C26], comprising an Fc region having at least one property selected from the following (a) to (f): (a) increased binding affinity for FcRn under acidic pH conditions relative to the binding affinity of the native Fc region for FcRn; (b) reduced binding affinity for pre-existing ADA relative to the binding affinity of the native Fc region for pre-existing ADA; (c) increased plasma half-life relative to the plasma half-life of the native Fc region; (d) the plasma clearance of the Fc region is reduced relative to the plasma clearance 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] The anti-IL-8 antibody of [C26] or [C27], comprising an Fc region comprising one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E according to EU numbering method. [C29] The anti-IL-8 antibody of [C28], comprising an Fc region comprising one or more amino acid substitutions selected from the group consisting of: (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E under EU numbering; or (b) L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E. [C30] The anti-IL-8 antibody of [C26], comprising: 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], comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:80 and a light chain comprising the amino acid sequence of SEQ ID NO:82. [C32] An isolated nucleic acid encoding the anti-IL-8 antibody of any one of [C26] to [C31]. [C33] A vector comprising the nucleic acid as [C32]. [C34] A host cell comprising a vector as [C33]. [C35] A method of producing an anti-IL-8 antibody, comprising culturing a host cell such as [C34]. [C36] A method of producing the anti-IL-8 antibody of any one of [C26] to [C31], further comprising the step of isolating the antibody from a host cell culture. [C37] A pharmaceutical composition, comprising: the anti-IL-8 antibody of any one of [C26] to [C31], and a pharmaceutically acceptable carrier. [C38] A method of treating a patient having a condition in which excess IL-8 is present, comprising the step of administering to the individual the 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 of any one of [C26] to [C31]. [C40] A method of inhibiting IL-8, wherein the method comprises 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 according to [C40], wherein the method inhibits the biological activity of IL-8.

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

Non-limiting embodiments disclosing A, B or C are described below. All the embodiments 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 countries that want to obtain the patent right of this application.

Reveal A or Reveal B In some embodiments, it is disclosed that line A relates to an antibody comprising an antigen-binding domain whose antigen-binding activity varies according to ionic 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. and increase (in the category of revealing A, also called "ion concentration-dependent antibody with increased isoelectric point value"; and the antigen-binding domain of this antibody is also called "ion concentration-dependent antigen-binding domain with increased isoelectric point value" "). The present invention is based in part on the inventors' unexpected discovery that the elimination of antigens from plasma can be facilitated by ion concentration-dependent antibodies whose isoelectric point (pi) is modified by modifying at least one amino acid that may be exposed on the antibody surface residues (eg, when the antibody is administered in vivo); and binding of the antibody to the extracellular matrix can be increased using an ion concentration-dependent antibody with an increased (rising) isoelectric point value. The present invention is also based in part on the inventors' unexpected discovery that this beneficial effect is brought about by combining two completely different concepts: an ion concentration-dependent antigen binding domain or an ion concentration-dependent antibody; and the isoelectric point (pi ) antibodies that are increased by modification of at least 1 amino acid residue that may be exposed on the surface of the antibody (also referred to as "antibodies with increased isoelectric point values" in the context of Disclosure A; and the isoelectric point (pI) is derived from An antibody that is reduced (reduced) by modification of at least one amino acid residue that may be exposed on the surface of the antibody (also referred to as an "isoelectric point-reduced antibody" in the context of Disclosure A. It belongs to the present invention of Disclosure A and thus Classified as pioneering inventions that can lead to significant technological innovations in this technical field (eg, in 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 further modified so that the antigen-binding activity of the antigen-binding domain depends on ion concentration conditions Changes are also included within the scope of the disclosure A described herein (in the scope of the disclosure A 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 at least one amino acid residue that may be exposed on the surface of the antibody has a different charge than before the antibody is modified (natural antibodies (such as natural Ig antibodies, preferably is a natural IgG antibody), or at least 1 amino acid residue in the corresponding position of a control or parent antibody (for example, before antibody modification or before antibody library construction or construction, etc.), and its net antibody pI is increased, also Included in Disclosure A are also included 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 that before modification of the antibody (natural antibodies (such as natural Ig) by modifying at least one amino acid residue that may be exposed on the surface of the antibody. Antibodies, preferably native IgG antibodies or control or parental antibodies (e.g., before antibody modification or prior to antibody library construction or construction, etc.), are also included in disclosure A (such antibodies are also referred to as "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 to increase the isoelectric point value of the antibody, which is also included in the disclosure A (so Antibodies that are also referred to as "Ion concentration-dependent antibodies with increased isoelectric point values" are within the scope of Disclosure A described herein).

Within the scope of disclosures A and B described herein, "amino acid" includes not only natural but also unnatural amino acids. Within the scope of disclosures A and B described herein, amino acids or amino acid residues can be expressed using a single letter (eg A) or a 3 letter code (eg Ala) or both (eg Ala(A)).

When used in the context of disclosing A and B, "modified amino acid", "modified amino acid residue", or equivalent terms may be understood as, but not limited to, chemical modification of a molecule to one of the antibody amino acid sequences or Multiple (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) specific amino acids (residues), or additions, deletions, substitutions to antibody amino acid sequences Or one or more (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) amino acids are inserted. 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 chain drag using antibody heavy or light chains; or by 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 modifications are not limited but are preferably by substituting one or more amino acid residues of the antibody amino acid sequence with a different amino acid (individually). Amino acid addition, deletion, substitution or insertion and modification of amino acid sequences by humanization or chimerization can be carried out using methods known in the art. Engineering or modification of amino acids (residues), such as amino acid additions, deletions, substitutions, or insertions, can also be performed on antibody variable regions or antibody constant regions of antibodies for the preparation of recombinant antibodies to reveal A or B.

In one embodiment, within the scope of disclosures A and B described herein, a substituted amino acid (residue) refers to substitution into a different amino acid (residue), which can be designed to modify eg (a) to (c) ) of: (a) the structure of the polypeptide backbone within the region of the sheet or helical conformation; (b) the charge or hydrophobicity of the target site; or (c) the size of the side chain.

Amino acid residues are classified into, for example, the following groups according to the nature of the side chains in the structure: (1) Hydrophobicity: n-leucine, Met, Ala, Val, Leu, and Ile; (2) Neutral, hydrophilic: Cys , Ser, Thr, Asn, and Gln; (3) Acidic: Asp and Glu; (4) Basic: His, Lys, and Arg; (5) Residues that affect chain orientation: Gly and Pro; and (6) Aromatic Properties: 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 can be conservative substitutions, non-conservative substitutions, or a combination thereof. There are several suitable methods available for substituting amino acids with amino acids other than the natural amino acids. (Wang et al., Annu. Rev. Biophys. Biomol. Struct. 35:225-249 (2006); Forster et al., Proc. Natl. Acad. Sci. USA 100(11):6353-6357 (2003) ). For example, cell-free translation systems can be used, including tRNAs in which the unnatural amino acid is linked to an amber suppressor tRNA (Clover Direct (Protein Express)) complementary to the UAG codon which is a stop codon.

Within the scope of disclosures A and B described herein, it should be understood that an "antigen" structure is not limited to a particular structure, so long as the antigen includes an epitope that will bind to an antibody. This antigen can be an inorganic or organic material. Antigens can be any ligand, including various interleukins, such as interleukins, chemokines, and cell growth factors. Alternatively, receptors presented in soluble form or modified to be soluble in biological fluids such as plasma can of course be used as antigens. Non-limiting examples of such soluble receptors include the soluble IL-6 receptor, described in Mullberg et al., J. Immunol. 152(10):4958-4968 (1994). In turn, the antigen can be monovalent (eg, soluble IL-6 receptor) or multivalent (eg, IgE).

In one embodiment, it is disclosed that the antigens to which the antibodies of A and B can bind are preferably those present in the biological fluid of the subject (for example, WO2013/125667 exemplifies the biological fluid, preferably plasma, interstitial fluid, lymph fluid, ascites fluid, or pleural fluid). Soluble antigen (within the scope of disclosure A and B described herein, the subject to which the antibody is to be administered (administered) can be any animal such as human, mouse, etc.); but the antigen can also be a membrane antigen.

Within the scope of disclosures A and B described herein, the term "prolonged half-life in plasma" or "shortened half-life in plasma" of a target molecule (which may be an antigen or antibody) or equivalent terms may also be used in addition to the term "half-life 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, the clearance rate in plasma (CL), and the area under the concentration curve (AUC) (Pharmacokinetics: Enshuniyoru Rikai (Understanding) through practice) Nanzando). These parameters can be specifically assessed by non-compartmental analysis using the accompanying experimental procedures of 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.

In the context of Disclosures A and B described herein, "epitope" 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. An epitope can thus be defined, for example, by structure. Alternatively, an epitope can be defined by the antigen-binding activity of an antibody that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that make up the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be designated by its specific sugar chain structure. It is revealed that the antigen binding domains of A and B can bind to a single epitope on the antigen or to different epitopes.

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

In the configuration of an epitope, generally the amino acids constituting the epitope do not exist consecutively in the 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 herein, "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 native immunoglobulins (abbreviated "Ig")), and molecules and variants derived therefrom, such as Fab, Fab', F(ab') ₂ , diabodies, 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, one-armed antibodies (including all the embodiments of one-armed antibodies described in WO2005/063816), Multispecific antibodies (eg, bispecific antibodies: antibodies specific for 2 different epitopes, including antibodies that recognize different antigens, and antibodies that recognize different epitopes on the same antigen). Within the scope of disclosures A and B described herein, the "double-specific antibody" is not limited, but can be prepared as an antibody molecule with a common L chain described in WO2005/035756, or by using the method described in WO2008/119353, 2 Mixing of antibodies of a general type with constant regions similar to IgG4 results 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, an antibody may have a structure in which the variable region of the heavy chain and the variable region of the light chain are linked together into a single chain (eg, sc(Fv) ₂ ). Or it can be an antibody-like molecule (eg, scFv-Fc) obtained by linking an Fc region (the constant region lacking the CH1 domain) to a scFv (or sc(Fv) ₂ ), the variable heavy (VH) region being linked in the variable region (VL) of the light chain. The multispecific antibody is composed of a 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 in which a single domain antibody is linked to an Fc region (Marvin et al., Curr. Opin. Drug Discov. Devel. 9(2):184-193 (2006)), Fc fusion proteins (eg immunoadhesin) ) (US2013/0171138), functional fragments thereof, functionally equivalent substances, and sugar chain modified variants thereof. Here, native IgG (eg native IgG1) refers to a polypeptide having the same amino acid sequence as naturally occurring IgG (eg native IgG1), belonging to the class of antibodies substantially encoded by immunoglobulin gamma genes. Natural IgG may be its spontaneous mutant or the like.

Generally, if the structure of the antibody is substantially the same or similar to that of native IgG, its basic structure can be a Y-shaped structure of 4 chains (2 heavy chain polypeptides and 2 light chain polypeptides). Generally heavy and light chains can be linked together by disulfide bonds (SS bonds) to form heterodimers. Such heterodimers can be linked together via disulfide bonds and form Y-shaped heteroquadruplexes. The 2 heavy or light chains can be the same or different from each other.

For example, an IgG antibody can be cleaved into a 2-unit Fab (region) and a 1-unit Fc (region) by digestion with papain, which cleaves the hinge region (also referred to as this in the context of A and B disclosed herein). "hinge"), the heavy chain Fab region is linked to the Fc region. Typically, the Fab region contains an antigen binding domain. Phagocytic cells such as leukocytes and macrophages have receptors that bind to the Fc region (Fc receptors) and can be recognized by Fc receptor antibodies that have bound to an antigen and phagocytose the antigen (opsonization) . At the same time, the Fc region is involved in mediating immune responses, such as ADCC or CDC, and has an effector function, ie, when an antibody binds to an antigen, it induces a response. This antibody effector function is known to vary by immunoglobulin (constructive isotype) type. The Fc region of the IgG class can be indicated, for example, as the range from cysteine at position 226 or proline at position 230 (EU numbering) to the C-terminus; but the Fc region is not limited thereto. The Fc region can be appropriately obtained by partially digesting monoclonal antibody IgG1, IgG2, IgG3 or IgG4 antibody or others with a protease such as pepsin, and extracting the adsorbed fraction from a protein A or protein G column.

Within the scope of disclosures A and B described herein, the positions of amino acid residues in the variable regions (CDRs and/or FRs) of antibodies are shown according to Kabat, and the positions of amino acid residues in the constant or Fc regions are Expressed according to EU numbering based on Kabat's amino acid positions (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991).

Within the scope of Disclosures A and B described herein, a "library" refers to a molecule (population) of sequence variability, such as a polybody, each of which may be the same or different from each other in sequence; polyfusion polypeptides comprising such antibodies; or encoding Nucleic acids or polynucleotides of such amino acid sequences are described in detail in WO2013/125667 (eg paragraphs 0121-0125). The 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 can be a phage library. The term "comprising essentially" means that the antibodies may have different antigen-binding activities, corresponding to some portion of a majority of independent colonies of different sequences in the library. In one embodiment, the antibody can be derived from an animal that has been immunized with a specific antigen, an infected patient, a person whose blood antibody titers are elevated due to vaccination, or lymphocytes from cancer or autoimmune disease. An immune repertoire constructed from antibody genes can be suitably used as a random variable region repertoire. In an alternative embodiment, an unaltered library constructed from antibody genes from healthy human lymphocytes containing unaltered sequences (unbiased in the stocked antibody sequence) can be suitably 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)). Amino acid sequences containing unaltered sequences may refer to those obtained from unaltered libraries. In an alternative embodiment, the CDR sequences of the V gene or reconstituted functional V gene from genomic DNA may be replaced by a synthetic library containing a set of synthetic oligonucleotides encoding sequences for a codon set of appropriate length as appropriate. Random variable region library. In this case, sequence variation is also observed in the CDR3 gene, and only the heavy chain CDR3 sequence may be substituted. A standard method for generating amino acid diversity in the variable region of an antibody may be to increase variation in the positions of amino acid residues that may be exposed on the antibody surface.

In one embodiment, if an antibody of A or B is disclosed, for example, to have a structure substantially equivalent or similar to that of a native 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 subdivided into 3: CH1 to CH3. Generally, the Fab region of the heavy chain contains VH region and CH1, and the Fc region of the heavy chain generally contains CH2 and CH3. Generally, the hinge region is located between CH1 and CH2. The variable region generally has complementarity Determining Regions ("CDR") and Framework Regions ("FR"). Typically, the VH and VL regions each have 3 CDRs (CDR1, CDR2, and CDR3) and 4 FRs (FR1, FR2, FR3, and FR4). Generally, the 6 CDRs of 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 capable of recognizing and binding to antigens.

Ig antibodies are divided into several classes (constructive isotypes) based on structural differences in the constant regions. In many mammals, five immunoglobulins are classified according to structural differences in the constant regions: IgG, IgA, IgM, IgD and IgE. Also, in the case of humans, IgG has four subclasses: IgG1, IgG2, IgG3, and IgG4; and IgA has two subclasses: IgA1 and IgA2. Heavy chains are divided into γ chain, μ chain, α chain, δ chain and ε chain according to the difference in the constant region, and based on these differences, there are 5 immunoglobulin classes (construction isotypes): IgG, IgM, IgA, IgD and IgE . On the other hand, there are two types of light chains: lambda chains and kappa chains, and all immunoglobulins have one of these two.

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

Within the scope of disclosures A and B described herein, an "antigen-binding domain" can be of any structure as long as it binds to the antigen of interest. Such domains may include, for example, the variable regions (eg, 1 to 6 CDRs) of antibody heavy and light chains; a module of about 35 amino acids called the A domain, which is included in Avimer, the cell membrane present in the body Proteins (WO2004/044011 and WO2005/040229); Adnectin, which contains a 10Fn3 domain, and binds to a protein in the glycoprotein fibronectin expressed on the cell membrane (WO2002/032925); Affibody, which has 58 amino groups as constituting protein A Scaffolds for IgG binding domains of acid 3-helix bundles (WO1995/001937); Designed Ankyrin Repeat Protein (DARPin), a region exposed on the surface of Ankyrin repeat (AR) molecules, with the following Structure: primary unit with turns comprising 33 amino acid residues, 2 antiparallel helices, and repeating stacked loops (WO2002/020565); Anticalin et al., which are 4 loop regions supporting 8 Anti-parallel strands on one side of the central torsion barrel structure are highly conserved in lipocalin molecules such as neutrophil gelatinase-associated apolipoprotein (NGAL) (WO2003/029462); and by horse A cavity region formed by a parallel plate structure within a shoe-like structure formed by stacked repeats of modules of leucine-rich repeats (LRR) of variable lymphocyte receptors (VLRs) that do not have immunoglobulins structure, used as an innate immune system for jawless vertebrates such as lampreys and hagfish (WO2008/016854). It is revealed that the A or B antigen-binding domains preferably 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, "ionic 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 other than boron, carbon and silicon group IV Elements, elements of group VIII, such as iron and platinum group elements, elements belonging to subgroup A of groups V, VI and VII, and ions of metal elements such as antimony, bismuth, polonium. Metal atoms have the property of releasing electrons to become cations. called dissociation tendency. Metals with strong free hydrogenation are presumed to be chemically reactive.

In one embodiment disclosed in A, the metal ion is preferably calcium ion, which is described in detail in WO2012/073992 and WO2013/125667.

In one embodiment of Disclosure A, "ionic concentration conditions" may focus on ion concentration-dependent differences in the biological behavior of the antibody between low and high ion concentrations. Also, "its antigen-binding activity changes depending on ion concentration conditions" may refer to revealing that the ion concentration-dependent antigen-binding domain of A or B or the antigen-binding activity of an ion concentration-dependent antibody changes 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 a hydrogen ion concentration (pH) or a calcium ion concentration. When the ion concentration is the hydrogen ion concentration (pH), the ion concentration-dependent antigen-binding domain may also be referred to as a "pH-dependent antigen-binding domain"; and when the ion concentration is the calcium ion concentration, it may also be referred to as a "calcium ion concentration-dependent domain". 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 increased isoelectric point values, and an ion concentration-dependent antibody with increased isoelectric point values , can be obtained from a library consisting mainly of antibodies with different sequences (variable), the antigen-binding domain of which contains at least one amino acid residue that causes the antigen-binding domain or the antigen-binding activity of the antibody to change according to ionic 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 again, such a light or heavy chain variable region can be combined with a heavy or light chain variable region that has been constructed as a library of random variable region sequences. If the ion concentration is hydrogen or calcium ion concentration, non-limiting examples of the library include, for example: heavy chain variable regions constructed as a library of random variable region sequences, and a library of combinations of light chain variable region sequences, wherein the light chain variable region 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) amino acid residues are substituted with At least one amino acid residue that changes antigen-binding activity depending on ionic concentration. Also if the ion concentration is calcium ion concentration, the library includes heavy chain variable region sequences such as SEQ ID NO:5 (6RL#9-IgG1) or SEQ ID NO:6 (6KC4-1#85-IgG1) and constructed Light chain variable regions of a random variable region sequence library or a library of combinations of light chain variable regions 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 between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 5 mM. 3 mM, between 200 μM and 2 mM, or between 400 μM and 1 mM. It is preferably selected from 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. Preferably, the concentration is selected between 1 μM and 5 μM, which approximates the calcium ion concentration in the early endosome.

Whether the antigen-binding activity of an antigen-binding domain or an antibody containing this domain changes depending on the metal ion concentration (eg, calcium ion concentration) can be easily determined according to known methods, such as 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 the domain can be determined and compared at low and high calcium ion concentrations. In this case, the conditions other than the calcium ion concentration should preferably be the same. Further, 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, under conditions in HEPES buffer at 37°C, or using BIACORE (GE Healthcare) or other equipment.

In one embodiment in the context of Disclosure A, the ion concentration-dependent antigen binding domain, the ion concentration-dependent antibody, the ion concentration-dependent antigen-binding domain with increased isoelectric point value, or the ion concentration-dependent antibody with increased isoelectric point value. The antigen-binding activity was higher under conditions of high calcium ion concentration than under conditions of low calcium ion concentration. In this case, the ratio of the antigen-binding activity under the low calcium ion concentration condition to the antigen-binding activity under the high calcium ion concentration condition is not limited; but the KD (dissociation constant) of the antigen under the low calcium ion concentration condition is relative to the high calcium ion concentration The ratio of KD of the conditions, ie, KD (3 μM Ca)/KD (2 mM Ca), is preferably 2 or more, more preferably 10 or more, and 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. Dissociation constant (KD) and apparent dissociation constant (KD) can be determined by known methods, such as BIACORE (GE healthcare), Scatchard plot or flow cytometry.

Alternatively for example the dissociation rate constant (kd) can also 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 representing the ratio of antigen-binding activity, the ratio of the dissociation rate constant (kd) under the condition of low calcium ion concentration to the dissociation rate constant (kd) under the condition of high calcium ion concentration , that is, kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) is preferably 2 or more, more preferably 5 or more, more preferably 10 or more, more preferably 30 or more . The upper limit value of kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) is not limited, and may be any value, for example, 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. Dissociation rate constants (kd) and apparent dissociation rate constants (kd) can be determined using known methods, such as BIACORE (GE healthcare) or flow cytometry.

In one embodiment, the method for making or screening for antigen-binding activity at high calcium ion concentration conditions is higher than at low calcium ion concentration conditions. . Methods include eg those described in WO2012/073992 (eg paragraphs 0200-0213).

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

Or the method could include, for example: (a) contacting the antigen and the antigen binding domain or antibody or library thereof under conditions of high calcium ion concentration; (b) incubating the antigen binding domain or antibody bound to the antigen in step (a) under conditions of low calcium ion concentration; and (c) Dissociating the antigen binding domain or antibody dissociated in step (b).

Or the method could include, for example: (a) contacting an antigen with an antigen binding domain or antibody or library thereof under conditions of low calcium ion concentration; (b) selecting an antigen-binding domain or antibody that does not bind to this antigen or has low antigen-binding capacity in step (a); (c) allowing the antigen binding domain or antibody selected in step (b) to bind to the antigen under conditions of high calcium ion concentration; and (d) isolating the antigen binding domain or antibody bound to the antigen in step (c).

Or the method could include, for example: (a) contacting the antigen-binding domain or antibody or library thereof with a column on which the antigen has been immobilized under conditions of high calcium ion concentration; (b) washing out 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) Isolation of the antigen binding domain or antibody eluted in step (b).

Or the method could include, for example: (a) passing the antigen-binding domain or antibody or its library under low calcium ion concentration conditions through the column to which the antigen has been immobilized to collect the antigen-binding domain or antibody that is not bound to the column and eluted; (b) binding the antigen-binding domain or antibody collected in step (a) to the antigen under conditions of high calcium ion concentration; and (c) isolating the antigen binding domain or antibody that binds to this antigen in step (b).

Or the method could include, for example: (a) contacting the antigen with the antigen binding domain or antibody or library thereof under conditions of high calcium ion concentration; (b) obtaining an antigen binding domain or antibody that binds to this antigen in step (a); (c) incubating the antigen binding domain or antibody obtained in step (b) at a low calcium ion concentration; and (d) an isolated antigen-binding domain or antibody 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 appropriately combined to obtain the most suitable 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 as 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 alters the binding activity of an ion-concentration-dependent antigen-binding domain is introduced into the sequence, at least one amino acid residue that can and may be exposed on the surface of the antigen-binding domain or antibody The charge modifiers are imported together to increase the isoelectric point value.

Alternatively, in the context of the present disclosure A, 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 from immunized animals, or libraries thereof; or antigen-binding domains, antibodies or libraries obtained by introducing mutations of natural or unnatural amino acids capable of sequestering calcium (described below) (e.g., libraries with increased calcium chelatable amino acids, Or a library of calcium-chelatable amino acids was introduced at a specific site).

In one embodiment in the context of Disclosure A, if the ion concentration is calcium ion concentration, the type of amino acid that alters the binding activity of the ion concentration-dependent antigen-binding domain or the ion concentration-dependent antigen-binding domain with increased isoelectric point value is different. It is limited as long as a calcium-binding motif can be formed. 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 an arbitrary 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, those described above, including SEQ ID NO: 7 (corresponding to "Vk5-2"). The calcium-binding motifs contained in the antigen-binding domains are shown.

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

The amino acid having metal chelating activity is not limited to a specific position in the antigen-binding domain. In one embodiment, the amino acid can be located anywhere in the heavy chain variable region and/or the light chain variable region that can form the antigen binding domain. Can include at least 1 amino acid residue based on the heavy and/or light chain, e.g., CDRs (one or more of CDR1, CDR2, and CDR3) and/or of the heavy chain and/or light chain, that will result in a change in the calcium concentration-dependent antigen-binding activity of the antibody FR (one or more of FR1, FR2, FR3, and FR4). Such amino acid residues may be placed at one or more positions such as positions 95, 96, 100a and 101 according to the Kabat numbering of the heavy chain CDR3; positions 30, 31 and 32 according to the Kabat numbering of the light chain CDR1 one or more positions; position 50 according to the Kabat numbering of the light chain CDR2; and/or position 92 according to the Kabat numbering of the light chain CDR3. These amino acid residues can be placed individually or in combination.

Troponin C (Troponin C), calmodulin (calmodulin), parvalbumin (parvalbumin), and myosin (myosin) light chain are known to have multiple calcium-binding sites. In one embodiment, one or more of the light chain CDRl, CDR2, and CDR3 can be designed to include a binding motif thereof. For the above purpose, for example, cadherin domain; EF hand contained in calmodulin; C2 domain contained in protein kinase C; Gla domain contained in coagulation protein factor IX; asialoglycoprotein receptor or mannose C-type lectin of carbohydrate-binding receptor; A domain contained in LDL receptor; Annexin; thrombospondin type-3 domain; and EGF-like domain.

In one embodiment, if the ion concentration is the hydrogen ion concentration (pH), the concentration condition of protons, ie, hydrogen nuclei, is used synonymously with the hydrogen index (pH). 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 (eg, less than 10 ^-3 ), aH ⁺ is almost equal to the hydrogen ionic 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 may focus on the difference in the biological behavior of the pH-dependent antibody against the hydrogen ion concentration condition or pH condition at high hydrogen ion concentration (acidic pH range) and at low hydrogen ion concentration (neutral pH range). For example, in the context of Disclosure A, "the antigen-binding activity under conditions of high hydrogen ion concentration (acidic pH range) is lower than the antigen-binding activity under conditions of low hydrogen ion concentration (neutral pH range)", meaning an 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 should be selected from pH 4.0 to pH 6.5, preferably pH 4.5 to pH 6.5, preferably pH 5.0 to pH 6.5, more preferably pH 5.5 to pH 6.5, preferably pH 6.7 to pH 9.5, more preferably pH 7.0 than pH selected from pH 6.7 to pH 10.0 to pH 9.0, and more preferably pH 7.0 to pH 8.0. Ideally, the above expression means: at the pH in the early endosome in vivo, the antigen-binding activity is weaker than the activity at the plasma pH in vivo; and may especially mean: the antigen-binding activity of the antibody is, for example, weaker at pH 5.8 than at, for example, pH 5.8. pH 7.4.

Whether or not 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 according to known methods, eg, using the context of Disclosure A or the assay described in WO2009/125825. For example, the antigen-binding activity of an antigen-binding domain or an antibody containing this domain to an antigen of interest can be measured and compared at low and high hydrogen ion concentrations. In this case, the conditions other than the hydrogen ion concentration are preferably the same. Further, 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, under conditions in HEPES buffer at 37°C, or using BIACORE (GE Healthcare) or other equipment.

Within the scope of Disclosure A, unless the context specifies otherwise, the "neutral pH range" (also referred to as "low hydrogen ion concentration", "high pH", "neutral pH conditions" or "neutral pH") is not particularly limited is a specific value; but 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, pH 7.0, for example, can be used.

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

In one embodiment in the context of Disclosure A, if the ion concentration is the hydrogen ion concentration, the ion concentration dependent antigen binding domain, the ion concentration dependent antibody, the ion concentration dependent antigen binding domain with increasing isoelectric point value, or the isoelectric point value The increased ion concentration-dependent antigen-binding activity of the antibody was higher at neutral pH than at acidic pH. In this case, the ratio of the antigen-binding activity at neutral pH to the antigen-binding activity at acidic pH is not limited; however, the difference between the dissociation constant (KD) of antigen at acidic pH relative to the KD at neutral pH The ratio, ie KD (acid 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 (acid 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 indicator to represent the ratio of binding activity, the ratio of the dissociation rate constant (kd) for the high hydrogen ion concentration condition to the dissociation rate constant for the low hydrogen ion concentration condition (kd), That is, kd (acid pH range)/kd (neutral pH range) can be 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit value of kd (acid pH range)/kd (neutral pH range) is not limited, and can be any value, for example, 50, 100, or 200.

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

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

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

Or the method could include, for example: (a) contacting the antigen with the antigen binding domain or antibody or library thereof at neutral pH; (b) incubating the antigen binding domain or antibody bound to the antigen in step (a) under acidic pH conditions; and (c) Isolation of the antigen binding domain or antibody dissociated in step (b).

Or the method could include, for example: (a) contacting the antigen with the antigen binding domain or antibody or pool thereof at acidic pH conditions; (b) selecting an antigen-binding domain or antibody that does not bind to this antigen or has low antigen-binding capacity in step (a); (c) allowing the antigen to bind to the antigen binding domain or antibody selected in step (b) at neutral pH; and (d) isolating the antigen binding domain or antibody bound to the antigen in step (c).

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

Or the method could include, for example: (a) allowing the antigen-binding domain or antibody or library thereof to pass through the column to which the antigen has been immobilized under acidic pH conditions to collect the eluted antigen-binding domain or antibody that is not bound to the column; (b) allowing the antigen binding domain or antibody collected in step (a) to bind to the antigen at neutral pH; and (c) isolating the antigen binding domain or antibody bound to the antigen in step (b).

Or the method could include, for example: (a) contacting the antigen with the antigen binding domain or antibody or library thereof at neutral pH conditions; (b) obtaining the antigen binding domain or antibody that binds to this antigen in step (a); (c) incubating the antigen binding domain or antibody obtained in step (b) under acidic pH conditions; and (d) an isolated antigen-binding domain or antibody 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 appropriately combined to obtain the most suitable molecule. The above conditions can be appropriately selected for acidic and neutral pH conditions. An ideal pH-dependent antigen binding domain or pH-dependent antibody can thus be obtained.

In the context of Disclosure A, in one embodiment, the antigen binding domain or antibody as 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 alters the binding activity of an ion-concentration-dependent antigen-binding domain is introduced into the sequence, at least one amino acid residue that can and may be exposed on the surface of the antigen-binding domain or antibody The charge modifiers are imported together to increase the isoelectric point value.

Alternatively, in the context of the present disclosure A, 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 from immunized animals, or Libraries thereof; or antigen-binding domains, antibodies or libraries obtained by introducing mutations of natural or non-natural amino acids having side chains with pKa 4.0-8.0 (described below) (e.g. with side chains having 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 sites). Such preferred antigen binding domains 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 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 in the context of revealing A, the site of introducing the mutation of an amino acid with a side chain pKa of 4.0-8.0 is not limited, and the mutation can be introduced at any site as long as the antigen-binding activity is greater than before substitution or insertion. become weaker in the acidic pH range than in the neutral pH range (increase in KD (acidic pH range)/KD (neutral pH range) value or increase in kd (acidic pH range)/kd (neutral pH range) value) . If the antibody has a variable region or CDR, the site may be within the variable region or CDR. The number of substituted or inserted amino acids can be appropriately determined by those having ordinary knowledge in the technical field; and the number can be one or more. Also, in addition to the above-mentioned substitution or insertion, other amino acids may be deleted, added, inserted, and/or substituted or modified. Substitution or insertion 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, wherein the propylamine in the alanine scanning known to those of ordinary skill in the art Acid replacement to histidine, and/or selected from the antigen binding domain or antibody due to random substitution or insertion mutation of these amino acids, KD (acidic pH range)/KD (neutral pH range) value compared to before 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 is not significantly reduced, not substantially reduced, substantially equal, or increased before and after these mutations; and in other words, the activity can be maintained at least 10% or higher, preferably. 50% or more, more preferably 80% or more, more preferably 90% or more or more. 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 substituted, deleted, added, or inserted with one or more amino acids outside the above-mentioned substitution or insertion site. the site to restore or increase.

In an alternative embodiment, an amino acid with a side chain pKa of 4.0-8.0 can be placed anywhere within the variable region of the heavy and/or light chain forming the antigen binding domain. At least one amino acid residue with a side chain pKa of 4.0-8.0 may be located, for example, in the CDRs (one or more of CDR1, CDR2, and CDR3) and/or FRs (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 region CDR2 according to the Kabat numbering; and/or 89 of the light chain variable region CDR3 according to the Kabat numbering , 90, 91, 92, 93, 94 and 95A at one or more of the amino acid residues. These amino acid residues may be placed individually or in combination as long as the antigen-binding activity of the antibody changes according to hydrogen ion concentration conditions.

In one embodiment, within the scope of Disclosure A, arbitrary amino acid residues can be appropriately used as the amino acid residues that alter the antigen-binding activity of the antigen-binding domain or antibody as a condition of hydrogen ion concentration. In particular such amino acid residues may include those with side chain pKa 4.0-8.0. Electron-donating amino acids 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 a side chain pKa of 6.0-7.0, especially His(H).

Within the scope of Disclosure A recited, 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. Theoretical pi values can be calculated using gene and amino acid sequence analysis software (Genetyx et al.).

In one embodiment, whether compared to the antibody before modification (a native antibody (eg, a native Ig antibody, preferably a native IgG antibody) or a reference antibody (eg, an antibody before antibody modification or library construction or construction)) , the increase in the isoelectric point value of the antibody or the antibody revealing A can be determined by the above method, in addition to or in place of the above method, can be used from, for example, mice, rats, rabbits, dogs. Antibody pharmacokinetic assays in , monkey or human plasma, determined by combinations such as BIACORE, cell proliferation assay, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA) or fluorescent immunoassay.

Within the scope of Disclosure A, "amino acid residues that may be exposed on the surface" may generally refer to amino acid residues located on the surface of the polypeptide constituting the antibody. "Amino acid residues on the surface of a polypeptide" may refer to amino acid residues whose side chains are in contact with solvent molecules (usually mostly water molecules). However, the side chain does not necessarily have to completely contact the solvent molecule. If even a part of the side chain contacts the solvent molecule, the amino acid residue is still defined as "surface-positioned amino acid". 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 interact with amino acid residues even if only part of their side chains are in contact with solvent molecules. One of ordinary skill in the art can use commercial software to create homology models of polypeptides or antibodies, eg, by homology modeling. Alternatively methods such as X-ray crystallization can be used. The amino acid residues likely to be exposed on the surface can be determined using, for example, the axes of a three-dimensional model of the antibody obtained with a computer program such as the InsightII program (Accelrys). Surface exposure sites can be determined using algorithms known in the art (e.g. Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558) 1983). The part that can be exposed to the surface can be determined using software suitable for protein simulation and the three-dimensional structural information obtained from this antibody. The 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. Also, Pacios describes software for determining surface exposure and area for personal computers (Pacios, Comput. Chem 18(4): 377- 386 (1994); J. Mol. Modelling. 1:46-53 (1995)). Based on the above information, as described above, appropriate amino acid residues on the surface of the polypeptide constituting the antibody can be selected.

Methods of increasing the isoelectric point of proteins, such as reducing the number of amino acids with negatively charged side chains at neutral pH (eg, aspartic acid and glutamic acid) and/or increasing the number of amino acids with positively charged side chains Acid numbers (eg, arginine, lysine, and histidine). Amino acid residues with negatively charged side chains, expressed negatively at pH conditions as -1, which is sufficiently greater than the pKa of their side chains, is a theory well known to those of ordinary skill in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the negative charge of the side chain at neutral pH conditions (eg, solutions at pH 7.0) is expressed as -1. Conversely, amino acid residues with positively charged side chains have a positive expression of +1 at pH conditions, which is sufficiently lower than the pKa of their side chains. For example, the theoretical pKa of the side chain of arginine is 12.5, and the positive charge of the side chain at neutral pH conditions (eg, a solution at pH 7.0) is expressed as +1. Amino acid residues with uncharged side chains at neutral pH conditions (eg, solutions at pH 7.0) are known as 15 types of natural amino acids, namely alanine, cysteine, phenylalanine, glycine, Isoleucine, leucine, methionine, aspartic acid, proline, glutamic acid, serine, threonine, valine, tryptophan, and tyrosine. Of course it should be understood that the amino acid for changing the isoelectric point value may be an unnatural amino acid.

From the above, as a method of increasing the isoelectric point of a protein at neutral pH conditions (such as a solution at pH 7.0), a charge modification +1 can be imparted to the protein of interest, for example, by the amino acid sequence of the protein, aspartic acid. (residue) or glutamic acid (residue) (whose side chain has a negative charge of -1) is substituted with an amino acid (residue) with an uncharged side chain. In turn, a charge modification +1 can be imparted to the protein of interest, for example by substituting an amino acid (residue) with an uncharged side chain to arginine or lysine (the side chain of which has a positive charge +1) for the amino group acid (residue). Also, a charge modification +2 can be imparted to the protein of interest, for example by substituting substituted aspartic acid or glutamic acid (which has a negative side chain -1) with arginine or lysine (which has a positive side chain) +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 or inserted into the amino acid sequence of the protein, or deleted from the amino acid sequence. An amino acid with an uncharged side chain and/or an amino acid with a negatively charged side chain of the amino acid sequence of a protein. It should be understood that, in addition to the charge from the side chain, for example, the amino acid residues at the N-terminus and C-terminus of the protein have charges from the main chain (NH ³⁺ of the amino group at the N-terminus and COO ^- of the carbonyl group at the C-terminus) . Therefore, the isoelectric point of the protein can also be increased by some addition, deletion, substitution or insertion of 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 strength of the charge of the amino acid (residues) or the effect of the isoelectric point of a protein is not only (or substantially) dependent on the amino acid sequence constituting the antibody itself or the type of target antigen, but rather on the addition, deletion, substitution or insertion of amino acid residues type and number.

Antibodies that increase the isoelectric point by modifying at least 1 amino acid residue that may be exposed on the surface of the antibody ("antibodies with increased isoelectric point" or "antibodies with increased isoelectric point") can enter more rapidly Elimination of antigens from plasma may be facilitated for cellularity, as eg described or taught in WO2007/114319, WO2009/041643, WO2014/145159 or WO2012/016227.

Among several antibody structural isotypes, for example, IgG antibodies have significantly larger molecular weights and their primary metabolic pathway is not renal excretion. IgG antibodies, the molecule being part of the Fc region, are known to be recovered in a salvage pathway via FcRn, and thus have long in vivo half-lives. IgG antibodies are presumed to be primarily metabolized by endothelial cells via metabolic pathways (He et al., J. Immunol. 160(2):1029-1035 (1998)). Specifically, it is believed that IgG antibodies are recovered by binding to FcRn, and that IgG antibodies that cannot bind are metabolized unless they enter endothelial cells exclusively. If the Fc region is modified to reduce FcRn binding activity, the plasma half-life of the IgG antibody 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 correlated manner, as described, for example, in WO2007/114319 and WO2009/041643. Specifically, the plasma half-life of the antibody whose isoelectric point has been recorded in the above-mentioned literature is reduced, and the amino acid sequence constituting Fc, which may lead to immunogenicity, will still be reduced without modification. Any type of antibody molecule excreted by the kidneys of the pathway, 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, due to the increase in the isoelectric point value, the net positive charge of the antibody with increased isoelectric point increases, so the antibody will be more strongly affected by the net charge than the antibody with no increased isoelectric point. Negative physiochemical Coulomb interaction on the endothelial cell surface; binds the antibody through nonspecific binding and enters the cell, resulting in a shortened half-life of the antibody in plasma or enhanced elimination of the antigen from the plasma. In turn, increasing the isoelectric point of the antibody enhances the uptake of the antibody (or antigen/antibody complex) into cells and/or intracellular permeability, which is thought to decrease the concentration of the antibody in plasma, decrease the bioavailability of the antibody, and/or Shorten the half-life of this antibody in plasma; and these phenomena are expected to generally occur in vivo, regardless of cell type, tissue type, organ type, etc. Furthermore, if the antibody and the 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 affect the decrease or increase of uptake into the cell.

In one embodiment, methods of making or screening for antibodies with increased isoelectric point 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) modifying nucleic acid encoding an antibody that includes 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) culturing host cells to express the nucleic acid; and (c) Collect antibodies from host cell cultures.

Or the method could include, for example: (a') modifying the 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 needed and) select an antibody with an increased isoelectric point compared to the antibody before modification. Here, the antibody or the antibody before modification or the reference antibody as a starting material may be, for example, an ion concentration-dependent antibody. Alternatively, when amino acid residues are modified, amino acids that alter the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence.

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

In an alternative embodiment, the method can be, for example, a method of making a multispecific antibody comprising a first polypeptide and a second polypeptide and optionally a third polypeptide and a fourth polypeptide, comprising: (A) Modification of nucleic acids 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 includes at least one amine group that may be exposed on the surface of the polypeptide acid residue to modify the charge of this amino acid residue to increase the isoelectric point value of this antibody; (B) culturing host cells to express the nucleic acid; and (C) Collection of polyspecific antibodies from host cell cultures.

Or the method could include, for example: (A') Modification of nucleic acids encoding the 1st polypeptide and/or the 2nd polypeptide and optionally the 3rd polypeptide and/or the 4th 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 this amino acid residue to increase the charge of this amino acid residue; (B') culturing host cells to express the nucleic acid; (C') collection of polyspecific antibodies from host cell cultures; and (D') (confirmed and if necessary) selected antibodies with increased isoelectric points compared to those before antibody 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 alter the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence.

Alternatively the method may simply comprise culturing the host cells obtained in step (B) or (B') and collecting the antibody from the cell culture. In this case, the polypeptide to be modified is preferably a homomultimer of the first polypeptide, a homomultimer of the second polypeptide, or a heteromultimer of the first and second polypeptides (if necessary, the third polypeptide). The homomultiplex of the 4th polypeptide, or the heteromultiplex of the 3rd and 4th polypeptides).

In an alternative embodiment, the method may be, for example, a method of making a humanized or human antibody with reduced half-life in plasma, comprising: in the group comprising CDRs selected from human derived CDRs, CDRs derived from animals other than humans, and synthetic CDRs CDRs of the group; FRs of human origin; and antibodies of human constant regions, (1) modifying at least one amino acid residue that may be exposed on the surface of at least one region selected from the group consisting of CDRs, FRs and constant regions The amino acid residue at the corresponding position before modification becomes an amino acid residue with a different charge, so that the isoelectric point of the antibody is increased.

Alternatively the method may comprise, for example, in an antibody comprising CDRs selected from the group consisting of CDRs of human origin, CDRs of animal origin other than human, and synthetic CDRs; FRs of human origin; and antibodies of human constant regions, (I') modification is selected from the CDRs . At least one amino acid residue in at least one region of the group consisting of FR and constant region that may be exposed on the surface becomes an amino acid residue with different charges in the corresponding position of the amino acid residue before modification; and (II') (Confirm and if necessary) 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 alter the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence.

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

In one embodiment of the antibody of Disclosure A, the isoelectric point value may suitably be increased, eg, 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 increase, 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 this antibody before modification or modification (native antibody (such as a natural Ig antibody, preferably a natural IgG antibody) or a control or parent antibody ( For example antibodies before antibody modification or before or during library construction)). Those with ordinary knowledge in this technical field can, according to the application, consider the balance between the pharmacological effect and toxicity, such as the number of antibodies or antigen-binding domains or the isoelectric point of the antigen, and determine the optimal isoelectricity of the antibody that reveals A according to the appropriate routine. point value. Without being bound by a particular theory, it is believed that it would be beneficial in one embodiment to disclose antibodies to A, not just shuttle between plasma and cellular endosomes and repeatedly bind with a single antibody molecule due to the presence of an ion concentration-dependent antigen binding domain In the case of multiple antigens, there is also an increase in the net positive charge of the antibody due to an increase in the isoelectric point, allowing rapid uptake of the antibody into cells. These properties can shorten the half-life of the antibody in plasma, increase the extracellular matrix binding activity of the antibody, or enhance the elimination of antigens from plasma. The optimum isoelectric point value can be determined to benefit from these characteristics.

In one embodiment disclosed in the context of A, the antibody is compared before modification or modification of at least 1 amino acid residue to increase the isoelectric point value (a native antibody (e.g. native Ig antibody, preferably native IgG). antibody) or a control or parent antibody (such as the antibody before antibody modification or library construction or construction), which can be an ion concentration-dependent antibody), an antibody that reveals an increase in the ion concentration-dependent isoelectric point value should be enhanced. Eliminate antigen from plasma, eg, at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold , 4.75 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 in vivo) or Its extracellular matrix-binding activity may preferably be increased by, for example, at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25 times, 4.5 times, 4.75 times or 5 times or more.

In one embodiment in the context of Disclosure A, a pre- or control or parental antibody is compared to an ion concentration-dependent antigen binding domain (a native antibody (eg, a native Ig antibody, preferably a native IgG antibody) for this antibody) (eg antibody before antibody modification or library construction or construction), which can be an antibody with increased isoelectric point), ion concentration-dependent isoelectric point increase Revealing A antibody should enhance the elimination of antigen from plasma, eg, 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 may be should 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 times or 5 times or more.

In one embodiment, the evaluation is to evaluate whether the extracellular matrix binding activity of the antibody of A is compared to that of the antibody before modification or modification (a natural antibody (eg, a natural Ig antibody, which can be a natural IgG antibody) or a control or parent antibody) (eg antibody before antibody modification or library construction or construction), can be an ion concentration-dependent antibody or antibody that increases in isoelectric point) The analytical method for the increase is not limited. For example, the assay can be performed using an ELISA system, which detects the binding of an antibody to the extracellular matrix, by adding the antibody to the extracellular matrix immobilized plate, and adding a labeled antibody against the antibody. Alternatively, as described in Examples 1 to 4 and WO2012/093704 herein, electrochemiluminescence (ECL) can also be used, which can detect extracellular matrix binding capacity with high sensitivity. This method can be carried out, for example, using the ECL system, in which a mixture of antibody and ruthenium antibody is added to an extracellular matrix immobilized plate, and the binding of this antibody to the extracellular matrix is measured based on ruthenium-based electrochemiluminescence. The concentration of the antibody to be added can be appropriately set; the added concentration can be high to increase the sensitivity for detecting extracellular matrix binding. Such extracellular matrix can be derived from animals or plants, as long as it contains glycoproteins such as collagen, proteoglycan, fibronectin, laminin, entactin, fibrin, and perlecan ; Suitable for extracellular matrix from animals. For example extracellular matrices from animals such as human, mouse, rat, monkey, rabbit or dog can be used. For example, natural extracellular matrix of human origin can be used as an antibody pharmacodynamic indicator of human plasma. Conditions for assessing extracellular matrix binding of antibodies are preferably in the neutral pH range, about pH 7.4, which are physiological conditions; however, conditions do not necessarily have to be in the neutral range, and binding can also be in the acidic pH range (eg, about pH 6.0) Evaluate. Alternatively, when assessing extracellular matrix binding of an antibody, the assay can be performed in the presence of the antibody-bound antigen molecule 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 the antibody before modification or modification of at least 1 amino acid residue to increase the pI (a natural antibody (such as a natural Ig antibody, preferably a natural antibody). IgG antibodies) or reference antibodies (eg, antibodies prior to antibody modification or prior to library construction or at the time of 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, more preferably 70% of the binding activity of the antibody before modification or modification. % or 75% or more, preferably 80%, 85%, 90% or 95% or more of the activity. Alternatively, the antibody to reveal A only needs to maintain the binding activity to a degree that allows it to function when it binds 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 "modifying at least 1 amino acid residue that may be exposed on the surface of this antibody" or an equivalent expression may refer to at least 1 amino acid residue that may be exposed on the surface of the antibody One or more additions, deletions, substitutions and insertions are performed. Such modifications preferably include substitution of at least one amino acid residue.

Such substituted amino acid residues may include, for example, substitution of amino acid residues with negatively charged side chains in the amino acid sequence of the antibody of interest to amino acid residues with uncharged side chains, Amino acid residues in side chains are replaced with amino acid residues with positively charged side chains, and amino acid residues with negatively charged side chains are replaced with amino acid residues with positively charged side chains , which can be implemented individually or in appropriate combination. Insertion or addition of amino acid residues can include, for example, insertion or addition of an amino acid with an uncharged charge and/or insertion or addition of an amino acid with a positively charged side chain to the amino acid sequence of the antibody of interest , which can be implemented individually or in appropriate combination. Deletion of amino acid residues can include, for example, deletion of amino acids with an uncharged charge and/or deletion of amino acids with negatively charged side chains from the amino acid sequence of the antibody of interest, which can be implemented alone or in suitable combination.

One or more of these additions, deletions, substitutions and insertions can be appropriately combined by one of ordinary skill in the art to the amino acid sequence of the antibody of interest. Modifications that result in a reduction in the local charge of the amino acid residues are also acceptable, as only the net isoelectric point increase of the antibody that reveals A is sufficient. For example, antibodies that have increased isoelectric point values (too much) can be modified to decrease isoelectric point (slightly), if desired. It is also acceptable to perform the modification of at least 1 amino acid residue simultaneously or at different times for other purposes (eg, increasing antibody stability or reducing immunogenicity) to reduce the local charge of the amino acid residue. 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 an antibody, the natural amino acids are as follows: The amino acid with a negatively charged side chain can be Glu (E) or Asp (D); amino acids with uncharged side chains can be Ala (A), Asn (N), Cys (C), Gln (Q), Gly (G), His (H), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) or Val (V); Amino acids with positively charged side chains can be His (H), Lys (K) or Arg (R).

As detailed in Examples 1 to 4, in solutions of neutral pH (eg pH 7.0), lysine and arginine are almost 100% positively charged when present in the antibody as residues, while histidine is It exists in the antibody in the form of residues, and is only about 9% positively charged, and most of the rest are presumed to have no charge. Therefore, Lys(K) or Arg(R) should be selected as the amino acid with a positively charged side chain.

In one embodiment, the antibody of disclosure A preferably has variable and/or constant regions. In addition, the variable region preferably has a heavy chain variable region and/or a light chain variable region, and/or preferably has CDRs (eg, one or more of CDR1, CDR2, and CDR3) and/or FRs (eg, FR1, FR2, one or more of FR3 and FR4). The constant region preferably has 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 the human lambda chain constant region). It is also possible to use modified variants of these constant regions.

In one embodiment, modifying at least one amino acid residue that may be exposed on the surface of the antibody can modify a single amino acid or a combination of amino acids. An ideal approach would be to introduce a combination of multiple amino acid substitutions at the site of the amino acid that can be exposed on the surface of the antibody. Again, without limitation, such a plurality of amino acid substitutions are preferably 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 substituted with a positively charged side chain (such as Lys ( K) or Arg (R)); or if a positively charged pre-existing amino acid is used (such as Lys (K) or Arg (R)), for example, a sterically close to the amino acid can be used. or multiple amino acids (which, depending on the state, can include amino acids embedded within the antibody molecule) are substituted with positively charged amino acids to create a dense state with locally positive charges at the site of steric proximity. "Site" is not particularly limited; it may mean that one or more amino acid substitutions are introduced, for example, within 20 angstroms, preferably within 15 angstroms, and more preferably within 10 angstroms. Whether the amino acid substitution site of interest is exposed on the antibody molecule The surface or whether the amino acid substitution site is in close proximity to other amino acid substitution sites or pre-existing amino acids as described above can be assessed using known methods such as X-ray crystallography.

In addition to the methods described above, methods for providing multiple positive charges at sites that are sterically close to each other can include the use of amino acids that are otherwise positively charged in native IgG constant regions. Such amino acids include, for example: arginine at positions 255, 292, 301, 344, 355 and 416 according to EU numbering; and 121, 133, 147, 205, 210, 213, 214, Lysine acids 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 positively charged amino acids for sites that are sterically close to these positively charged amino acids.

If the antibody of A is revealed to have variable regions (which can be modified), amino acid residues that are not shielded from antigen binding (ie, those that may still be exposed on the surface) can be modified, and/or no amino acid modifications can be introduced Amino acid modifications that do not (substantially) inhibit antigen binding may be performed at sites shielded from antigen binding. 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 acid of the antigen binding domain can be modified such that the modification does not (substantially) reduce the ability to change in accordance with ion concentration conditions The binding activity of amino acid residues (eg, at calcium binding motifs or histidine insertion sites and/or histidine substitution sites) or amino acid residues can be modified in accordance with ionic concentration conditions A site other than an amino acid residue that alters the antigen-binding activity of an 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 type or location so that the isoelectric point of the antibody does not fall below acceptable levels. If the isoelectric point of the antibody is below acceptable levels, the overall isoelectric point of the antibody can be increased by modifying at least one amino acid residue of the antibody molecule that may be exposed on the surface.

Without limitation, FR sequences with a high electrical point are preferably selected from human germline FR sequences or sequences of equivalent regions, and these amino acids may sometimes be modified.

If the antibody disclosed in A has an FcγR binding domain (which can be a binding domain for any of the following FcγR isotypes and paratypes) and/or a constant region (which can be modified) of an FcRn binding domain, at least 1 of the constant regions The sites for modification of amino acid residues that may be exposed on the surface can optionally be amino acid residues of amino acid residues other than the FcγR binding domain and/or the 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 is desirable to select a site that does not (substantially) affect binding activity or binding affinity for FcγR and/or FcRn, or if it will. The effect is the biologically or pharmacologically acceptable site.

In one embodiment, the modification of at least 1 amino acid of the antibody of disclosure A with increased isoelectric point is made by modifying at least 1 amino acid residue of the variable region (which may be modified) that may be exposed on the surface The positions of residues are not limited; however, such positions may be selected from the group consisting of the following according to the Kabat numbering: (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 CDRs of the heavy chain variable region; (c) of 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) 24 of the CDRs 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, such as Lys (K), Arg (R), GIn (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 themselves have the effect of increasing the isoelectric point value of the antibody sufficiently to assist in increasing the isoelectric point value increase of the antibody revealing A. Such positions to assist 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 the Kabat numbering.

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

When selecting a modification site of at least one amino acid residue, for example, from the above-mentioned group, 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 Bit: 39K, 39R, 39Q, 39G, 39S or 39N; (g) For 41 bit: 41K, 41R, 41Q, 41G, 41S or 41N; (h) For 43 bit: 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, a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of any of the foregoing amino acid positions are modified. In some embodiments, a combination of 1-20, 1-15, 1-10, or 1-5 of any of the foregoing amino acid positions is modified.

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

77R/82aN/82bR; 77R/82aG/82bR; 77R/82aS/82bR; 77R/85G; 41R/44R; 82aN/82bR; 82aG/82bR; ; 82K/82bR; 82bR/83R; 77R/85R; or 63R/64K.

Similarly, the modified amino acid type of the light chain variable region is for example: (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, combinations of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of any of the foregoing amino acid positions are modified. In some embodiments, a combination of 1-20, 1-15, 1-10, or 1-5 of the foregoing amino acid positions is modified.

Non-limiting examples of combinations of amino acid position modifications in the light chain variable region are, for example, positions 24 and 27; 25 and 26; 41 and 42; 42 and 76; 52 and 56 according to the Kabat numbering; Bits 65 and 79; Bits 74 and 77; Bits 76 and 79; Any 2 or more positions selected from the group consisting of positions: 16, 24, and 27; Any 2 or more positions: 24, 27, and 37 positions; any 2 or more positions selected from the group of the following constituting positions: 25, 26, and 37 positions; selected from the following group of constituting positions Any 2 or more positions: positions 27, 76, and 79; any 2 or more positions selected from the group of the following positions: positions 41, 74, and 77; selected from the group of the following positions Any 2 or more positions: 41, 76, and 79; any 2 or more positions selected from the following group of constituting positions: 24, 27, 41, and 42; selected from the following group of constituting positions Any 2 or more positions of: 24, 27, 52, and 56; any 2 or more positions selected from the group consisting of: 24, 27, 65, and 69; selected from the following Any 2 or more positions from the group of positions: 24, 27, 74, and 77 positions; any 2 or more positions selected from the following group of constituting positions: 24, 27, 76, and 79 positions; Any 2 or more positions selected from the group consisting of the following positions: 25, 26, 52, and 56; any 2 or more positions selected from the group consisting of the following positions: 25, 26, 65, and 69 bits; selected from any 2 or more of the following group of positions: 25, 26, 76, and 79; selected from any 2 or more of the following group of positions: 27, Positions 41, 74, and 77; selected from any 2 or more of the following group of positions: Positions 27, 41, 76, and 79; selected from any 2 or more of the following group of positions Positions: Positions 52, 56, 74, and 77; selected from any 2 or more positions of the following group of positions: Positions 52, 56, 76, and 79; selected from any of the following group of positions 2 or more positions: 65, 69, 76, and 79; selected from any 2 or more of the following group of positions: 65, 69, 74, and 77; selected from the following group of positions Any 2 or more positions from the group: positions 18, 24, 45, 79, and 107; any 2 or more positions selected from the group consisting of positions: 27, 52, 56, 74, and 77 positions ; any 2 or more positions selected from the group consisting of the following positions: 27, 52, 56, 76, and 79 positions; any 2 or more positions selected from the group consisting of the following positions: 27, 65 , 69, 74, and 77; selected from any 2 of the following groups of positions or more positions: 27, 65, 69, 76, and 79; selected from any 2 or more positions from the group consisting of: 41, 52, 56, 74, and 77; selected from the following Any 2 or more positions from the group of positions: 41, 52, 56, 76, and 79; any 2 or more positions selected from the following group of positions: 41, 65, 69, 74, and 77; selected from any 2 or more of the following group of positions: 41, 65, 69, 76, and 79; selected from any 2 or more of the following group of positions: Positions 24, 27, 41, 42, 65, and 69; selected from any 2 or more positions from the group of the following constituent positions: 24, 27, 52, 56, 65, and 69; selected from the following constituent positions Any 2 or more positions from the group of positions: 24, 27, 65, 69, 74, and 77; any 2 or more positions selected from the following group of positions: 24, 27, 65, 69 , 76, and 79; selected from any 2 or more of the following group of positions: 24, 27, 41, 42, 74, and 77; selected from any 2 of the following group of positions or more positions: 24, 27, 52, 56, 74, and 77 positions; any 2 or more positions selected from the group consisting of the following positions: 24, 27, 41, 42, 76, and 79 positions; Any 2 or more positions selected from the group consisting of the following positions: 24, 27, 52, 56, 76, and 79; any 2 or more positions selected from the group consisting of the following positions: 24, Positions 27, 74, 76, 77, and 79; selected from any 2 or more positions from the group of the following constituent positions: positions 52, 56, 65, 69, 74, and 77; or selected from any of the following constituent positions Any 2 or more positions in the group: positions 52, 56, 65, 69, 76 and 79, wherein the modified amino acid at each position can be selected from any of the above amino acids in terms of side chain charge For example, but not limited to Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S) or Asn (N).

24R/27R; 24K/27K; 25R/26R; 25K/26K; 41R/42K; 42K/76R; 52R/56R; 65R/79K; 74K/77R; 76R/79K; 16K /24R/27R; 24R/27R/ 37R; 25R/26R/37R; 27R/76R/79K; 41R/74K/77R; 41R/76R/79K; 24R/27R/41R/42K; 24R/27R/52R/56R ; 24R/27R/52K/56K; 24R/27R/65R/69R; 24R/27R/74K/77R; 24R/ 27R/76R/79K; 25R/26R/52R/56R; 25R/26R/52K/56K; 25R /26R/65R/69R; 25R/26R/ 76R/79K; 27R/41R/74K/77R; 27R/41R/76R/79K; 52R/56R/74K/77R; 52R/56R/76R/ 79K; 65R/69R /76R/79K; 65R/69R/74K/77R; 18R/24R/45K/79Q/107K; 27R/52R/56R/74K/77R; /77R; 27R/65R/69R/76R/79K; 41R/52R/56R/74K/77R; 41R/52R/56R/76R/79K; 41R/65R/69R/74K/77R; 41R/65R/ 69R/76R /79K; 24R/27R/41R/42K/65R/69R; 24R/27R/52R/56R/65R/69R; 24R/27R/ 65R/69R/74K/77R; 24R/27R/65R/69R/76R/79K ; 24R/27R/41R/42K/74K/77R; 24R/ 27R/52R/56R/74K/77R; 24R/27R/41R/42K/76R/79K; 24R/27R/52R/56R/76R/79K; 24R /27R/74K/76R/77R/79K; 52R/56R/65R/69R/74K/77R; or 52R/56R/65R/69R/76R/79K.

In WO2007/114319 or WO2009/041643, the effect of increasing the isoelectric point by modifying some amino acid residues of the variable region has been explained or demonstrated by theoretical evidence, homology models or experimental techniques not exclusively (or substantially). above) depends on the type of the amino acid sequence constituting the antibody itself or the 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, antigen-binding activity against several types of antigens is (substantially) maintained, or at least can be expected to be maintained with a high probability by one of ordinary skill in the art.

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

In an alternative or further embodiment, for example, known methods such as X-ray crystallography or from antibody constant regions (preferably human constant regions, more preferably human Ig-type constant regions, still more preferably human IgG-type constant regions) can be used, for example. The homology simulation method of the homology model constructed by the region, but not limited thereto) to identify the amino acid residues of the constant region of the antibody that may be exposed on the surface to determine the antibody used to make the revealed A with increased isoelectric point. 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; but this site is preferably selected from the group consisting of: 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 following groups 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 should also be selected from the group consisting of: 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, the amino acid of each position can be selected from above-mentioned amino acid after modification, for example Lys (K), Arg (R), Gln (Q) or Asn (N) but not limited in terms of side chain charge here. When selecting a modification site of at least one amino acid residue, for example, selected from the above-mentioned group, for example, the amino acid type after modification of each site can be as follows: In 254-bit: 254K, 254R, 254Q or 254N; in 258-bit, 258K, 258R, 258Q or 258N; In 281-bit: 281K, 281R, 281Q or 281N; In 282-bit: 282K, 282R, 282Q or 282N; At 285-bit: 285K, 285R, 285Q or 285N; At 309-bit: 309K, 309R, 309Q or 309N; In 311-bit: 311K, 311R, 311Q or 311N; In 315-bit: 315K, 315R, 315Q or 315N; On 327-bit: 327K, 327R, 327Q or 327N; On 330-bit: 330K, 330R, 330Q or 330N; On 342-bit: 342K, 342R, 342Q or 342N; On 311-bit: 343K, 343R, 343Q or 343N; On 345-bit: 345K, 345R, 345Q or 345N; On 356-bit: 356K, 356R, 356Q or 356N; On 358-bit: 358K, 358R, 358Q or 358N; On 359-bit: 359K, 359R, 359Q or 359N; On 361-bit: 361K, 361R, 361Q or 361N; On 362-bit: 362K, 362R, 362Q or 362N; On 384-bit: 384K, 384R, 384Q or 384N; On 385-bit: 385K, 385R, 385Q or 385N; On 386-bit: 386K, 386R, 386Q or 386N; On 387-bit: 387K, 387R, 387Q or 387N; At the 389th position: 389K, 389R, 389Q or 389N; At the 399th position: 399K, 399R, 399Q or 399N; In 400-bit: 400K, 400R, 400Q or 400N; In 401-bit: 401K, 401R, 401Q or 401N; In 402 bit: 402K, 402R, 402Q or 402N; In 413 bit: 413K, 413R, 413Q or 413N; On 418-bit: 418K, 418R, 418Q or 418N; On 419-bit: 419K, 419R, 419Q or 419N; In 421 bits: 421K, 421R, 421Q or 421N; In 433 bits: 433K, 433R, 433Q or 433N; In 434-bit: 434K, 434R, 434Q or 434N; and in 443-bit: 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 of Disclosure A, the net isoelectric point of this antibody can be obtained by modifying at least one amine that may be exposed on the surface of the variable region (may be modified) and constant region (may be modified) as described above base acid residues.

Within the scope of disclosures A and B described herein, when the antibody of A or B is disclosed as an IgG-type antibody or a molecule derived therefrom, the heavy chain constant region of the antibody may include an IgG1-type, IgG2-type, IgG3-type or IgG4-type. constant region. For disclosure of A or B, the heavy chain constant region may be, but is not limited to, a human heavy chain constant region. 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 between individuals (Methods Mol. Biol. 882:635-80 (2012); Sequences of proteins of immunological interest, NIH Publication No. 91-3242) . Examples include human IgGl 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 are aspartic acid at position 356 and leucine at position 358 according to the EU numbering method, while G1m3 is glutamic acid at position 356 and 358 according to the EU numbering method. Methionine. However, there are no reports suggesting that these reported subtypes differ significantly in primary antibody function and properties. Therefore, a person with ordinary knowledge in the technical field can easily predict various evaluations using a specific subtype, and the results are not limited to the subtype used, 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 IgGl", "human IgG2", "human IgG3" or "human IgG4", this subtype is not limited to a particular 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 suitably be, but is not limited to, a human light chain constant region. There are reports, such as Sequences of proteins of immunological interest, NIH Publication No. 91-3242, that some paratype sequences are derived from genetic polymorphism of the human kappa chain constant region and the human lambda chain constant region. Such subtypes include, for example, the human kappa chain constant region (SEQ ID NO:22) and the human lambda chain constant region (SEQ ID NO:23). However, no reports suggest that these reported subtypes differ significantly in primary antibody function and properties. Therefore, those skilled in the art can easily understand that any subtype can be expected to have the same effect (hereinafter also collectively referred to as native (human) IgG) when referring to specific subtypes within the scope of disclosure A and B described herein. (type) constant region).

In addition, the Fc region of a natural IgG antibody constitutes a part of the constant region of a natural IgG antibody, so if it is revealed that the antibody of 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 native (human) IgG (type) Fc region) contained in the constant region of the Fc region. The Fc region of native IgG may refer to an Fc region having the same amino acid sequence as the Fc region derived from naturally occurring IgG. Specific examples of the Fc region of native 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 IgG1 constant region (SEQ ID NO: 19) The Fc region (the Fc region of the IgG class) contained in the IgG4 constant region (SEQ ID NO: 21) may refer to, for example, cysteine 226 to C-terminal according to EU numbering or proline 230 to position 230 according to EU numbering. C terminal).

In one embodiment, 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). and/or light chain constant region) or native (preferably the Fc region of human IgG).

Within the scope of Disclosure A, WO2013/081143 reports that, for example, it is possible to form a multivalent immune complex with multimeric antigens and ion concentration-dependent antibodies (multivalent antigen-antibody complexes) and by recognizing 2 or more of the monomeric antigens. Multiple epitopes can form multiple specific ion concentration-dependent antibodies of multivalent immune complexes or multiple antibody binding site ion concentration-dependent antibodies (multivalent antigen-antibody complexes), which can bind more strongly to FcγR, FcRn, Complement receptors, due to avidity (avidity) (between multiple epitopes and The sum of the binding strengths of the binding sites), so the antibody can be taken up into the cell more quickly. Therefore, when the isoelectric point is increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody, the above-mentioned ion concentration-dependent antibody can form a multivalent immune complex with a multimeric antigen or a unitary antigen, which can As an antibody revealing A (ion concentration-dependent antibody with increased isoelectric point value). Those of ordinary skill in the art will understand that an ion concentration-dependent antibody that can form a multivalent immune complex with a multimeric or monomeric antigen has an increased isoelectric point value compared to an antibody that cannot form a multivalent immune complex The ion concentration-dependent antibody with increasing value can be taken up into the cell faster. Those of ordinary skill in the art will also appreciate that, in one embodiment, the activity of the antibody revealing A to FcRn and/or FcγR may be increased under neutral pH conditions, in this case, with multimeric antigens or monomeric antigens. Antigens form multivalent immune complexes with increased ion concentration-dependent antibody uptake into cells with increased isoelectric point values.

In one embodiment, the antibody disclosed in A may be a one-armed antibody (including all embodiments of the one-armed antibody described in WO2005/063816). In general, a one-armed antibody is an antibody lacking the two Fab regions that are usually present in an IgG antibody, and can be produced, for example, by the method described in WO2005/063816, without limitation. Without limitation, in an IgG-type antibody with a heavy chain whose structure is, for example, VH-CH1-hinge-CH2-CH3, if the Fab region is cleaved at a position closer to the N-terminus than the hinge (such as VH or CH1), the antibody will Expressed in a way that includes additional sequences, when the Fab region is cleaved closer to the C-terminus than the hinge (eg CH2), the Fc region will have an incomplete form. Therefore, without limitation, from the viewpoint of the stability of the antibody molecule, the one-armed antibody is preferably produced by cleaving the hinge region (hinge) of one of the two Fab regions of the IgG antibody. More preferably, the cleaved heavy chain is linked to the uncleaved heavy chain by intramolecular disulfide bonds. WO2005/063816 has reported that such one-armed antibodies have better stability than Fab molecules. Antibodies with increased or decreased isoelectric point values can also be produced using such one-armed antibodies. Also, if the antibody with increased isoelectric point value introduced into the ion concentration-dependent antigen binding domain is a one-armed antibody, the half-life of the antibody in plasma is compared with that of the antibody without the increased isoelectric point value of the ion concentration-dependent antigen binding domain. can be further shortened, cellular uptake of the antibody can be further enhanced, antigen elimination from plasma can be further enhanced, or the antibody's affinity for the extracellular matrix can be further increased.

Without being bound by a particular theory, if a cellular uptake-accelerating effect of a one-armed antibody is expected in one embodiment, it can be envisioned, but not limited to, that the isoelectric point value of the soluble antigen is lower than that of the antibody. The net isoelectric point of a complex consisting of antibody and antigen can be calculated using known methods by considering the complex as a single molecule. 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-type IgG antibody molecule (with 2 Fabs) binds to a single low isoelectric point soluble antigen, the latter complex has a lower net isoelectric point than to two low isoelectric point soluble antigens. When such a normal-type antibody is converted into a one-armed antibody, only one antigen can bind to a single molecule of the antibody; the decrease in the isoelectric point of the complex due to binding of the second antigen can be suppressed. 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, the conversion to a one-armed antibody allows an increase in the isoelectric point of the complex compared to normal antibodies, thereby accelerating uptake into cells.

Without limitation, when the isoelectric point of the Fab of an IgG-type antibody molecule (with 2 Fabs) is usually lower than that of Fc, the conversion to a one-armed antibody will increase the amount of friction between the one-armed antibody and the antigen complex. net isoelectric point. Also, when implementing such conversion to a one-armed antibody, from the viewpoint of the stability of the one-armed antibody, one of the Fabs should be cleaved at the hinge region located at the junction between the Fab and the Fc. In this case, by selecting a site that increases the isoelectric point value of the one-armed antibody to a desired level, an effective increase in the isoelectric point value can be expected.

Therefore, those with ordinary knowledge in the technical field can understand that it is not excluded (or substantially) dependent on the amino acid sequence of the antibody itself and the type of soluble antigen, by calculating the theoretical isoelectric point value of the antibody (the theory of Fc, etc. Electric point value and Fab theoretical isoelectric point value) and the theoretical isoelectric point value of soluble antigen, and predict the relationship between the difference of the theoretical isoelectric point value, in order to convert this antibody into a one-arm antibody, the isoelectric point value of the antibody can be increased, and consequent cellular uptake of this antigen can be accelerated.

In one embodiment, the antibody that discloses A or B can be a multispecific antibody, and the multispecific antibody can be, but is not limited to, a bispecific antibody. The multispecific antibody may be a multispecific antibody comprising a first polypeptide and a second polypeptide. Herein, "a polyspecific antibody comprising a first polypeptide and a second polypeptide" refers to an antibody that binds 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, each of the first polypeptide and the second polypeptide preferably contains a heavy chain constant region. In another embodiment, the multispecific antibody may comprise a third polypeptide and a fourth polypeptide, each comprising a light chain variable region and preferably a light chain constant region. In this case, polypeptides 1 to 4 can be assembled together to form a multispecific antibody.

In one embodiment, if the antibody of A is revealed to be a polyspecific antibody and the polyspecific antibody contains a heavy chain constant region, reducing its isoelectric point value, for example the following sequence can be used: IgG2 or IgG4 sequence at position 137; at 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; IgG4 sequence at position 274; IgG2, IgG3 or IgG4 sequence at position 274; IgG1, IgG2 or IgG4 sequence at position 276; IgG4 sequence at position 355; IgG3 sequence at position 392; IgG4 sequence at position 419; IgG1, IgG2 or IgG4 sequence at position 435. At the same time to increase its isoelectric point value, 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; IgG2 sequence at position 217; IgG2 sequence at position 233; IgG1, IgG2 or IgG3 sequence at position 268; IgG1 sequence at position 274; IgG3 sequence at position 276; IgG1, IgG2 or IgG3 sequence at position 355; IgG1, IgG2 or IgG4 sequence at position 392; IgG1, IgG2 or IgG3 sequence at position 419; or IgG3 sequence at position 435.

In one embodiment, if the antibody of A is revealed to have two heavy chain constant regions, the isoelectric point values of the two heavy chain constant regions may 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, isoelectric point differences can be introduced between the two heavy chain constant regions. The modification site of at least one amino acid residue in order to introduce such a difference in isoelectric point in the constant region may be as above or according to EU numbering, and the selection of the heavy chain constant region described in WO2009/041643 consists of the following The position of the group: 137-bit, 196-bit, 203-bit, 214-bit, 217-bit, 233-bit, 268-bit, 274-bit, 276-bit, 297-bit, 355-bit, 392-bit, 419-bit and 435-bit. Alternatively, the amino acid residue at position 297 of the glycation site may be modified to remove the sugar chain, since removal of the sugar chain from the heavy chain constant region results in a difference in isoelectric point.

In one embodiment, the antibody disclosed in A or B may be a polyclonal antibody or a monoclonal antibody, and preferably a mammalian-derived monoclonal antibody. Monoclonal antibodies include those produced by fusion tumors or by host cell makers genetically engineered with expression vectors bearing the antibody genes. The antibody of this disclosure 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 antibodies that disclose A or B may be derived from, without limitation, any animal species (e.g., human; or non-human animals, such as mouse, rat, hamster, rabbit, monkey, macaque, rhesus, arabian baboon). , chimpanzee, goat, sheep, dog, cow or camel) or any bird; and this antibody is preferably from 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 scope of Disclosures A and B described herein, an Fc receptor (also referred to as "FcR") refers to a receptor protein or a molecule or Fc derived therefrom that binds to the Fc region of an immunoglobulin (antibody). region variant. Within the scope of Disclosure A described herein, eg, Fc receptors for IgG, IgA, IgE, and IgM are known to be FcγR, FcαR, FcεR, and FcμR each, and Fc within the scope of Disclosure A and B described herein The receptor can also be, for example, FcRn (also known as a "nascent Fc receptor").

Within the scope of Disclosure A, "FcyR" may refer to a receptor protein that binds to the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a molecule or Fc region variant derived therefrom, and may include substantially 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, FcyRI (CD64) including the isotypes FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32) including the isotypes FcyRIIa (including the subtypes H131 (H-type) and R131 (R-type)) , FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including the isotypes FcγRIIIa (including paratypes V158 and F158) and FcγRIIIb (including paratypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and All unidentified human FcyRs and FcyR isoforms and paratypes. Also FcyRIIb1 and FcyRIIb2 have been reported to be cleavage variants of human FcyRIIb (hFcyRIIb). There is also a report on a cleavage variant called FcyRIIb3 (Brooks et al., J. Exp. Med, 170: 1369-1385 (1989)). In addition to the methods described above, hFcyRIIb includes, for example, all cleavage variants registered with 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 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.

FcyRs can be from any organism, but not limited to, human, mouse, rat, rabbit or monkey. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16) and FcyRIII-2 (CD16-2) and all unidentified mouse FcyRs, and FcyR isotypes and paratypes. Such desirable FcyRs include, for example, human FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16) or FcyRIIIB (CD16). Since FcγR is in a membrane form in vivo, it can be artificially converted into an appropriate soluble form and used in an experimental system.

For example, as shown in WO2014/163101, the polynucleotide sequence and amino acid sequence of FcγRI may be the sequences shown in NM_000566.3 and NP_000557.1, respectively; the polynucleotide sequence and amino acid sequence of FcγRIIA may be BC020823.1 and BC020823.1 The sequences shown respectively by AAH20823.1; the polynucleotide sequence and amino acid sequence of FcγRIIB can be the sequences shown respectively by BC146678.1 and AAI46679.1; the polynucleotide sequence and amino acid sequence of FcγRIIIA can be The sequences shown in each of BC033678.1 and AAH33678.1; the polynucleotide sequence and amino acid sequence of FcγRIIIB can be the sequences shown in each of 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 form) or arginine (R form) (J. Exp. Med. 172:19-25, 1990).

In FcγRIa including FcγRIa, FcγRIb (CD64), with FcγRIc, with FcγRIII (CD16) including FcγRIIIa (including subtypes V158 and F158), the α chain that binds to the Fc region of IgG is linked to a common γ chain with ITAM , the ITAM transmits activation signals intracellularly. FcyRIIIb (including paratype FcyRIIIb-NA1 and FcyRIIIb-NA2) are GPI anchor proteins. In contrast, the cytoplasmic domain of FcyRII (CD32), which includes FcyRIIa (including subtypes H131 and R131) and FcyRIIc isoforms, contains ITAM. These receptors are expressed on many immune cells such as macrophages, adipocytes and antigen presenting cells. Activation signals transmitted by these receptors bound to the Fc region of IgG promote macrophage phagocytosis, production of inflammatory interleukins, degranulation of adipocytes, and increased antigen-presenting cell function. Within the scope of A and B disclosed herein, FcyRs capable of transmitting activation signals as described above are also referred to as activated FcyRs.

The cytoplasmic domain of FcyRIIb (including FcyRIIb-1 and FcyRIIb-2) contains ITIM, which transmits inhibitory signals. In B cells, the cross-linking between FcγRIIb and B cell receptor (BCR) inhibits the activation signal from BCR, resulting in inhibition of BCR production of antibodies. In macrophages, cross-linking of FcγRIII to FcγRIIb inhibits phagocytic ability and the ability to produce inflammatory interferons. Within the scope of A and B disclosed herein, FcyRs capable of transmitting inhibitory signals as described above are also referred to as inhibitory Fcy receptors.

Within the scope of disclosure A, whether the binding activity of an antibody or Fc region (variant) to various FcγRs is increased, (substantially) maintained or decreased compared to that of the antibody or Fc region (variant) before modification, depending on the There are method evaluations known to those of ordinary skill in the art. Such a method is not particularly limited, and the methods described in the examples can be used, for example, BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010, which is based on surface plasmon resonance (SPR) phenomenon ). Alternatively, eg ELISA and Fluorescence Activated Cell Sorting (FACS) and ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) can be used. For these assays, the extracellular domain of human FcyR can be used as eg WO2013/047752).

For pH conditions for measuring the binding activity between an FcγR-binding domain contained in an antibody or an Fc region (variant) and an FcγR, acidic or neutral pH conditions can be appropriately used. For the temperature used for the measurement conditions, the binding activity (binding affinity) between the FcyR binding domain and the FcyR can be assessed at, for example, a temperature between 10°C and 50°C. The ideal temperature for determining the binding activity (binding affinity) of the human FcγR-binding domain to FcγR is, for example, 15°C to 40°C. More desirably, in order to determine the binding activity (binding affinity) of the FcγR binding domain to FcγR, any temperature from 20°C to 35°C can be used, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, Any of 29, 30, 31, 32, 33, 34, and 35°C. 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). variants), and within the scope of Disclosures A and B described herein, preferably the FcγR binding domain and the FcRn binding domain.

In one embodiment, when the antibody of A is revealed 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 may be a domain having the activity of directly or indirectly binding to FcγR.

In one embodiment, when the antibody of A is revealed to have FcγR-binding activity, it is desirable that the FcγR-binding activity of the antibody at neutral pH is increased compared to that of a reference antibody containing a native IgG constant region. Considering the point of view of comparing the FcγR binding activities between the two, it is not limited but appropriate to reveal that the antibody of A and the reference antibody containing the native IgG constant region have the same amino acid sequence in this region (eg, variable region), rather than this One or more amino acid residues are modified in the constant region of the antibody of A.

In one embodiment, when the antibody of A is revealed to have FcγR binding activity or increased FcγR binding activity at neutral pH conditions (eg, pH 7.4), without being bound by theory, it is believed that this antibody combination possesses the following properties: The property of shuttling with cellular endosomes is the property of repeatedly binding to multiple antigens with a single antibody molecule by having an ion concentration-dependent antigen-binding domain; by the increase of the isoelectric point and the increase of positive charge of the whole antibody, it is rapidly absorbed. 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 antigen from plasma can be further promoted; thus revealing the antibody of A is beneficial. Those of ordinary skill in the art can routinely determine the optimal isoelectric point value for such an antibody to benefit from these properties.

In one embodiment, the FcyR-binding activity of an FcyR-binding domain is higher than the FcyR-binding activity of the Fc region or constant region of native human IgG, wherein the sugar chain attached at position 297 according to EU numbering is a fucose-containing sugar Chains can be prepared by modifying amino acid residues in the Fc region or constant region of native human IgG (see WO2013/047752). A domain that binds to any structure of FcyR can also be used as an FcyR-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 can 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 FcγRI-binding cyclic peptides as described by Bonetto et al., FASEB J. 23(2):575-585 (2009). Does an FcγR-binding domain have higher FcγR-binding activity than those linked at position 297 according to EU numbering The sugar chain is a fucose-containing sugar chain, and the FcγR-binding activity of the Fc region or constant region of natural human IgG can be appropriately evaluated using the above-described method.

In one embodiment of Disclosure A, the starting FcyR binding domain preferably comprises, for example, a (human) IgG Fc region or a (human) IgG constant region. Any Fc region or constant region can be used as the starting Fc region or starting constant region as long as the variant of the starting Fc region or starting constant region can bind to human FcγR in the neutral pH range. An Fc region or constant region obtained by further modification of an Fc region or a constant region from which amino acid residues have been modified from the Fc region or constant region can also be obtained as an Fc of Reveal A. region or constant region. 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 comprise a known Fc region or 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 turn, the starting FcrR binding domain 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 IgGl, IgG2, IgG3 or IgG4 can be used as the appropriate starting FcγR binding domain, and also means that within the scope of Disclosure A described herein, the IgG class or subclass from any organism An Fc region or constant region of a subclass can be used as the starting Fc region or starting constant region. Examples of native IgG variants or publicly known and documented modified forms such as Strohl, Curr. Opin. Biotechnol. 20(6):685-691 (2009); Presta, Curr. Opin. Immunol. 20(4): 460-470 (2008); Davis et al., Protein Eng. Des. Sel. 23(4):195-202 (2010); WO2009/086320, WO2008/092117; WO2007/041635; and WO2006/105338, but not limited to this.

In one embodiment, the amino acid residues of the starting FcγR binding domain, starting Fc region, or starting constant region may include, for example, one or more mutations: such as substitutions that are different from the starting Fc region or starting constant region. amino acid residues; insertion of one or more amino acid residues into the starting Fc region or amino acid residues of the starting constant region; or deletion of one or more amines from the starting Fc region or starting constant region acid residues. The amino acid sequence of the modified Fc region or constant region preferably contains a portion of the amino acid sequence of the Fc region or constant region that does not occur in nature. Such variants must have less than 100% sequence identity or similarity to the starting Fc region or starting constant region. For example, the variant is about 75% less than 100%, more preferably about 80% less than 100%, more preferably about 85% less than 100%, relative to the amino acid sequence of the starting Fc region or starting constant region. %, still more preferably about 90% less than 100%, still more preferably about 95% 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 differs from the starting Fc region or starting constant region by at least 1 amino acid.

In one embodiment, an Fc region or constant region having FcγR binding activity in the acidic pH range and/or in the neutral pH range, which can be included in the antibody of Disclosure A, can be obtained by any method. Specifically, a variant of an Fc region or constant region having FcγR-binding activity in the neutral pH range can be obtained from the amino acid of a human IgG antibody that can be used as the starting Fc region or starting constant region. Suitable IgG antibody Fc regions or IgG antibody constant regions for modification may include, for example, the Fc regions or constant regions of human IgG (IgGl, 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 gene polymorphism are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, can be Use any of them for revealing A. In particular, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM for human IgGl sequences.

Within the scope of disclosure A, in yet 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 for example in: WO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072, WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635, WO20058/09 070963、WO2006/020114、WO2006/116260、WO2006/023403、WO2013/047752、WO2006/019447、WO2012/115241、WO2013/125667、WO2014/030728、WO2014/163101、WO2013/118858，與WO2014/030750。

The site 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 the Fc region or constant region of a human IgG antibody according to EU numbering position, 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 described in WO2013/047752L. Modification of such amino acid residues may increase FcγR binding in the Fc region or constant region of an IgG antibody under neutral pH conditions. WO2013/047752 describes that a desirable modification of an IgG-type constant region or Fc region is, for example, one or more amino acid residues selected from the group consisting of: According to the EU numbering system, 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 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; 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 The amino acid becomes any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 232 becomes any one of toGlu, 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 any one of Tyr; the amino acid at position 234 becomes Ala, Asp, Any one 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 any of Tyr; the amino acid at position 236 becomes Ala, Asp, Glu, Any one 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 one 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 , any of Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; 240 The amino acid at position becomes any one of toAla, Ile, Met, and Thr; the amino acid at position 241 becomes any one of Asp, Glu, Leu, Arg, Trp, and Tyr; the amino acid at position 243 The acid becomes any one of toGlu, Leu, Gln, Arg, Trp, and Tyr; the amino acid at the 244-position becomes His; the amino acid at the 245-position becomes Ala; the amino acid at the 246-position becomes Asp, Any one of Glu, His, and Tyr; the amino acid at position 247 becomes any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; the amino acid at position 249 Becomes any one of Glu, His, GIn, and Tyr; The amino acid at position 250 becomes Glu or GIn; The amino acid at position 251 becomes Phe; The amino acid at position 254 becomes Phe, Met, and Any of 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 any one of Asp, Glu, His, and Tyr; The amino acid at position 262 becomes Ala, Glu, Any one of Phe, Ile, and Thr; the amino acid at position 263 becomes any one of Ala, Ile, Met, and Thr; the amino acid at position 264 becomes Asp, Glu, Phe, Gly, His, Any one of Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 265 becomes Ala, Leu, Phe, Gly, His, Ile, Lys , Leu, Met, Asn, Pro, GIn, Arg, Ser, Thr, Val, Trp, and any one of Tyr; the amino acid at position 266 becomes any one of Ala, Ile, Met, and Thr; 267 The amino acid at position becomes any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 268 Become any one of 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 any of Tyr; the amino acid at position 270 becomes Glu, Phe, Gly, His, Ile, Any of Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 becomes Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met , Asn, Gln, Arg, Ser, Thr, Val, Trp, and any of Tyr; the amino acid at position 272 becomes Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Any of Ser, Thr, Val, Trp, and Tyr; the amino acid at position 273 becomes Phe or Ile; the amino acid at position 274 becomes Asp, Glu, Phe, Gly, His, Ile, Leu , Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 275 becomes Leu or Trp; the amino acid at position 276 becomes Asp, Glu, Any one of Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 278 becomes Asp, Glu, Gly, His, Ile, Lys , Leu, Met, Asn, Pro, GIn, Arg, Ser, Thr, Val, and any one of Trp; the amino acid at the 279 position becomes Ala; the amino acid at the 280 position becomes Ala, Gly, His, Any one of Lys, Leu, Pro, Gln, Trp, and Tyr; the amino acid at position 281 becomes any one of Asp, Lys, Pro, and Tyr; the amino acid at position 282 becomes Glu, Gly, Any one of Lys, Pro, and Tyr; the amino acid at position 283 becomes any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid at position 284 Change to any of Asp, Glu, Leu, Asn, Thr, and Tyr; amino acid at position 285 becomes any of Asp, Glu, Lys, Gln, Trp, and Tyr; amino acid at position 286 Become any one of Glu, Gly, Pro, and Tyr; The amino acid at position 288 becomes any one of Asn, Asp, Glu, and Tyr; The amino acid at position 290 becomes Asp, Gly, His, Any of Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid at position 291 becomes any one of Asp, Glu, Gly, His, Ile, Gln, and Thr; the amino acid at position 292 Change to any of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid at position 293 becomes toPhe, Gly, His , Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and any of Tyr; the amino acid at position 294 becomes Phe, Gly, His, Ile, Lys, Leu, Met, Any of Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Met, 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 any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr The amino acid at position 298 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 The acid becomes any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; amino acid at position 300 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 Asp, Glu, His , and any one of Tyr; the amino acid at the 302 position becomes Ile; the amino acid at the 303 position becomes any one of Asp, Gly, and Tyr; the amino acid at the 304 position becomes 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, Thr, Val, and Any of Tyr; the amino acid at the 313 position becomes Phe; the amino acid at the 315 position becomes Leu; the amino acid at the 317 position becomes Glu or Gln; the amino acid at the 318 position becomes His, Any of Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 becomes Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val Any one of , Trp, and Tyr; the amino acid at position 322 becomes Ala, Asp, Phe, Gly, His, I Any one of le, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 323 becomes Ile; the amino acid at position 324 becomes Asp, Phe, Gly, His, Ile, Leu, Met , any of Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 325 becomes Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Any of Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 326 becomes Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val , Trp, and any of Tyr; the amino acid at position 327 becomes Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and any one of Tyr; the amino acid at position 328 becomes Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Any of Tyr; the amino acid at position 329 becomes any of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr One; the amino acid at position 330 becomes any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 331 becomes any one of 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, Gln, Arg, Ser, Thr, Val, Trp, and any of Tyr; the amino acid at position 333 becomes Ala, Asp, Glu, Any of Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 becomes Ala, Glu, Phe, Ile, Leu, Pro, and Thr Any; the amino acid at position 335 becomes any of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 The acid becomes any one of Glu, Lys, and Tyr; the amino acid at position 337 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 group at position 376 The acid 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 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 396 becomes Leu; amino acid at position 421 becomes Lys; amino acid at position 427 becomes Asn; amino acid at position 428 becomes Phe or Leu; amino acid at position 429 becomes Met; amine at position 434 The base acid 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 the A-revealing antibody.

In one embodiment, this reveals that the (FcyR binding domain) of the antibody of A may have higher binding activity to (human) FcyRs, eg, to any one or more of FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, and FcyRIIIb, than native The binding activity of the IgG (the Fc region or constant region) or the reference antibody containing the starting Fc region or the starting constant region. For example, the FcyR-binding activity of the antibody (FcyR-binding domain) of A disclosed may be 55% or more, 60% or more, 65% or more, 70% or more, compared to the FcyR-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 greater than the FcγR binding activity of the reference antibody by 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 7.5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or More, 80 times or more, 90 times or more, or 100 times or more.

In yet another embodiment, the increased level of binding activity (in the neutral pH range) for inhibitory FcyRs (FcyRIIb-1 and/or FcyRIIb-2) may be greater than for activating FcyRs (FcyRIa: FcyRIb; FcyRIc; FcyRIIIa, including Paratype V158; FcyRIIIa, including paratype F158; FcyRIIIb, including paratype FcyRIIIb-NA1; FcyRIIIb, including paratype FcyRIIIb-NA2; FcyRIIa, including paratype H131; or FcyRIIa, including paratype R131) are increased 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, the ideal FcyR-binding domain of Reveal A also includes FcyR-binding domains that have greater binding activity for a particular FcyR than for other FcyRs (FcyR-binding domains, selective FcyR-binding activity). If an antibody (or its Fc region as the FcyR binding domain) is used, a single antibody molecule can only bind to a single FcyR molecule. Therefore, a single antibody molecule bound to an inhibitory FcγR cannot bind to other activating FcγRs, and a single antibody molecule bound to an activating FcγR cannot bind to other activating FcγRs or inhibitory FcγRs.

As mentioned above, activating FcyRs preferably include, for example, FcyRI (CD64), such as FcyRIa, FcyRIb, or FcyRIc; and FcyRIII (CD16), such as FcyRIIIa (such as paratype V158 or F158) or FcyRIIIb (such as paratype FcyRIIIb-NA1 or FcyRIIIb-NA2) . Inhibitory FcyRs preferably include, for example, FcyRIIb (such as FcyRIIb-1 or FcyRIIb-2).

In one embodiment, an FcyR binding domain having a greater binding activity for an inhibitory FcyR than an activating FcyR can be used as the selective FcyR binding domain contained in the antibody of Disclosure A. Such selective FcγR binding domains can include, for example, FcγRs that have higher binding activity for FcγRIIb (such as FcγRIIb-1 and/or FcγRIIb-2) than an activating FcγR selected from the group consisting of one or more of the following: Binding domains: FcyRI (CD64) such as FcyRIa, FcyRIb or FcyRIc; FcyRIII (CD16) such as FcyRIIIa (such as paratype V158 or F158) or FcyRIIIb (such as FcyRIIIb-NA1 or FcyRIIIb-NA2); FcyRII (CD32) such as FcyRIIa (including paratypes) type H131 or R131); and FcγRIIc.

In addition, whether an FcγR-binding domain has selective binding activity can be assessed by comparing the respective binding activities for FcγRs determined by the above method, for example, by comparing the KD value for activating FcγRs divided by the KD value for inhibitory FcγRs. The value (ratio), more specifically comparing 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, a KD value for an activating FcyR refers to a KD value for one or more of: FcyRIa; FcyRIb; FcyRIc; FcyRIIIa, including paratype V158 and/or F158; - NA2; FcyRIIa, including paratypes H131 and/or R131; and FcyRIIc; and KD values for inhibitory FcyRs refer to KD values for FcyRIIb-1 and/or FcyRIIb-2. The activating FcγR and the 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 thereto.

This FcγR selectivity indicator 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 greater, 30 or greater, 35 or greater, 40 or greater, 45 or greater, 50 or greater, 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, 110 or greater, 120 or greater, 130 or greater, 140 or greater, 150 or greater, 160 or greater, 170 or greater, 180 or greater, 190 or greater, 200 or greater, 210 or greater, 220 or greater, 230 or greater, 240 or greater, 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 greater, 1500 or greater, 2000 or greater, 2500 or greater, 3000 or greater, 3500 or greater, 4000 or greater, 4500 or greater, 5000 or greater, 5500 or greater, 6000 or greater, 6,500 or greater, 7,000 or greater, 7,500 or greater, 8,000 or greater, 8,500 or greater, 9,000 or greater, 9,500 or greater, 10,000 or greater, or 100,000 or greater; but Not limited to this.

In one embodiment, the amino acid at position 238 or 328 according to EU numbering of a human IgG (IgG1, IgG2, IgG3 or IgG4), each of which is an Fc region-containing variant or a constant region variant of Asp or Glu, is preferably used ( Antibodies) antibodies of Disclosure A as Fc-region-containing variants or constant-region variants, because as specifically disclosed in WO2013/125667, WO2012/115241, and WO2013/047752, they are very specific to FcyRIIb-1 and/or FcyRIIb-2 Compared to FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, including paratype V158, FcyRIIIa, including paratype F158; FcyRIIIb, including paratype FcyRIIIb-NA1; FcyRIIIb, including paratype FcyRIIIb-NA2; FcyRIIa, including paratype H131; FcyRIIa, Including the subtype R131, and/or FcγRIIc have higher binding activity. In such an embodiment, the antibody of this disclosure A has binding activity to all activating FcγRs (here, 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 native IgG Fc region is maintained or increased, and/or its binding activity for all activating FcγRs is decreased.

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

Within the scope of Disclosure A, the degree of "reduction in 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 "maintain or increase the binding activity to FcγRIIb", the "maintain or increase the binding activity to FcγRIIb" or "maintain or increase the binding activity to FcγRIIb" may be, but not limited to, 55% or more, 60% or more. % or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 87% or greater, 88% or greater, 89% or Greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater , 98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101% or greater, 102% or greater, 103% or greater, 104% or greater, 105 % or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 112% or greater, 114% or greater, 116% or Greater, 118% or greater, 120% or greater, 122% or greater, 124% or greater, 126% or greater, 128% or greater, 130% or greater, 132% or greater , 134% or greater, 136% or greater, 138% or greater, 140% or greater, 142% or greater, 144% or greater, 146% or greater, 148% or greater, 150 % or greater, 155% or greater, 160% or greater, 165% or greater, 170% or greater, 175% or greater, 180% or greater, 185% or greater, 190% or greater Greater, 195% or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more , 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more Greater, 800 times or more, 900 times or more, 1000 times or more, 10,000 times or more, or 100,000 times or more.

Within the scope described in Disclosure A, the degree of "reduced binding activity to FcyRIIa (H type) and FcyRIIa (R type)" or "reduced binding activity to FcyRIIa (H type) and FcyRIIa (R type)" may be, but 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 more less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less or 0.005% or less.

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

The above modifications may be in a single position alone or in combination in 2 or more positions. Alternatively such desirable modifications may include, for example, variants of human constant regions or human Fc regions 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 , wherein the amino acid at position 238 according to the EU numbering method is Asp, the amino acid at position 271 according to the EU numbering method is Gly, and can be substituted for 233, 234, 237, 264, 265, 266 according to the EU numbering method , 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396 in one or more positions. In this case, such variants may include, but are not limited to, variants of a human constant region or a human Fc region containing amino acids according to one or more of EU numbering: 233 is Asp, 234 is Tyr, 237 is Asp, 264 is Ile, 265 is Glu, 266 is any one of Phe, Met, and Leu; 267 is in Ala, Glu, Gly and Gln Any; Position 268 is Asp or Glu; Position 269 is Asp; Position 272 is any of Asp, Phe, Ile, Met, Asn and GIn; Position 296 is Asp; Position 326 is Ala or Asp; Position 327 Gly; 330 is Lys or Arg; 331 is Ser; 332 is Thr; 333 is any of Thr, Lys, and Arg; 396 is Asp, Glu, Phe, Ile, Lys, Leu, Met , any of Gln, Arg and Tyr.

In an alternative embodiment, the antibody of Disclosure A containing the Fc region variant or constant region variant is comparable to a reference antibody containing the constant region or Fc region of native IgG, with maintained or increased binding activity for FcγRIIb and for FcγRIIa (H type) and FcγRIIa (R type) binding activity decreased. Desirable amino acid substitution sites for such variants may be as described in WO2014/030728, eg 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 is Asp at position 238 according to EU numbering and has at least 1 amino acid selected from the following amino acid group: Asp at position 233, Tyr at position 234, Tyr at position 235 according to EU numbering Position 237 is Asp, 264 is Ile, 265 is Glu; 266 is Phe, Leu or Met; 267 is Ala, Glu, Gly or Gln; 268 is Asp, Gln or Glu; 269 is 271 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 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 ; 356 is Glu; 358 is Met; 396 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Position 409 is Arg; position 419 is Glu.

In an alternative embodiment, the antibody of Disclosure A containing the Fc region variant or constant region variant maintains binding activity for FcγRIIb compared to a reference antibody containing the constant region or Fc region of native IgG and for all The binding activity of activating FcγRs, especially to FcγRIIa (R-type), is reduced. Desirable amino acid substitution sites for such variants may 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 Laws 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330. More preferably this variant is Asp at position 238 according to EU numbering and has at least 1 amino acid selected from the group of amino acids: Phe at position 235 according to EU numbering; Gln or Asp at position 237; 241 is Met or Leu; 268 is Pro; 295 is Met or Val; 296 is Glu, His, Asn or Asp; 298 is Ala or Met; 323 is Ile; 324 is Asn or His; and 330 bits are His or Tyr.

Within the scope of Disclosure A, the level of "maintained 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 greater, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101% or greater, 102% or greater, 103% or greater Greater, 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 more, 140% or more, 150% or more, 175% or more or 2 times or more.

Within the scope of Disclosure A, the above-mentioned "reduced binding activity for all activating FcγRs, especially for FcγRIIa (R type)" may be, but not limited to, 74% or less, 72% or less, 70% or Less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less , 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15 % or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

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

Within the scope of disclosures A and B described herein, unlike FcγRs, which belong to the immunoglobulin superfamily, "FcRn", particularly human FcRn, are 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 line is expressed as a heterodimer, consisting of a soluble beta or light chain (Beta2 microglobulin) complexed with a transmembrane alpha or heavy chain. Like MHC, the α chain of FcRn contains three extracellular domains (α1, α2, and α3) whose short cytoplasmic domains tether the protein to the cell surface. The alpha1 and alpha2 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 mammalian maternal placenta and yolk sac and is involved in maternal-to-fetal IgG transmission. Furthermore, FcRn is expressed in the small intestine of neonatal rodents, and FcRn is involved in the transport of maternal IgG from digested colostrum or milk across the bristle edge 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. In general, FcRn binding to IgG molecules is strictly pH-dependent. Optimal binding was 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 signal sequences) each (RefSeq access numbers are shown in parentheses).

This precursor forms a complex with human Beta2-microglobulin in vivo. Thus, by known recombinant expression techniques, soluble human FcRn complexed with human Beta2-microglobulin for appropriate use in various experimental lines can be produced. Such soluble human FcRn can be used to evaluate the FcRn binding activity of an antibody or Fc region variant. In Disclosure A or B, FcRn is not particularly limited as long as it is in a form capable of binding to the FcRn binding domain; but ideally FcRn may be human FcRn.

Within the scope of disclosures A and B described herein, when an antibody or Fc region variant has FcRn binding activity, it may have an "FcRn binding domain", preferably a human FcRn binding domain. The FcRn-binding domain is not particularly limited as long as the antibody has binding activity or affinity for FcRn at acidic pH and/or at neutral pH; or may be a domain that has an activity to directly or indirectly bind 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 Molecules of IgG or albumin that have the activity of indirectly binding to FcRn. For disclosure of 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 weak or no FcRn binding activity in the acidic pH range and/or in the neutral pH range, the amino acid residues of the antibody or Fc region variant in the FcRn binding domain can be modified to be in the acidic pH range and /or FcRn binding activity in the neutral pH range. Alternatively, amino acids of domains that originally have FcRn-binding activity in the acidic pH range and/or in the 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 binds directly to FcRn. Such desirable FcRn binding domains include, for example, the constant and Fc regions of antibodies. However, it can bind to a region of a polypeptide having FcRn-binding activity such as albumin and IgG, and can indirectly bind to FcRn via albumin and IgG. Therefore, the FcRn-binding region may be a region that binds to a polypeptide having binding activity to albumin or IgG. Without limitation, in order to promote the elimination of the antigen from the plasma, the FcRn-binding domain with a high FcRn-binding activity at neutral pH is preferred, and in order to promote antibody retention in plasma, an FcRn-binding domain with a high FcRn-binding activity at an acidic pH is preferred. area. For example, an FcRn-binding domain that originally has a 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 an antibody or Fc region (variant) is increased, (substantially) maintained or decreased compared to that of the antibody or Fc region (variant) before modification, It can be assessed using known methods, such as those described in the Examples herein and eg BIACORE, Scatchard plots and flow cytometry (see WO2013/046722). The extracellular domain of human FcRn can be used as a soluble antigen in these assays. Conditions other than pH when measuring the FcRn-binding activity of the antibody or Fc region (variant) can be appropriately selected by those skilled in the art. The assay can be performed eg in MES buffer at 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 wafer as an analyte.

The FcRn binding activity of an antibody or Fc region (variant) can be assessed in terms of dissociation constant (KD), apparent dissociation constant (apparent KD), dissociation rate (kd), apparent dissociation rate (apparent kd).

For pH conditions for measuring the binding activity of FcRn to an FcRn-binding domain contained in an antibody or an 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 appropriate Use a temperature of 15 ° C to 40 ° C. More preferably any temperature from 20 ° C to 35 ° C, Any of 33, 34, and 35° C. A non-limiting example of such a temperature may be 25° C.

In one embodiment, when the antibody of A or B is revealed to have 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 may be a domain having an activity of directly or indirectly binding to FcRn. In a specific embodiment, it is disclosed that antibodies of A or B preferably have increased FcRn binding activity at neutral pH conditions, eg, compared to a reference antibody containing the constant region of native IgG (see WO2013/046722). From the standpoint of comparing the FcRn binding activities between the two, it is desirable, but not limited, to disclose that an antibody of A or B and a reference antibody containing the constant region of a native IgG, except that one or more amino acid residues have been modified Regions other than the constant region of the base (eg, variable regions) have the same amino acid sequence.

In one embodiment, within the scope of Disclosure A described herein, when antibodies of Disclosure A have increased FcRn binding activity at neutral pH conditions, without being bound by a particular theory, it is possible that antibodies of Disclosure A may have 2 or more in combination. Multiple of the following properties: shuttle between plasma and cellular endosomes, repeat binding to multiple antigens with a single antibody molecule by having an ion concentration-dependent antigen-binding domain; by increasing the isoelectric point value and increasing the positive charge of the whole antibody, and It is rapidly taken up into cells; and by increasing FcRn binding activity under neutral pH conditions, it is rapidly taken up into cells. 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 promoted. One of ordinary skill in the art can determine the optimal isoelectric point value of the antibody of this disclosure A to benefit from such properties.

Within the scope of disclosures A and B described herein. According to Yeung et al. (J. Immunol. 182:7663-7671 (2009)), the activity of native human IgG1 binding to human FcRn is KD 1.7 μM in the acidic pH range (pH 6.0), 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 advisable to use the following antibody as 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 stronger, its human FcRn binding activity in the neutral pH range is comparable to or stronger than that of natural human IgG; preferably an antibody or constant region variant or Fc region variant, whose human FcRn binding activity is in The acidic pH range is KD 2.0 μM or more, and its human FcRn binding activity is KD 40 μM or more in the neutral pH range; and more preferably an antibody or constant region variant or Fc region variant whose human FcRn The binding activity was KD 0.5 μM or more in the acidic pH range, and its human FcRn binding activity was KD 15 μM or more in the neutral pH range. KD values were determined according to the method described by Yeung et al. (J. Immunol. 182:7663-7671 (2009) (by immobilizing antibodies on a wafer and loading human FcRn as analyte)).

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

Within the scope of Disclosures A and B described herein, the starting FcRn binding domain may comprise, for example, the Fc region or constant region of (human) IgG. Any Fc region or constant region can be used as the starting Fc region or starting constant region as long as the starting Fc region or variant of the starting constant region can bind to FcRn in the acidic pH range and/or in the neutral pH range . Or an Fc region or constant region obtained by modifying a starting Fc region or starting constant region whose Fc region or constant region has been modified can also be appropriately used as the Fc region or constant region. The starting Fc region or starting constant region may comprise a known Fc region using recombinant manufacturing. 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 encoded as the starting Fc region or starting constant region. The origin of the starting Fc region or starting constant region is not limited and can be obtained from any non-human animal or human organism. In turn, the starting 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 IgGl, but is not limited to any particular IgG class. This means that the Fc region of human IgGl, IgG2, IgG3 or IgG4 can be used as a suitable starting FcRn binding domain, and the Fc region or constant region of an IgG class or subclass from any organism can be used as the starting Fc region or as the starting constant region. Examples of native IgG variants or modified forms are described, for example, in Strohl, Curr. Opin. Biotechnol. 20(6):685-691 (2009); Presta, Curr. Opin. Immunol. 20(4):460 -470 (2008); Davis et al., Protein Eng. Des. Sel. 23(4):195-202 (2010), WO2009/086320, WO2008/092117; WO2007/041635; and WO2006/105338).

Within the scope of Disclosures A and B described herein, the amino acid residues of the starting FcRn binding domain, starting Fc region, or starting constant region may include, for example, one or more mutations: such as substitution into and starting Fc region Or the amino acid residues of the starting constant region are mutations of different amino acid residues; Insert 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 to the starting Fc region or starting constant region. For example, the amino acid sequence of the variant and the amino acid sequence of the starting Fc region or the starting constant region are about 75% less than 100%, more preferably about 80% less than 100%, more preferably about 85% At least 100%, more preferably about 90% less than 100%, still more preferably about 95% less than 100% identical or similar. 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 1 amino acid.

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

One of the embodiments of A or B is disclosed, and 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 , an amino acid modification site that increases FcRn-binding activity under neutral pH conditions, described in, for example, WO2013/046722. Such modified sites include, for example, one or more positions selected from the group consisting of: the Fc region or 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 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442, as described in WO2013/046722. WO2013/046722 also describes that part of the ideal modification of the Fc region or constant region is, for example, one or more amino acids selected from the group consisting of: according to EU numbering, 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 individually for a single position 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 introduced into antibodies revealing A or B as appropriate.

In further or alternative embodiments, appropriate amino acid modification sites can be used to increase the FcRn binding activity under acidic pH conditions. Among such modification sites, one or more modification sites that allow the FcRn to bind in the neutral pH range can also be appropriately used in disclosure A or B. Such modified sites include, for example, those reported in WO2011/122011, WO2013/046722, WO2013/046704, and WO2013/046722. The permissible amino acids and modified amino acid types of the constant regions or Fc regions of human IgG antibodies are reported in Table 1 of WO2013/046722, and WO2013/046722 also describes that particularly desirable constant regions or Fc regions Modification sites in the region, e.g. according to EU numbering, one or more amino acid positions selected from the group consisting of: 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 these amino acid residue positions can also facilitate human FcRn binding in the neutral pH range of the FcRn binding domain. WO2013/046722 also describes that it is desirable to modify as part of an IgG-type constant region or Fc region, for example, modifying one or more amino acid residues selected from the group consisting of: According to EU numbering, (a) at position 237 The amino acid becomes Met; (b) the amino acid at the 238 position becomes Ala; (c) the amino acid at the 239 position becomes Lys; (d) the amino acid at the 248 position becomes Ile; (e) the amino acid at the 250 position 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 Thr becomes Thr; (h) The amino acid at position 255 becomes Glu; (i) The amino acid at position 256 becomes any of Asp, Glu, and Gln; (j) The amino acid at position 257 Become 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 the 270 position becomes Phe; (n) the amino acid at the 286 position becomes Ala or Glu; (o) the amino acid at the 289 position becomes His; (p) the amino acid at the 297 position becomes Ala; (q) ) The amino acid at the 298 position becomes Gly; (r) The amino acid at the 303 position becomes Ala; (s) The amino acid at the 305 position becomes Ala; (t) The amino acid at the 307 position 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, Leu , Met, Pro, GIn, and any of Thr; (v) the amino acid of 309 becomes any one of Ala, Asp, Glu, Pro, and Arg; (w) The amino acid of 311 becomes Any one of Ala, His, and Ile; (x) the amino acid at the 312 position becomes Ala or His; (y) the amino acid at the 314 position becomes Lys or Arg; (z) the amino acid at the 315 position becomes Ala or His; (aa) the amino acid at the 317th position becomes Ala; (ab) the amino acid at the 325th position becomes Gly; (ac) the amino acid at the 332nd position becomes Val; (ad) the amino acid at the 334th position becomes Leu; (ae) the amino acid at the 360 position becomes His; (af) the amino acid at the 376 position becomes Ala; (ag) the amino acid at the 380 position becomes Ala; (ah) the amino acid at the 382 position becomes Ala; (ai) Amino acid at position 384 becomes A la; (aj) the amino acid at the 385 position becomes Asp or His; (ak) the amino acid at the 386 position becomes Pro; (al) the amino acid at the 387 position becomes Glu; (am) the amino acid at the 389 position becomes Ala or Ser; (an) amino acid at position 424 becomes Ala; (ao) amino acid at position 428 becomes Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser , Thr, Val, Trp, and any of Tyr; (ap) amino acid at position 433 becomes Lys; (aq) amino acid at position 434 becomes Ala, Phe, His, Ser, Trp, and Tyr Any one of; and (ar) the amino acid at position 436 becomes His. The number of amino acids to be modified is not particularly limited, and the modification can be carried out 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, the FcRn-binding activity of the FcRn-binding domain of the antibody of A or B is revealed as compared to a reference antibody containing a native IgG Fc region or constant region, or containing 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 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, preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more , 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 more, 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, or 5 times or more.

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

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

FR sequences may advantageously include, for example, fully human FR sequences as shown for example in V-Base (vbase.mrc-cpe.cam.ac.uk/). These FR sequences can be used to disclose A or B as appropriate. The germline sequences can be classified according to their similarity (Tomlinson et al. (J. Mol. Biol. 227:776-798 (1992); Williams et al. (Eur. J. Immunol. 23:1456-1461 (1993) (Nat. Genetics 7:162-168 (1994)). Desirable germline sequences may be suitably selected from: Vκ, which is classified into 7 subgroups; Vλ, which is classified into 10 subgroups; and VH, which is 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 (e.g. VH2-5, VH2-26, and VH2-70); subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3- 66, VH3-72, VH3-73, and VH3-74); subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, and VH4-61); Subgroup VH5 (VH5-51); Subgroup VH6 (VH6-1); or Subgroup VH7 (VH7-4 and VH7-81). These are also described in, for example, Matsuda et al. (J. Exp. Med. 188:1973-1975 (1998)), and those of ordinary skill in the art can appropriately design antibodies based on such sequence information. Other fully human FR sequences or regions equivalent thereto may desirably be used.

Fully human VK 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 is classified as subgroup Vk4; B2 (also known as "Vk5-2"), which is 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, classified as 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)).

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

Within the scope of disclosures A and B described herein, "flexible residues" may refer to amino acid residue variations that exist in positions that exhibit high amino acid diversity, where either the light or heavy chain is variable The regions have some amino acids that differ when compared to known amino acid sequences and/or native antibody or antigen binding domains. Positions showing high diversity are generally located in the CDRs. The information provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) is useful for determining positions of high diversity in known and/or natural antibodies. Still other online databases (vbase.mrc-cpe.cam.ac.uk/, bioinf.org.uk/abs/index.html) provide collections of many human light and heavy chain sequences and their positions. Such sequence and positional information is useful for determining flexible residue positions. There is no limitation, for example, when the amino acid residues at a specific position have variability, it is suitable for 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 appreciated that when an antibody of A or B is disclosed to contain all or a portion of a light chain variable region and/or a heavy chain variable region, the antibody may optionally include one or more suitable flexible residues. For example, the variable region sequence of the heavy chain and/or light chain with FR sequence, which originally contains amino acid residues that will change the antigen-binding activity of the antibody according to the ion concentration (hydrogen ion concentration or calcium ion concentration), can be It is designed to contain other amino acid residues in addition to these amino acid residues. In this case, for example, the number and position of flexible residues can be determined without being limited to a specific embodiment, as long as the antigen-binding activity of the antibody of this disclosure A or B changes according to ion concentration conditions. Specifically, the CDR sequences and/or FR sequences of the heavy chain and/or light chain may include at least one flexible residue. For example, if the ion concentration is the 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 can 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 heavy chain variable region, in which the antibody may be exposed At least one amino acid residue on the surface is modified to increase the isoelectric point value.

(位置依照Kabat編號法顯示。) [Table 1]

(Locations are shown according to Kabat numbering.)

(位置依照Kabat編號法顯示。) [Table 2]

(Locations 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 create a Humanization of chimeric antibodies without such modifications with shorter plasma half-lives revealed A or B antibodies. Modification of amino acid residues that may be exposed on the surface of a humanized antibody can be performed before or concurrently with the humanization of the antibody. Alternatively, by using a humanized antibody as a starting material, amino acid residues that may be exposed on the surface can be modified to further engineer the isoelectric point value of the humanized antibody.

Adams et al. (Cancer Immunol. Immunother. 55(6):717-727 (2006)) report humanized antibodies, trastuzumab (antigen: HER2), bevacizumab (antigen: VEGF) and pertuzumab (antigen: HER2), using the same human Antibody FR sequences are humanized with roughly comparable pharmacokinetics in plasma. In particular, when humanization can be performed using the same FR sequences, it is understood that the plasma pharmacokinetics are roughly comparable. According to one embodiment of Disclosure A, in addition to the humanization step, the concentration of this antigen in plasma is decreased by modifying the amino acid residues that may be exposed on the antibody surface to increase the isoelectric point value of the antibody. In an alternative embodiment disclosing one of A or B, human antibodies can be used. By modifying the amino acid residues that may be exposed on the surface of human antibodies produced from human antibody libraries, it is possible to increase the production of human antibodies in mice, recombinant cells, etc. 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), antibodies lacking fucose (WO00/61739; WO02/31140, WO2006/067847; WO2006/067913), and Antibody to the sugar chain of GlcNAc (WO02/79255).

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

In an alternative embodiment, A or B is disclosed for 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 relates 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 particular embodiment, the nucleic acid can be obtained using suitable known methods. For specific embodiments, reference may be made, for example, to WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241, WO2013/0456752, WO2013/0456752, WO2013/0456752 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 of A or B disclosed may be an isolated or purified nucleic acid. The nucleic acid encoding the antibody for which A or B is disclosed herein can be any gene, and can be DNA or RNA or other nucleic acid analogs.

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

Sensitive antigens for obtaining nucleic acids encoding the above heavy and light chains include, but are not limited to: immunogenic complete antigens; and incomplete antigens, including non-immunogenic haptens. For example, the entire protein of interest, or a partial peptide of this protein can be used. Also included are substances consisting of polysaccharides, nucleic acids, lipids and other compositions known to have antigenic potential. Therefore, in some embodiments, the antigens of the antibodies of this disclosure A or B are not particularly limited. This antigen can be produced using, for example, baculovirus-based methods (see, eg, 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 carried out by linking the antigen to an immunogenic macromolecule such as albumin. Alternatively, soluble antigens can be prepared by linking this antigen to other molecules, if desired. When a transmembrane molecule such as a membrane antigen (eg, a receptor) is used as the antigen, a portion of the extracellular region of the membrane antigen can be used as a fragment, or a cell expressing the transmembrane molecule on its surface can be used as the immunogen.

In some embodiments, antibody-producing cells can be obtained by immunizing an animal with an appropriate sense antigen as described above. Alternatively, antibody-producing cells can be prepared by in vitro immunization of antibody-producing lymphocytes. A variety of mammals are available for immunization, and other routine antibody manufacturing procedures are available. Commonly used animals include rodents, lagomorphs, and primates. Animals can include, for example, rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates including monkeys such as macaques, rhesus monkeys, baboons, and chimpanzees. In addition, transgenic animals with human antibody gene stocks are known, and these animals can be used to obtain human antibodies (see, eg, WO 96/34096; Mendez et al., Nat. Genet. 15:146-156 (1997); WO93/12227, WO92/03918, WO94/02602, WO96/34096, and WO96/33735). Instead of using such transgenic animals, human lymphocytes can also be induced in vitro using, for example, a desired antigen or cells expressing the antigen, and the induced lymphocytes can be fused with human myeloma cells such as U266 to produce a tumor. A desired human antibody with antigen-binding activity was obtained (JP Pat. Publ. No. H01-59878).

Animal immunization can be carried out, for example, by appropriately diluting and suspending the antigen in phosphate-buffered saline (PBS), physiological saline, or others, and mixing with adjuvants as needed to emulsify; and then injecting the animal intraperitoneally or subcutaneously. Then it is advisable to administer the sense antigen that has been mixed with Freud's incomplete adjuvant every 4 to 21 days for several times. Antibody production can be determined, for example, by measuring the titer of the antibody of interest in animal serum.

Antibody-producing cells obtained from lymphocytes or animals immunized with the desired antigen can be produced using conventional fusion agents such as polyethylene glycol (Goding, Antibodies: Principles and Practice, Academic Press, 1986, 59-103) and myeloma cells fused to produce a fusion tumor. If desired, fusion tumors can be cultured and expanded, and the binding specificity of antibodies produced by fusion tumors can be assessed by, for example, immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA). Antibody-producing fusion tumors that have been determined for specificity, affinity, or activity of interest can then be sub-colonized, eg, by limiting dilution, if desired.

Nucleic acids encoding the selected antibodies can be obtained from fusion tumors or antibody-producing cells (sensory lymphocytes, etc.) Breeding. Alternatively this 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 that constitutes the heavy chain (all or a portion thereof) and/or light chain (all or a portion thereof) of the antibody revealing A or B is modified, eg, using genetic engineering techniques. Modifications to, for example, recombinant antibodies with artificial sequences that reduce heteroantigenicity against humans, such as chimeric or humanized antibodies, can be suitably obtained by, for example, encoding and antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies The nucleotide residues of the amino acid sequence associated with the antibody, goat antibody or camelid antibody component are prepared. Chimeric antibodies can be obtained by, for example, ligating DNA encoding the variable region of a mouse-derived antibody and DNA encoding the constant region of a human antibody, encapsulating the ligated DNA coding sequence into an expression vector, and then obtaining The recombinant vector is introduced into the host to express the gene. Humanized antibodies, also known as reshaped human antibodies, are antibodies in which human antibody FRs are joined in frame to antibody CDRs isolated from a non-human mammal, such as a mouse, to form the coding sequence. DNA sequences encoding such humanized antibodies can be synthesized by overlap extension PCR using some oligonucleotides as templates. The materials and experimental methods of overlap extension PCR are described in WO98/13388 and the like. For example, DNA encoding the amino acid sequence of an antibody variable region revealing A or B can be obtained by using overlap extension PCR using some oligonucleotides designed to have overlapping nucleotide sequences. The overlapping DNA is then ligated in frame with the DNA encoding the constant region to form the coding sequence. The DNA ligated in the above-described manner can be inserted into an expression vector so that the DNA can 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 culturing host cells. The expressed antibody can be suitably purified from the culture medium of the host or the like (EP239400; WO96/02576). The FRs of the humanized antibody, in turn linked via a CDR, may be selected, eg, to allow this CDR to form an antigen-binding site suitable for the antigen. If necessary, the amino acid residues constituting the FRs of the variable regions of the selected antibodies can be modified with appropriate substitutions.

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

Methods for introducing desired amino acid modifications into antibodies are well established in the art. For example, libraries can be prepared by introducing modifications 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 alters the antigen-binding activity of the antibody depending on ionic concentration conditions to make. In addition, flexible residues can be added if desired using the method of Kunkel et al. (Methods Enzymol. 154:367-382 (1987)).

In an alternative embodiment, A relates to a vector comprising a nucleic acid encoding the above-mentioned increased ion concentration-dependent antigen binding domain, or the above-mentioned ion concentration-dependent antibody of increased isoelectric point value. 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, WO2012/115241 , WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, or WO2012/016227 or WO2012/0 by reference in their entirety.

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

In one embodiment, the expression vector can be used as a vector containing nucleic acid for Reveal A or B. Expression vectors can be used to allow expression of the polypeptide in vitro, in E. coli, in cultured cells, or in vivo. For example, pBEST vector (Promega) for in vitro expression; pET vector (Invitrogen) for E. coli expression; pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cell expression; and pME18S vector (Takebe et al. , Mol. Cell Biol. 8:466-472 (1988)) for in vivo performance. DNA can be inserted into the vector using known methods, such as by ligase reactions using restriction enzyme sites (see Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.4-11.11).

In an alternative embodiment, it is disclosed that line A relates to a host or host cell comprising a vector comprising a nucleic acid encoding the above-mentioned increased ion concentration-dependent antigen binding domain or the above-mentioned increased ion concentration-dependent isoelectric point value Sexual antibodies. In one embodiment, the host or host cell can be prepared by a method described in: e.g. WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012 /115241, WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, or WO2012/016227 or WO2012/016227 are cited in their entirety.

The type of host cell disclosed for A or B is not particularly limited, and the host cell includes, for example, bacterial cells such as E. coli and various animal cells. Host cells can be suitably 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; amphibian cells such as Xenopus 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 liposomal DOTAP (Boehringer-Mannheim), electroporation, and lipid staining.

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

To use host cells to produce A or B revealing antibodies, the host cells are transformed with expression vectors containing nucleic acids encoding the A or B revealing antibodies, 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, suitably 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 line for the production of antibodies that reveal A or B, for example, a nucleic acid encoding an antibody revealing A or B can be introduced into such animals or plants to produce such antibodies in vivo, This antibody can then be collected from the animal or plant.

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

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

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

When plants are used to produce antibodies revealing A or B, tobacco can be used. When tobacco is used, 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 obtained bacteria can be used to infect tobacco, such as Nicotiana tabacum (Ma et al., Eur. J. Immunol. 24:131-138 (1994)), and the desired antibody is 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 germ cells of infected duckweed (Cox et al. Nat. Biotechnol. 24(12):1591-1597 (2006) ).

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

Antibodies of Disclosure A or B produced as described above can be isolated from host cells or from within or outside the host (such as culture medium and milk) and refined into substantially pure and homogeneous antibodies. The antibody can be suitably isolated and purified, for example, by suitably selecting and combining chromatography as column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polyacrylamide gel electrophoresis , isoelectric point focusing, dialysis, recrystallization, etc. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobicity chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. Such chromatography can be carried out, 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 modification enzyme to partially delete the peptide before or after purification of the antibody. As such a protein modification enzyme, trypsin, chymotrypsin, lysine endopeptidase, protein kinase, and glucosidase can be used, for example.

In an alternative embodiment, a method for producing an antibody containing an antigen-binding domain whose antigen-binding activity changes depending on ionic concentration conditions disclosed in Line A may comprise culturing host cells or feeding a host, from cultures of such cells, host secretions , or other methods known in the art to collect antibodies.

In one embodiment, Disclosure A relates to a production method that can 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 having maintained or enhanced 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 at neutral pH; (g) select antibodies with pI; (h) confirm the isoelectric point (pI) of the collected antibodies, and then Antibodies with increased isoelectric point (pi) are selected; and (i) antibodies with altered or increased antigen-binding activity are selected according to ionic concentration conditions.

The reference antibodies herein include, but are not limited to, natural antibodies (such as natural Ig antibodies, preferably natural IgG antibodies) and antibodies before antibody modification (before or during construction of antibody libraries, such as increasing the isoelectric point value before ion concentration dependence. antibodies or antibodies that have an increased isoelectric point value before conferring an ion concentration-dependent antigen-binding domain).

After generating the antibody revealing A, the antibody obtained can utilize antibody pharmacokinetic assays using, for example, mouse, rat, rabbit, dog, monkey, human plasma to select for antibodies that have improved elimination of the antigen from plasma compared to a reference antibody .

Alternatively, after the antibody of Revelation A is produced, the obtained antibody can be compared with a reference antibody for extracellular matrix binding ability using electrochemiluminescence or other methods to select an antibody with increased binding to the extracellular matrix.

Alternatively, after making the antibody of Revelation A, the obtained antibody can be compared with a reference antibody for binding activity to various FcγRs at neutral pH using BIACORE (registered trademark) or other tools to select binding to various FcγRs at neutral pH. Antibodies with increased activity. In this case, various FcyRs may be of interest to the type of FcyR, eg, FcyRIIb. Likewise, antibodies can also be selected that maintain or increase FcyRIIb binding activity (at neutral pH conditions) and that reduce their binding activity to one or more activating FcyRs selected from the group consisting of: FcyRIa, FcyRIb, FcyRIc, FcyRIIIa , FcγRIIIb and FcγRIIa and so on. In such a case, the FcyR can be a human FcyR.

Alternatively, after making the antibody of Reveal A, the obtained antibody can be compared with a reference antibody for the FcRn binding activity at neutral pH conditions using BIACORE or other known techniques to select antibodies with increased FcRn binding activity at neutral pH conditions . In this case, the FcRn can be human FcRn.

Alternatively, after making the antibody of Reveal A, the obtained antibody can be evaluated for its isoelectric point by isoelectric focusing or other methods to select antibodies with increased isoelectric point values compared to the reference antibody. In this case, an antibody may be selected that has an increase in the isoelectric point value by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 or more, or at least 0.6, 0.7, 0.8, or 0.9 or more; or the isoelectric point The value increases by at least 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 or more or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 or more or 3.0 or more antibodies.

Alternatively, after making the antibody of Revelation A, the obtained antibody can be compared with a reference antibody for the 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 the ion concentration conditions. The ion concentration can be, for example, a hydrogen ion concentration or a metal ion concentration. When the ion concentration is a metal ion concentration, it can be, for example, a calcium ion concentration. Whether binding activity is altered or increased can be assessed based on the presence of, for example: (a) altered or enhanced cellular uptake of antigen; (b) altered or enhanced ability to bind multiple times to different antigen molecules; (c) reduced antigen concentration in plasma Altered or enhanced; or (d) altered plasma retention of the antibody. Alternatively, any two or more of these selection methods can be combined.

In an alternative embodiment, Disclosure A relates to a method of making or screening an antibody comprising an antigen-binding domain whose antigen-binding activity is altered according to ion concentration conditions and whose pI may be exposed on at least 1 of the surface of the antibody by modification amino acid residues ("Ion concentration-dependent antibodies with increased isoelectric point values"). The production methods can, for example, optionally combine the embodiments described in the context of Disclosure A, such as the aforementioned embodiments for the production or screening of antibodies for increased isoelectric point values, and for the production or screening of calcium ion concentrations. Embodiments of the method of an antigen-binding domain-dependent or calcium concentration-dependent antibody or library of the foregoing, the antigen-binding activity of which is higher under conditions of high calcium concentration than under conditions of low calcium concentration, and/or for manufacture or screening for pH dependence Embodiments of the method of an antigen binding domain or pH-dependent antibody or library thereof, the antigen binding activity of which is higher in neutral pH conditions than in acidic pH conditions.

In an alternative embodiment, Disclosure A provides a method of making or screening an antibody comprising an antigen-binding domain with increased extracellular matrix-binding activity, wherein its antigen-binding activity is altered according to ion concentration conditions, and the pi is determined by Modifications may increase by exposing at least one amino acid residue on the surface of the antibody ("ion concentration-dependent antibodies with increased isoelectric point values"). Ion concentration-dependent increases in the isoelectric point value of antibodies can be considered in this method. Such a method may be carried out, for example, by suitably optionally combining the relevant embodiments described in the context of Disclosure A, such as embodiments for the manufacture or screening of the aforementioned methods of antibodies with increased isoelectric point values, and for manufacture or Embodiments of methods of screening for calcium concentration-dependent antigen-binding domains or calcium concentration-dependent antibodies or libraries thereof, the antigen-binding activity of which is higher at high calcium concentration than at low calcium concentration, and/or for manufacture or Embodiments of the methods of screening for pH-dependent antigen binding domains or pH-dependent antibodies or libraries thereof have higher antigen-binding activity at neutral pH conditions than at acidic pH conditions. For example, the antibody obtained can be compared with a reference antibody for extracellular matrix binding ability using electrochemiluminescence or other known techniques to select antibodies with increased extracellular matrix binding.

The reference antibody here can include, but is not limited to, a natural antibody (such as a natural Ig antibody, preferably a natural IgG antibody) and an antibody before modification (before or during the construction of the antibody library, such as the ion concentration before the isoelectric point value is increased) dependent antibodies, or antibodies with an increased isoelectric point value prior to conferring an ion concentration-dependent antigen-binding domain).

In an alternative embodiment, Disclosure A relates to a method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity varies according to ionic concentration conditions, the method comprising the steps of: modifying at least one surface that may be exposed on the antibody. amino acid residues to increase the isoelectric point (pI). In some embodiments, the amino acid residue modification comprises a modification selected from the group consisting of: (a) substituting a negatively charged amino acid residue into an uncharged amino acid residue; (b) substituting a charged amino acid residue A negatively charged amino acid residue is a positively charged amino acid residue; and (c) substituting an uncharged amino acid residue into a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is substituted into a histidine. In a further embodiment, the antibody comprises variable and/or constant regions, and the amino acid residues are modified in the variable and/or constant regions. In a further embodiment, at least one amino acid residue modified according to this method is at a position in a CDR or FR selected from the group consisting of: (a) in the FR of the heavy chain variable region according to 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) according to Kabat numbering in the CDRs of the heavy chain variable region 31, 61, 62, 63, 64, 65, and 97; (c) 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, 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 at a position in a CDR or FR selected from the group consisting of: (a) 8 of the FRs 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) between the heavy chain variable regions 31, 61, 62, 63, 64, 65, and 97 in CDR; (c) 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, positions 77, 79, and 107; and (d) positions 24, 25, 26, 27, 52, 53, 54, 55, and 56 of the CDRs of the light chain variable region. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody made in accordance with the method against the corresponding antigen at an acidic pH (eg, pH 5.8) and a neutral pH (eg, pH 7.4). In a further embodiment, the method comprises selecting an antibody against the antigen with a KD (acid pH range (eg pH 5.8))/KD (neutral pH range (eg pH 7.4)) of 2 or higher. In some embodiments, the method further comprises comparing the antigen-binding activity of the antibody produced by the method under conditions of high ion concentration (eg, hydrogen ion or calcium ion concentration) and low ion concentration. In a further embodiment, the method further comprises selecting antibodies that have higher antigen-binding activity at high ionic concentrations (eg, 2-fold) than antibodies at low ionic concentrations. In some embodiments, if the ion concentration is calcium ion concentration, the high calcium ion concentration can 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 suitably selected from 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. Concentrations between 1 μM and 5 μM are also preferably chosen, which are close to the calcium ion concentration of early endosomes in vivo. In some embodiments, the lower limit value of KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (eg, KD (3 μM Ca)/KD (2 mM Ca)) is 2 or more, 10 or more or 40 or more, with a high limit of 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, with a high limit of 50 or less, 100 or less, or 200 or less. In some embodiments, if the ion concentration is the hydrogen ion concentration, the low hydrogen ion concentration (neutral pH range) can 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, pH 7.0, for example, 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, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The acidic pH range is preferably pH 5.8, which corresponds to the living hydrogen ion concentration in the early endosome, but for convenience of measurement, pH 6.0, for example, can be used. In some embodiments, the lower limit of KD (acid pH range)/KD (neutral pH range) (eg, KD (pH 5.8)/KD (pH 7.4)) 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 embodiments, the method further comprises comparing the elimination of the antigen from plasma prior to administration of the antibody produced by the method and administered only without including the reference antibody introduced by the method modified to be different. In a further embodiment, the method further comprises selecting an antibody made according to the method and an antibody that does not contain the modification introduced according to the method for promoting (eg, 2-fold) elimination of the antigen from plasma. In some embodiments, the method further comprises comparing the antibody produced by the method to an antibody with and without the modifications introduced by the method to be different for extracellular matrix binding of the antibody. In a further embodiment, the method further comprises selecting an antibody produced by the method that has increased extracellular matrix binding (eg, 2 when bound to an antigen) compared to an antibody that is different only without the modifications introduced by the method. times). In a further embodiment, the antibody produced by this method has an increased pI (a natural antibody (such as a natural Ig antibody, preferably a natural antibody) before modification or modification of at least one amino acid residue compared to the antibody. IgG antibody) or a reference antibody (eg, the antibody before antibody modification or library construction or at the time of 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, preferably 60% or more, compared to the binding activity of the antibody before or before modification. 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 of making an antibody comprising an antigen-binding domain whose antigen-binding activity varies according to ionic concentration conditions, wherein the method comprises modifying at least one surface that may be exposed on the surface of the antibody constant region. amino acid residues to increase the 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 of (c) is a positively charged amino acid residue; and (c) the substituted uncharged amino acid residue is a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is substituted with histidine. In a further embodiment, the antibody comprises variable and/or constant regions, and the amino acid residues are modified in the variable and/or constant regions. In a further embodiment, the at least 1 amino acid residue modified according to this method is at a position in the constant region 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 bits. In a further embodiment, the at least 1 amino acid residue modified according to this method is at a position in the constant region 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, the at least 1 amino acid residue modified according to this method is at a position in the constant region selected from the group consisting of: 282, 309, 311, 315, 342 according to EU numbering , 343, 384, 399, 401 bits. In some embodiments, the method further comprises comparing the antigen-binding activity of the antibody produced by the method under conditions of high ion concentration (eg, hydrogen ion or calcium ion concentration) and low ion concentration. In a further embodiment, the method further comprises selecting antibodies that have higher antigen-binding activity at high ionic concentrations than antibodies at low ionic concentrations. In some embodiments, the method further comprises comparing the elimination of the antigen from plasma prior to administration of the antibody produced by the method and administered only without including the reference antibody introduced by the method modified to be different. In a further embodiment, the method further comprises selecting an antibody made according to the method and an antibody that does not contain the modification introduced according to the method for promoting (eg, 2-fold) elimination of the antigen from plasma. In some embodiments, the method further comprises comparing the antibody produced by the method to an antibody with and without the modifications introduced by the method to be different for extracellular matrix binding of the antibody. In a further embodiment, the method further comprises selecting an antibody produced by the method that has increased extracellular matrix binding (eg, 2 when bound to an antigen) compared to an antibody that is different only without the modifications introduced by the method. times). In some embodiments, the method includes an antibody made in accordance with the method and a reference antibody comprising the constant region of native IgG directed against the Fc gamma receptor (FcyR) 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 native IgG. In some embodiments, antibodies made in accordance with this method are selected to have enhanced FcγRIIb binding activity at neutral pH. In some embodiments, antibodies made in accordance with this method are selected to have binding activity to one or more activating FcyRs (preferably selected from the group consisting of: FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa) and to FcyRIIb Alternatively, the FcγRIIb binding activity is maintained or enhanced, and the binding activity to activating FcγRs is reduced, compared to a reference antibody differing only in the point that the constant region is that of a native IgG constant region. In some embodiments, the method further comprises comparing the FcRn binding activity at neutral pH of an antibody made in accordance with the method to a reference antibody that differs only in that the constant region is that of a native IgG. In a further embodiment, the method further comprises selecting an antibody produced according to the method for FcRn binding activity at neutral pH compared to a reference antibody that differs only at the point that the constant region is that of a native IgG increase (eg 2x). In some embodiments, the antibody produced by this method has an increased pI (native antibody (e.g., native Ig antibody, preferably native IgG) before modification or modification of at least one amino acid residue) compared to the antibody. antibody) or a reference antibody (eg, the antibody before antibody modification or library construction or at the time of construction)), which (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, more preferably 70%, compared to the binding activity of the antibody before or before modification. % or 75% or more, preferably 80%, 85%, 90% or 95% or more of the activity.

In additional embodiments, the method comprises modifying at least one amino acid residue of the variable and constant regions of the antibody that may be exposed on the surface 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 above-disclosed constant region positions. In a further embodiment, at least one amino acid residue modified according to this method is located at one of the above-disclosed variable region positions. In a further embodiment, at least 1 amino acid residue modified according to this method is located at one of the positions of the constant regions disclosed above and at least 1 amino acid residue modified according to this method is located at a position disclosed above one of the variable regions. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody made in accordance with 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 an antibody that has a KD (acidic pH range)/KD (neutral pH range) of 2 or higher against the antigen. In some embodiments, the method further comprises comparing the antigen-binding activity of the antibody produced by the method under conditions of high ion concentration (eg, hydrogen ion or calcium ion concentration) and low ion concentration. In further embodiments, the method further comprises selecting antibodies that have higher antigen-binding activity at high ionic concentrations than at low ionic concentrations. In some embodiments, if the ion concentration is calcium ion concentration, the high calcium ion concentration can be selected from between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM. mM or a concentration between 400 μM and 1 mM. Concentrations selected between 500 μM and 2.5 mM are also ideal. In some embodiments, the low calcium ion 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 2 μM and 4 μM. Preferably, the concentration is selected from between 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) (eg, KD (3 μM Ca)/KD (2 mM Ca)) value is 2 or more, 10 or more or 40 or more, with a high limit of 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 the hydrogen ion concentration, the low hydrogen ion concentration (neutral pH range) can 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 Select. The low hydrogen ion concentration is preferably pH 7.4, which is close to the (blood) pH in plasma in vivo, for example pH 7.0 can be used for ease of measurement. 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, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The acidic pH range may be pH 5.8 or pH 6.0. In some embodiments, the lower limit of KD (acid pH range)/KD (neutral pH range) (eg, KD (pH 5.8)/KD (pH 7.4)) 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 embodiments, the method further comprises comparing the elimination of the antigen from plasma prior to administration of the antibody produced by the method and administered only without including the reference antibody introduced by the method modified to be different. In a further embodiment, the method further comprises selecting an antibody made according to the method and an antibody that does not contain the modification introduced according to the method for promoting (eg, 2-fold) elimination of the antigen from plasma. In some embodiments, the method further comprises comparing the antibody produced by the method to an antibody with and without the modifications introduced by the method to be different for extracellular matrix binding of the antibody. In a further embodiment, the method further comprises selecting an antibody produced by the method that has increased extracellular matrix binding (e.g., 5 when complexed with an antigen) compared to an antibody that does not include modifications introduced by the method that are different. times). In some embodiments, the method further comprises comparing the Fc gamma receptor (FcyR) binding activity at neutral pH (e.g., pH 7.4) of the antibody made in accordance with the method to a reference antibody comprising the constant region of native IgG. In a further embodiment, the method includes selecting an antibody made according to the method for enhanced FcγR binding activity at neutral pH (eg, pH 7.4) compared to an antibody comprising the constant region of a control native IgG. In some embodiments, antibodies made in accordance with this method are selected to have enhanced FcγRIIb binding activity at neutral pH. In some embodiments, an antibody produced according to this method is selected to have binding activity to one or more activating FcyRs (preferably selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa) and FcyRIIb, And optionally, the FcγRIIb binding activity is maintained or enhanced, and the binding activity to activating FcγRs is reduced. In some embodiments, the method further comprises comparing the antibody produced according to the method to the constant region of an IgG only in which the constant region is native The FcRn-binding activity of the reference antibodies at neutral pH conditions differed from one another. In a further embodiment, the method further comprises selecting an antibody produced according to the method for FcRn binding activity at neutral pH compared to a reference antibody that differs only at the point that the constant region is that of a native IgG increase (eg 2x). The antibody produced according to this method has an increased pI (natural antibody (such as a natural Ig antibody, preferably a natural IgG antibody) or a reference antibody before modification or modification of at least one amino acid residue compared to the antibody. (eg, the antibody before antibody modification or library construction or at the time of 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, more preferably 70% of the binding activity of the antibody before modification or modification. % or 75% or more, preferably 80%, 85%, 90% or 95% or more of the activity.

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

In an alternative embodiment, Disclosure A relates to a composition or pharmaceutical composition comprising the antibody of Disclosure A above. In one embodiment, the pharmaceutical composition disclosed in A may be a pharmaceutical composition for accelerating the elimination of antigens from a subject's biological fluid (preferably plasma, etc.) and/or for increasing extracellular matrix binding (if the antibody disclosed in A is administered). A pharmaceutical composition to (administered to) the subject (preferably in vivo). The pharmaceutical composition of Disclosure A may optionally contain a pharmaceutically acceptable carrier. A pharmaceutical composition herein generally refers to an agent used in the treatment, prevention, diagnosis or examination of a disease.

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

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

In some embodiments, the present disclosure provides antibodies with increased pI (antibodies with increased isoelectric point value) by modifying at least one amino acid residue that may be exposed on the surface; methods of producing such antibodies; or Use of such antibodies to enhance the elimination of antigens from plasma (when the antibodies are administered to a subject in vivo). It can be understood that the scope of the disclosure A described herein and the contents described in the examples can be appropriately applied to such an embodiment. In other embodiments, the present disclosure provides antibodies whose pI is reduced by modifying at least one amino acid residue that may be exposed on the surface ("antibodies with reduced isoelectric point values"); methods of producing such antibodies; Or the use of such antibodies to improve plasma retention (when the antibodies are administered in vivo to a subject). The inventors found that the cellular internalization of antibodies can be enhanced by increasing the isoelectric point value by introducing specific amino acid mutations into specific parts of the amino acid sequence of the constant region. Those skilled in the art can understand that: by introducing amino acids with different side chain charges into the above-mentioned sites, the cellular internalization of the antibody is inhibited due to the decrease of the isoelectric point value, and the plasma retention of the antibody can be prolonged. It can be understood that the content described in the category of A and the paired examples disclosed herein can be appropriately applied to such an implementation.

In one embodiment, the present disclosure provides a method of producing a modified antibody having increased or decreased half-life in plasma compared to the antibody prior to modification, the method comprising: (a) modifying a nucleic acid encoding the antibody prior to modification to alter 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 make the antibody; and (c) ) produced antibodies are collected from host cell cultures.

An additional embodiment provides a method of prolonging or reducing the half-life of an antibody in plasma, comprising modifying at least one amino acid residue selected from the group consisting of: According to EU Numbering Act 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.

These methods may further include determining whether the half-life in plasma of the collected and/or modified antibody is prolonged or shortened by comparing the antibody prior to modification.

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 not limited thereto: (a) Glu (E ) with 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, line B is disclosed with respect to Fc region variants, their uses and methods of making them.

In the context of disclosures A and B described herein, "Fc region variant" may refer to modification of the Fc region, for example, by modifying at least one amino acid of the Fc region of a native IgG antibody 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, the Fc region variant includes not only an Fc region into which amino acid modification has been introduced, but also an Fc region containing the same amino acid sequence as the aforementioned Fc region.

In an alternative embodiment, line B is disclosed for an Fc region variant comprising an FcRn binding domain containing Ala at position 434; any of Glu, Arg, Ser and Lys at position 438; and Glu, Asp and GIn at position 440 (disclosure B category disclosed herein, such Fc region variants are also referred to as "novel Fc region variants" for documentation purposes), according to EU numbering.

In practice, it is revealed that Fc region variants of B, regardless of the type of target antigen, can be introduced into almost any antibody (eg, multispecific antibodies such as bispecific antibodies). For example, the Fc region variants shown in Example 20 can be used to produce anti-Factor IXa/Factor X bispecific antibodies (eg, F8M-F1847mv [F8M-F1847mv1 (SEQ ID NO: 323) and F8M-F1847mv2 (SEQ ID NO: 323)] 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 light chain]; and F8M-F1927mv [F8M-F1927mv1 (SEQ ID NO:328) and F8M-F1927mv2 (SEQ ID NO:329) as heavy chain and F8ML (SEQ ID NO:325) as a light chain]).

As mentioned above, WO2013/046704 reports: Fc region variants that have introduced mutations that increase FcRn binding under acidic conditions and specific mutations (representative examples are double residue mutations, according to EU numbering Q438R/S440E), show binding to rheumatoid factor was significantly reduced. However, WO2013/046704 does not describe the Fc region variant with reduced rheumatoid factor binding due to Q438R/S440E modification. Compared with antibodies with natural Fc region, the plasma retention is better. Safe 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 safe and more favorable 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 (amino acid substitution at position 434 according to EU numbering to Ala (A)) and specific double residue mutations (representative examples are The Fc region variant of Q438R/S440E) is suitable for prolonging the retention of antibodies in plasma and maintaining significantly reduced binding to rheumatoid factors.

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

In one embodiment, Disclosure B provides a novel combination of amino acid substitutions of the FcRn binding domain that increases 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, the Fc region variant of B is disclosed to contain Ala at position 434; any of Glu, Arg, Ser, and Lys at position 438; and any of Glu, Asp, and Gln at position 440, according to EU and preferably Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering. Ideally, the Fc region variant of B is revealed to additionally contain Ile or Leu at position 428, and/or any of Ile, Leu, Val, Thr and Phe at position 436, according to EU numbering. More 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 revealed that the Fc region variant of B may be a natural Ig antibody Fc region variant, more preferably a natural IgG (IgG1, IgG2, IgG3 or IgG4 type) antibody Fc region variant. Parts of the native Fc region are described within the scope of disclosures A and B. More specifically, in the disclosure B, the native Fc region may refer to an unmodified or naturally occurring Fc region, preferably an unmodified or naturally occurring Fc region of a native Ig antibody, the Fc region of which is an amine group. Acid residues remain unmodified. The source of the antibody for the Fc region can be Ig, eg, IgM or IgG, eg, human IgGl, IgG2, IgG3 or IgG4. In one embodiment, it can be human IgGl. Whereas a (control) antibody comprising a native Fc region may refer to an antibody containing an unmodified or naturally occurring Fc region.

Positions 428, 434, 438, and 440 are common to the Fc region of all native human IgG1, IgG2, IgG3, and IgG4 antibodies. However, at position 436 of the Fc region, the native human IgG1, IgG2 and IgG4 antibodies are all Tyr (Y), and the native human IgG3 antibody is Phe (F). On the other hand, Stapleton et al. (Nature Comm. 599 (2011) reported that a human IgG3 subtype containing amino acid substitution R435H according to the EU numbering method has a plasma half-life comparable to IgG1. Therefore, the inventors also assumed that 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 bisamino acid residues of Q438R/S440E, Q438R/S440D, Q438K/S440E and Q438K/S440D according to the EU numbering method, which when combined with FcRn, which can increase in acidic conditions, will be combined. Significantly reduces rheumatoid factor binding.

Therefore, in a preferred embodiment, the FcRn binding domain of the Fc region variant of Disclosure B may comprise a combination of substituted amino acid positions selected from the group consisting of: (a) according to EU numbering, N434A/Q438R/ (b) N434A/Q438R/S440D; (c) N434A/Q438K/S440E; (d) N434A/Q438K/S440D; (e) N434A/Y436T/Q438R/S440E; (f) N434A/Y436T/Q438R/S440D ; (g) N434A/Y436T/Q438K/S440E; (h) N434A/Y436T/Q438K/S440D; (i) N434A/Y436V/Q438R/S440E; (j) N434A/Y436V/Q438R/S440D; (k) N434A/ Y436V/ Q438K/S440E; (l) N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/ S440E; (n) N434A/R435H/F436T/Q438R/S440D; (o) N434A/R435H/ F436T/Q438K/ S440E; (p) N434A/R435H/F436T/Q438K/S440D; (q) N434A/R435H/F436V/Q438R/ S440E; (r) N434A/R435H/F436V/Q438R/S440D; (s) N434A/ R435H/F436V/Q438K/ S440E; (t) N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v) M428L/N434A/Q438R/S440D; (w) M428L/N434A/ Q438K/S440E; (x) M428L/ N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/ Y436T/Q438R/S440D; (aa) M428L/N434A/Y436T Q438K/S440E; (ab) M428L/N434A/ Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad) M428L/N434A/ Y436V/Q438R/S4 40D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af) M428L/N434A/ Y436V/Q438K/S440D; (ag) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (ah ) L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

In a preferred embodiment, the FcRn binding domain of the Fc region variant of Disclosure B may comprise 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; (e) M428L/N434A/Y436T/Q438R/S440E; (f) M428L /N434A/ Y436V/Q438R/S440E; (g) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (h) L235R/G236R/A327G/A330S/P331S/M428L/Q434A/Y436R S440E.

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

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

In some embodiments, it is revealed that the Fc region variant of B has an increase in FcRn binding activity in the acidic pH range (eg, at pH 6.0 and 25°C), eg, 1.5-fold, 2-fold, 3-fold greater than the Fc region of native IgG. 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 increased FcRn binding activity of the 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 a native IgG.

Manipulation of the FcRn binding domain by introducing amino acid substitutions occasionally reduces antibody stability (WO2007/092772). Poorly stable proteins tend to aggregate easily during storage, and the stability of pharmaceutical proteins is very important in the manufacture of pharmaceutical agents. Therefore, the development of stable antibody preparations may be difficult due to the reduced stability caused by the substitution of the Fc region (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 did not contain significant amounts of high molecular weights, whereas manipulation of the FcRn binding domain by introducing substitutions may generate large amounts of high molecular weights. In this case, such high molecular weight species must be removed from the drug using a refining step.

Amino acid substitutions of antibodies may have negative effects, such as increasing the immunogenicity of therapeutic antibodies, thereby causing interferon storms and/or producing anti-drug antibodies (ADAs). The clinical utility and efficacy of therapeutic antibodies may be limited by ADAs because they affect the pharmacokinetics of therapeutic antibodies and sometimes cause severe side effects. There are many factors that affect 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 problematic. Such pre-existing antibodies, such as rheumatoid factor (RF), are autoantibodies (antibodies against autologous proteins) against the Fc portion of the antibody (ie, IgG). Rheumatoid factor is particularly found in patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis. In arthritic patients, RF binds to IgG to form an immune complex that contributes to disease progression. Recently, a humanized anti-CD4 IgG1 antibody with an Asn434His mutation was reported to elicit 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 increases the binding of the Fc region of the antibody to rheumatoid factors compared to the parental human IgG1.

RF is a polyclonal autoantibody against human IgG. The RF epitope of human IgG sequences will vary based on each colony; however, the RF epitope appears to be located at the CH2/CH3 junction and in the CH3 domain, overlapping with the FcRn binding epitope. Therefore mutations that increase FcRn binding activity at neutral pH may also increase binding activity to specific RF colonies.

In the context of Disclosure B, the term "anti-drug antibody" or "ADA" can refer to an endogenous antibody that has binding activity to an epitope located at a therapeutic antibody, and thus can bind to the therapeutic antibody. The terms "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 prior to administration of the therapeutic antibody to the patient. In some embodiments, the pre-existing ADA is a human antibody. In a further embodiment, the pre-existing ADA is a rheumatoid factor.

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

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 of 15°C to 40°C, such as between 20°C and 25°C or 25°C. In yet another embodiment, the interaction of a human pre-existing ADA with a human Fc region is measured at pH 7.4 (or pH 7.0) and 25°C.

Significantly increased binding activity to (pre-existing) ADA or equivalent performance may be indicative of a (pre-existing) ADA of the Fc region variant of Disclosure B or an antibody containing this variant within the scope of Disclosure B described herein Binding activity (binding affinity) (i.e., KD) compared to the measured (pre-existing) ADA binding activity (binding affinity) of a reference Fc region variant of a reference antibody containing this 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 can be observed in individual patients or groups of patients.

In one embodiment, the term "patients" or "patients" as used in the context of Disclosure B is not limited and includes 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 disease can include rheumatoid arthritis.

In one embodiment of Disclosure B, the significantly increased binding activity to pre-existing ADA in an individual patient may refer to the binding of an antibody (eg, a therapeutic antibody) containing an Fc region variant to pre-existing ADA detected in the patient The activity is increased 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 to this antibody is preferably 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 ECL response of less than 500 or 250 for the reference antibody. Specifically, between the binding activity of the reference antibody to pre-existing ADA and the binding activity of the antibody with the Fc region variant, the ECL response is preferably 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, in a group of patients, targeting including (a) increased binding activity to FcRn at acidic pH and (b) binding activity to pre-existing ADA at neutral pH The fraction of patients with a measured ECL response of at least 500 (preferably at least 250) for the antibody of the increased Fc region variant compared to the fraction of patients with an ECL response to the reference antibody of at least 500 (preferably at least 250 or more) Partially increased, eg, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% higher than the fraction of patients with an ECL response to the reference antibody.

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

Alternatively, in an individual patient, the significantly reduced binding activity of an antibody containing the Fc region variant to pre-existing ADA may refer to an ECL response of 500 or more (preferably to the original antibody) compared to the ECL response to the reference antibody. 1000 or more or 2000 or more) becomes less than 500, preferably less than 250.

In a preferred embodiment, the Fc region variant of B and the antibody containing the Fc region variant are revealed to have low binding activity to pre-existing ADA at neutral pH. In particular, it is desirable that the binding activity of the antibody containing the Fc region variant of Reveal B to pre-existing ADA at neutral pH is lower than that of the reference antibody containing the Fc region of native IgG at neutral pH (eg pH 7.4) to pre-existing ADA There was or no significant increase in ADA binding activity. Low binding activity (binding affinity) or affinity at baseline for pre-existing ADA may refer to, but is not limited to, an ECL response in an individual patient of less than 500 or less than 250. Low binding activity to pre-existing ADA in a population of patients may, for example, mean that the ECL response of the population of patients is 90%, preferably 95%, more preferably 98% less than 500.

An Fc region variant that discloses B, or an antibody containing it, is preferably selected for its binding activity to (pre-existing) ADA in plasma at neutral pH without a significant increase and for 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 for ADA at neutral pH conditions (eg pH 7.4) compared to the Fc region of native IgG, the ADA may be pre-existing ADA, preferably rheumatoid Sex Factor (RF).

In one embodiment, it is desirable to reveal 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, reduced clearance (CL) in plasma and retention in plasma Prolonged time or half-life (t1/2) in plasma. These correlations are known in the art.

In one embodiment, it is desirable to reveal that the Fc region variant of B has increased FcRn binding activity at acidic pH conditions but not significantly increased ADA binding activity at neutral pH conditions compared to the Fc region of native IgG. The clearance rate (CL) is reduced, the retention time in plasma is prolonged or the half-life in plasma (t1/2) is prolonged. The ADA may be pre-existing ADA, preferably rheumatoid factor (RF).

In one embodiment, the disclosure of the Fc region variant of B is advantageous because of its plasma retention 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 2 Fc region variants: Fc region variant F1718 described in WO2013/046704 (Fc region, mutations introduced at 4 sites: N434Y/Y436V/Q438R/S440E), and a novel Fc region variant Plasma retention of F1848m (mutated at 4 sites: N434A/Y436V/Q438R/S440E). The amino acid mutation of the two Fc region variants differs only in that according to EU numbering at position 434, the introduced amino acid is mutated to Y (tyrosine) for F1718 and to 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)). It is therefore desirable to reveal that the Fc region variant of B has 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) demonstrate that the various Fc region variants, ie, F1847m, F1886m, F1889m, and F1927m, have further improved plasma retention than F1848m. Therefore, those of ordinary skill in the art know that the Fc region variant of disclosure B including F1847m, F1886m, F1889m or F1927m and F1848m, compared to the reference Fc region variant including substitution N434Y/Y436V/Q438R/S440E, has Better plasma retention improvement.

Binding to FcyR or alpha 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 favorable. In some embodiments, the Fc region variant of B is revealed to have only weak effector receptor binding activity or no effector receptor binding. Examples of effector receptors include activating FcyRs, particularly FcyRI, FcyRII, and FcyRIII. FcyRI includes FcyRIa, FcyRIb, and FcyRIc, and subtypes thereof. FcyRII includes FcyRIIa (with 2 subtypes: R131 and H131) and FcyRIIb. 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 (eg, Fab, F(ab)' ₂ , scFv, sc(Fv) ) ₂ and diabody).

Fc regions that have only weak effector receptor binding activity or do not bind to effector receptors are described, for example, in Strohl et al. (Curr. Op. Biotech. 20(6):685-691 (2009)), including in particular For example, deglycosylated Fc regions (N297A and N297Q) and silencing Fc regions, which by manipulating the Fc region to silence its effector function (or suppress immunity) (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-L235A/G23A7A/E31 -L236E). WO2008/092117 describes an antibody containing a silent Fc region containing the substitutions G236R/L328R, L235G/G236R, N325A/L328R or N325L/L328R according to EU numbering. WO2000/042072 describes an antibody comprising a silent Fc region comprising 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 that 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, which contains the substitution D265A. Modifications of these amino acid residues can also be appropriately introduced into the Fc region variant of B-revealing.

Expressed as "weak binding to effector receptors" may refer to effector receptor binding activity that is less than, e.g., 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, 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 a variant of an Fc region that includes one or more amino acid substitutions, insertions, additions, deletions, etc., that has reduced binding to effector receptors compared to a native Fc region. Since the effector receptor binding activity can be significantly reduced, such a silent Fc region will no longer bind to the effector receptor. A silent Fc region can include, for example, an Fc region containing amino acid substitutions at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269 , EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332. Modifications of these amino acid positions can also be appropriately introduced into Fc region variants of B-revealing.

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: EU235, EU237, EU238, EU239, EU270, EU298, EU325, and EU329 The group of wherein, is substituted into amino acid residues selected from the group consisting of:

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

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

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

The amino acid at EU297 is preferably substituted as Ala.

The amino acid at 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 EU 300 is preferably substituted with an amino acid selected from the group consisting of Arg, Lys and Pro.

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

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

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

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

The amino acid at 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: Substitute Lys or Arg in EU235, Substitute Lys or Arg in EU237, Substitute Lys or Arg in EU238, Substitute Lys or Arg in EU239, Substitute Phe in EU270, Substitute Gly in EU298 , Gly at EU325, or Lys or Arg at EU329. More preferably the silent Fc region may comprise: substitution at EU235 for arginine or at EU239 for lysine. More preferably the silent Fc region may comprise the L235R/S239K substitution. Modifications of these amino acid residues can also be appropriately introduced into the Fc region variant of B-revealing.

In one embodiment, antibodies comprising Fc region variants that reveal B have only weak complement protein binding activity or do not bind complement proteins. In some embodiments, the complement protein is C1q. In some embodiments, weak complement protein binding activity refers to a 10-fold or more, 50-fold or more reduction in complement protein binding activity compared to the complement protein binding activity of native IgG or an antibody comprising a native IgG Fc region. 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, an Fc region variant of B revealing or an antibody comprising the Fc region variant can be assessed for its (human) FcRn binding activity in the neutral pH range and/or in the acidic pH range in the same manner as above .

In one embodiment, a method of modifying antibody constant regions to make B-revealing Fc region variants can be based on, for example, evaluating the structural isotypes of several constant regions (IgGl, IgG2, IgG3, and IgG4) to select antigens in the acidic pH range. Constructive isoforms with decreased binding activity and/or increased dissociation rates in the acidic pH range. Alternative methods can be based on introducing amino acid sequences that substitute amino acid sequences of the native IgG structural isotype to reduce antigen binding activity in the acidic pH range (eg, pH 5.8) and/or increase the dissociation rate in the acidic pH range. The hinge region sequence of the antibody constant region is significantly different in different structural isotypes (IgG1, IgG2, IgG3, and IgG4), and the difference in the amino acid sequence of the hinge region significantly affects the antigen-binding activity. Therefore, structural isotypes with reduced antigen-binding activity in the acidic pH range and/or increased dissociation rate in the acidic pH range can be selected by selecting an appropriate structural isotype according to the type of antigen or epitope. Since 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 structural homotype can be located in the hinge region.

In an alternative embodiment, Disclosure B provides the use of an antibody comprising an Fc region variant of Disclosure B above to accelerate the release of an antibody that has been internalized in the antigen-binding form into the cell and the antigen-free form outside the cell. Here, "the antibody that has been internalized into the cell in the antigen-binding form and released into the cell in the antigen-free form" does not necessarily mean that the antibody that has been internalized into the cell in the antigen-binding form is completely released into the cell in the antigen-free form. Extracellular. Comparing to before modification of the FcRn-binding domain (eg, before increasing the FcRn-binding activity of the antibody in the acidic pH range), it is acceptable for a portion of the antibody to be fully released outside the cell in the episomal form. Preferably, the antibody released extracellularly maintains its antigen-binding activity.

The term "capacity to eliminate antigen from plasma" or an equivalent term, may refer to the ability of an antibody to eliminate antigen from plasma when administered or secreted in vivo. Thus, "the ability of this antibody to eliminate antigen from plasma is increased" may refer to an increased rate of elimination of antigen from plasma if the antibody is administered compared to before modification of its FcRn binding domain. Increased activity of an antibody to eliminate the antigen from plasma can be assessed, for example, by administering a soluble antigen and the antibody in vivo, and measuring the concentration of soluble antigen in plasma after administration. The soluble antigen can be either bound antibody or antibody unbound antigen, and the concentration can be respectively "antibody bound antigen concentration in plasma" and "antibody unbound antigen concentration in plasma". The latter is synonymous with "free antigen concentration in plasma". "Total antigen concentration in plasma" may refer to the sum of the antigen concentration of bound antibody and the antigen concentration of unbound antibody.

In an alternative embodiment, Disclosure B provides a method of extending the plasma residence time of an antibody comprising an Fc region variant of Disclosure B. Native human IgG can bind to FcRn from non-human animals. For example, because native human IgG binds more strongly to mouse FcRn than human FcRn (Ober et al., Intl. Immunol. 13(12):1551-1559 (2001)), this antibody was administered to mice for evaluation. Quantify the properties of this antibody. Alternatively, for example, mice in which the native FcRn gene is disrupted but replaced and express the human FcRn gene as a transgenic gene (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 herein, the plasma concentration of free antigen not bound to antibody, or the ratio of free antigen concentration to total antigen concentration can be determined (eg Ng et al., Pharm. Res. 23 (1): 95-103 (2006)). Or if an antigen exhibits a specific function in vivo, whether the antigen binds to an antibody (antagonistic molecule) that neutralizes the function of the antigen can be assessed by testing whether the function of the antigen is neutralized. Whether the antigenic function is neutralized can be assessed by measuring specific in vivo markers reflecting the antigenic function. Whether an 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 specific limitations on the measurement, such as determining the concentration of free antigen in plasma, determining the ratio of free antigen in plasma to total antigen in plasma, and in vivo marker measurement; however, measurement should be performed during a certain period after antibody administration implemented later. In the context of disclosure B, "after a certain period of time after the antibody is administered" is not particularly limited, and this period can be appropriately determined by those of ordinary knowledge in the technical field according to the nature of the administered antibody, including, for example, 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 "total antigen concentration in plasma", which is the sum of the antigen concentration of bound antibody and that of unbound antibody, or "free antigen concentration in plasma", which is the unbound antigen concentration Antigen concentration of the antibody.

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

The smaller the C value, the higher the efficiency of antigen elimination per antibody, and the larger the C value, the lower the antigen elimination efficiency per antibody.

In some aspects, when the antibody of Disclosure B is administered, the antigen/antibody molar ratio is reduced by 2-fold, 5-fold, 10-fold compared to administration of a reference antibody comprising the native human IgG Fc region as the human FcRn binding domain , 20 times, 50 times, 100 times, 200 times, 500 times, or 1,000 times 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 Reveal B does not cross-react with the mouse paired antigen, mouse strains 32 or 276 ( Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010)) for evaluation. When the antibody cross-reacts with the mouse paired antigen, the antibody can be assessed simply by injecting the antibody into human FcRn transgenic mouse strains 32 or 276 (Jackson Laboratories). In the co-injection model, a mixture of antibody and antigen is administered to mice. For the steady-state antigen of the fusion model, the fusion pump filled with the antigen solution was transplanted into mice to achieve a certain antigen concentration in plasma, and then the antibody was injected into the mice. All test antibodies were administered at the same dose. Total antigen concentration in plasma, free antigen concentration in plasma, and antibody concentration in plasma can be measured at appropriate time points.

To assess the long-term effect of the B-revealing antibody, total or free antigen concentration or antigen/antibody ratio in plasma can be measured at 2, 4, 7, 14, 28, 56, or 84 days after administration Ear ratio. In other words, to assess the properties of the antibody, the total or free antigen concentration or the antigen/antibody ratio in plasma can be measured by 2 days, 4 days, 7 days, 14 days, 28 days, 56 days or 84 days after administration. molar ratio to determine the antigen concentration in plasma over a long period of time. Whether the antigen concentration or antigen/antibody molar ratio in plasma is decreased due to this antibody can be determined by assessing such a decrease at one or more time points as described above.

To assess the short-term effect of the B-revealing antibody, total or free antigen concentration or antigen/antibody ratio in plasma can be measured 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration Ear ratio. In other words, to assess the properties of the antibody, the total or free antigen concentration in plasma or the moles of antigen/antibody can be measured 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours or 24 hours after administration ratio to determine the antigen concentration in plasma over a short period of time. When it is difficult to determine plasma retention in humans, plasma retention can be predicted in mice (such as normal mice, human antigen-expressed mice or human FcRn-expressed mice) or monkeys (such as Malayan monkey).

In an alternative embodiment, Disclosure B relates to an antibody comprising an Fc region variant of Disclosure B as described above. Various embodiments of such antibodies are within the scope of Disclosures A and B described herein, unless inconsistent with the context, and may be employed without departing from ordinary skill in the art.

In one embodiment, an antibody comprising an Fc region variant that discloses B is useful as a therapeutic antibody for the treatment of patients with autoimmune disease, graft rejection (graft-versus-host disease), other inflammatory diseases, or allergic diseases. Patients, as described in WO2013/046704.

In one embodiment, the antibody comprising the Fc region variant 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 Antibody to the sugar chain of GlcNAc in the middle (WO02/79255). In one embodiment, the antibody may be deglycosylated. In some embodiments, the antibody includes, for example, a mutation at the heavy chain glycation site to inhibit glycation at this site, as described in WO2005/03175. Such unglycosylated antibodies can be produced by modifying the heavy chain glycation site, ie, introducing N297Q or N297A substitutions according to EU numbering, and expressing the protein in an appropriate host cell.

In an alternative embodiment, disclosure B relates to a composition or a pharmaceutical composition comprising an antibody comprising such an Fc region variant. The various embodiments of the compositions or pharmaceutical compositions described within the scope of disclosures A and B may be applied without departing from ordinary skill in the art, unless inconsistent with the context. Such compositions can be used to enhance plasma retention (in a subject, when the antibody system of Disclosure B is administered (administered) to the subject).

In an alternative embodiment, B is disclosed for a nucleic acid encoding an Fc region variant or an antibody comprising the Fc region variant. The various embodiments of nucleic acids within the scope of disclosure A and B described herein, unless inconsistent with the context, can be applied without departing from ordinary skill in the art. Alternatively, it is disclosed that B relates to a vector containing the nucleic acid. Various implementations within the scope of Disclosures A and B described herein, unless inconsistent with the context, may be employed without departing from ordinary technical knowledge in the art. Alternatively, it is revealed that B relates to the host or host cell containing the antibody. Various implementations within the scope of Disclosures A and B described herein, unless inconsistent with the context, may be employed without departing from ordinary technical knowledge in the art.

In an alternative embodiment, it is disclosed that line B relates to a method for making an Fc region variant comprising an FcRn binding domain or an antibody comprising the Fc region variant, comprising culturing the above-mentioned host cell or growing the above-mentioned host, from a cell culture, host 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 mutation that enhances FcRn binding activity under acidic pH conditions compared to the Fc region of native IgG (b) selection of Fc region variants whose binding activity to (pre-existing) ADA was not significantly improved at neutral pH compared to the Fc region of native IgG; (c) selection of Fc region variants, which Increased plasma retention compared to the Fc region of native IgG; and (d) selecting an antibody that includes an Fc region variant that promotes elimination of the antigen from plasma that enhances plasma clearance compared to a reference antibody that includes the Fc region of native IgG Eliminate antigens.

From the viewpoint of assessing the plasma retention of the Fc region variant of revealed B, without limitation, it is appropriate that the antibody containing the Fc region variant produced by the disclosure B and the "reference antibody containing the Fc region of the native IgG" are identical to each other, only The exception is the Fc region for comparison. The FcRn can be human FcRn.

For example, after an antibody comprising an Fc region variant of Reveal B is produced, BIACORE (registered trademark) can be used for the FcRn binding activity under acidic pH conditions (eg pH 5.8) for this antibody and a reference antibody comprising an IgG Fc region which may be native or other known techniques to select Fc region variants or antibodies containing the Fc region variants with increased FcRn binding activity under acidic pH conditions.

Alternatively, for example, after making an antibody comprising an Fc region variant of disclosure B, this antibody and a reference antibody comprising a native IgG Fc region can be assayed for ADA binding activity at neutral pH by electrochemiluminescence (ECL) Or compared 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 making an antibody comprising the Fc region variant of disclosure B, this antibody and a reference antibody comprising a native IgG Fc region can be obtained using plasma from, for example, mouse, rat, rabbit, dog, monkey or human. 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 a subject.

Alternatively, the antibody and a reference antibody comprising a native IgG Fc region can be prepared, for example, by using plasma from, for example, mouse, rat, rabbit, dog, monkey or human by Antibody pharmacokinetic assays are performed to select Fc region variants or antibodies containing the Fc region variants that enhance antigen elimination from plasma.

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 comprising the variant, the method comprising substituting amino acids such that the resulting Fc region variant or containing The antibody of this variant 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 a method comprises substituting amino acids such that the Fc region variant obtained or the antibody comprising the antibody further comprises, according to EU numbering, Ile or Leu at position 428 and/or Ile, Leu, Val, Thr or Phe at position 436. In yet another embodiment, the Fc region variant obtained by amino acid substitution according to this method or the antibody containing the variant further comprises 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 of producing an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant, the method comprising substituting amino acids such that the resulting Fc region variant or comprising the same 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 a method comprises substituting amino acids such that the Fc region variant obtained or the antibody containing the variant further comprises Ile or Leu at position 428 and/or Ile, Leu according to EU numbering , Val, Thr or Phe at bit 436. In yet another embodiment, the amino acid substitution according to this method to obtain an Fc region variant according to the present invention or an antibody containing this variant further comprises Leu at position 428 and/or Val or Thr at position 436 according to EU numbering .

In one embodiment, such a method comprises substituting all amino acids at positions 434, 438 and 440 to Ala; Glu, Arg, Ser or Lys; and Glu, Asp or Gln. In an additional embodiment, the method comprises substituting amino acids such that the Fc region variant obtained or the antibody containing the variant further comprises 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 into the Fc region variant obtained or the antibody containing the variant further comprises Leu at position 428 and/or Val or Thr at position 436 according to EU numbering according to this method.

In an alternative embodiment, disclosure of B for an Fc region variant or an antibody comprising the Fc region variant is obtained by any of the methods disclosed above for B.

In an alternative embodiment, Disclosure B provides a method for reducing (pre-existing) ADA binding activity of an Fc region variant with increased FcRn binding activity at acidic pH (eg, pH 5.8); and a method for making Fc region variants with increased FcRn binding activity at acidic pH and reduced preexisting ADA binding activity, comprising: (a) providing an antibody comprising an Fc region with increased FcRn binding activity at acidic pH compared to a reference antibody ( and (b) introduced into the Fc region according to EU numbering; (i) amino acid substitution to Ala at position 434; (ii) substitution of any of Glu, Arg, Ser and Lys at position 438 and (iii) amino acid substitution at position 440 to any of Glu, Asp and GIn; (iv) optionally, amino acid substitution at position 428 to Ile or Leu; and/ Or (v) alternatively, 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 preferably a human IgG Fc domain (variant). Also, in order to increase the FcRn-binding activity at acidic pH and decrease the (pre-existing) ADA-binding activity at a neutral pH range (eg, pH 7.4), the Fc region (variant) is to contain a member selected from the group consisting of Combination 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; (i) N434A/Y436V/Q438R/S440E; (j) ) N434A/Y436V/Q438R/S440D; (k) N434A/Y436V/ Q438K/S440E; (l) N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/S440E; (n) N434A/R435H /F436T/Q438R/S440D; (o) N434A/R435H/F436T/Q438K/S440E; (p) N434A/R435H/F436T/Q438K/S440D; (q) N434A/R435H/F436V/Q438R/S440E; (r) N434A /R435H/F436V/Q438R/S440D; (s) N434A/R435H/F436V/Q438K/S440E; (t) N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v) M428L /N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x) M428L/N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/Y436T /Q438R/S440D; (aa) M428L/N434A/Y436T/Q438K/S440E; (ab) M428L/N434A/ Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad ) M428L/N434A/ Y436V/Q438R/S440D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af) M428L/N434A/ Y436V/Q438K/S440D; Y436T/Q438R/S440E; and (ah) L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

This method can optionally further comprise: (c) assessing whether the antibody has reduced (pre-existing) ADA binding activity, including the antibody in which the Fc region variant has reduced binding activity compared to the reference antibody.

Alternatively this method can be used as a method to enhance the extracellular release of an antibody that has been internalized in the antigen-bound form into the cell in the antigen-free form 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 in the treatment of diseases associated with IL-8, as detailed below described. The meanings of the terms given below are applicable to the disclosure of C all, and are not inconsistent with the common technical knowledge of those of ordinary skill in the art and the embodiments known to those of ordinary skill in the art.

I. Reveal the definition within the category of C In the definition within the scope of disclosing C, "acidic pH" refers to 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.

In a definition within the scope of disclosing 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" as used in Disclosure C, unless otherwise specified, refers to a protein derived from any vertebrate, primate (such as humans, macaques, rhesus monkeys) and other mammals (such as dogs and rabbits). natural IL-8. The term "IL-8" includes full-length IL-8, untreated IL-8, and any form of IL-8 that results 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 an antibody that binds IL-8 with sufficient affinity to be useful as a diagnostic and/or therapeutic agent targeting IL-8 .

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

The term "affinity" as used in the context of disclosing C generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, the term "binding affinity" used in the context of disclosing C refers to intrinsic binding affinity, reflecting a 1:1 interaction of members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Binding affinity can be measured using methods known in the art, including those described in the disclosure of the C category.

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 (eg, ^10-8 M or less). , from 10 ^-8 M to 10 ^-13 M, from 10 ^-9 M to 10 ^-13 M).

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

"Binding to the same epitope" as a reference antibody refers to an antibody that blocks binding of a control antigen to its antigen by, for example, 50%, 60%, 70%, or 80% or more; and conversely, a reference antibody blocks This antibody binds, for example, 50%, 60%, 70%, or 80% or more to its antigen. Exemplary competitive assays can be used here without limitation.

"Chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is derived from one particular source or species, and the other portion is derived from a different source or species.

A "humanized" antibody system refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody can include substantially at least 1, and usually 2, variable regions, wherein all (or substantially all) HVRs (eg, CDRs) correspond to non-human antibodies, and all (or substantially all) this FR corresponds to a human antibody. A humanized antibody may optionally include at least a portion of the antibody constant region from a human antibody.

The term "monoclonal antibody" in the context of Disclosure C refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except where possible variations are possible. Monoclonal antibodies, eg, containing naturally occurring mutations or variants due to the manufacture of monoclonal antibodies, are generally present in trace amounts. In contrast, polyclonal antibody preparations generally include different antibodies directed to different determinants (epitopes), and each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. The modifier "monoclonal" thus represents the properties of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring a particular method of making the antibody. For example, monoclonal antibodies used in Disclosure C can be made using a variety of techniques, including but not limited to fusion tumor methods, recombinant DNA methods, phage display methods, and methods of transgenic animals that include all or a portion of the human immunoglobulin locus , such a method and other methods for making monoclonal antibodies will be described herein.

Within the scope of the recitations of Disclosure C, "native antibody" refers to immunoglobulin molecules having various naturally-occurring structures. In one embodiment, a native IgG antibody is, for example, a heteroquaternary 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-terminus, each heavy chain has a variable domain (VH), also called variable heavy chain domain or heavy chain variable domain, followed by three definite domains (CH1, CH2, and CH3 ). Likewise, in order from N- to C-terminus, each light chain has a variable region (VL), referred to as a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Antibody light chains can be assigned either of two types, kappa (κ) and lambda (λ), based on the amino acid sequence of their invariant domains. Such invariant domains as used by the present disclosure C include any reported subtype (dual) or any subclass/construction isotype. Heavy chain constant regions include, but are not limited to, the constant region antibodies of native IgG (IgGl, IgG2, IgG3, and IgG4). Known IgG1 counterpart genes include, for example, IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, and IGHG1*05 (see imgt.org), any of which may serve as the native human IgG1 sequence. Invariant domain sequences can be from a single paired gene or subclass/construction isotype or from multiple paired genes or subclasses/construction isotype. Specifically, such antibodies include, but are not limited to, CH1 from IGHG1*01, CH2 and CH3 from IGHG1*02 and IGHG1*01 antibodies.

The recited "effector function" within the scope of revealing C refers to the biological activity attributable to the Fc region of an antibody, which 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; ) down-regulated; and B cell activation, but not limited thereto.

The term "Fc region" disclosed in the category description 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. Native Fc region refers to the Fc region of a native antibody.

In one embodiment, the human IgG heavy chain Fc region extends from the amino acid residues of Cys226 or Pro230 to the carboxy 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 be present. Unless otherwise specified within the context of disclosing C, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

A "framework" or "FR" recited in the category revealing C refers to variable domain residues other than 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 sequences: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4 in VH (or VL).

The "human common framework" described in the category revealing C is the framework of the most commonly occurring amino acid residues in a selected human immunoglobulin VL or VH framework sequence. Typically, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Generally, the subgroups of sequences are in accordance with the subgroups 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.

An "acceptor human framework" as used to disclose the scope of C is a framework that includes amino acid sequences from a VL or VH framework of a human immunoglobulin framework or a human common framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human common framework may include its identical amino acid sequence or may include existing amino acid sequence substitutions. In some embodiments, the number of existing amino acid substitutions is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less. In one embodiment, the VL acceptor human framework and the VL human immunoglobulin framework sequence or human consensus framework sequence are identical.

Within the context of the recitation of Disclosure C, the term "variable region" or "invariant domain" refers to the domain of the antibody heavy or light chain that binds the antibody to the antigen. The variable domains (VH and VL) of the heavy and light chains of natural antibodies generally have similar structures, and each domain includes 4 conserved framework regions (FR) and 3 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 without limitation. Also, antibodies that bind to a particular antigen can use VH or VL domains from antibodies to screen for complementary VL or VH domains for isolation, see e.g. Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The terms "hypervariable regions" or "HVRs" used in the context of the recitations of disclosure C refer to sequences that are highly variable ("complementarity determining regions" or "CDRs") and/or form structurally defined loops (" "loops of hypervariability") and/or regions of antibody variable domains that contain antigen-contacting residues ("antigen contact points"). Typically, antibodies comprise 6 hypervariable regions: 3 in the VH (H1, H2, H3) and 3 in the VL (L1, L2, L3).

Without being limited thereto, here, HVR such as: (a) hypervariable loop, wherein amino acid residues are 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs wherein 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) antigenic contact points where amino acid residues 27c-36 (L1), 46-55 (L2 ), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)) and (d) combinations (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 (eg, FR residues) are numbered according to Kabat et al., supra.

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

Within the scope of the records disclosed in C, "isolated" antibody systems have been isolated from components of the natural environment. In some embodiments, the antibody has been refined by means of electrophoresis (eg, SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC) to be greater than 95% or 99% pure. %. For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

Within the scope of the recitations of Disclosure C, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule normally contained in a cell containing the nucleic acid molecule, but which is present extrachromosomally or is present on a chromosome at a location other than the natural chromosomal location.

An "isolated nucleic acid encoding an anti-IL-8 antibody" within the scope of the recitation of Disclosure C refers to nucleic acid molecule or molecules encoding one or more of the anti-IL-8 antibody heavy and light chains (or fragments thereof) , including nucleic acids in a single vector or in isolated vectors, and cells present in one or more locations on a host cell.

Within the scope of the recitations of Disclosure C, the terms "host cell", "host cell line" 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 the cell to be transformed and progeny therefrom regardless of generation. Progeny are not necessarily identical in nucleic acid content to their parent cells and may include mutations. Also included herein are screening or selection of mutant progeny that have the same function or biological activity as the original transformed cell.

Within the scope of the recitations of Disclosure C, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are introduced into a host cell and encapsulate its genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are called "expression vectors".

Within the scope of the recitation of Disclosure C, the term "package insert" is used to refer to instructions customarily included in the commercial package of a medicinal product, containing instructions for indications, usage, dosage, administration, combination Information on treatments, contraindications and/or warnings regarding the use of such medicinal products.

Within the scope of the recitations disclosed in C, the "percent (%) amino acid sequence identity" with respect to a control polypeptide sequence is defined as the amino acid residue in the candidate sequence and the amino acid residue in the control polypeptide sequence between the amino acid residues in the control polypeptide sequence The percent identical after sequence alignment, in order to achieve the maximum percent sequence identity, gaps can be introduced as needed, and any conservative substitutions are not considered as part of the sequence identity. To determine the alignment of percent amino acid sequence identity, various means are within the purview of those of ordinary skill in the art, 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 appropriate parameters for aligning sequences, including any algorithm required 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 comparative computer program ALIGN-2. ALIGN-2 was produced by Genentech, Inc., source code and user documentation have been filed with the U.S. Copyright Office, Washington D.C., 20559, and registered with U.S. Copyright Registration No. TXU510087. ALIGN-2 programs are publicly available from Genentech, Inc., South San Francisco, California or can be compiled from source code. ALIGN-2 programs should use the UNIX (registered trademark) operating system code, including digital UNIX (registered trademark) V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

When using ALIGN-2 for amino acid sequence comparison, a given amino acid sequence A has or includes a given amino acid sequence B against a given amino acid sequence B (it can also be said that a given amino acid sequence A corresponds to a given amino acid sequence B). Amino acid sequence identity %) is calculated in the following manner: 100 times the fraction X/Y, where X is the same match when A and B are 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 appreciated 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 scope of the disclosure C described.

Within the scope of the recitations of Disclosure C, a "pharmaceutical composition" generally refers to an agent for the treatment, prevention, detection or diagnosis of a disease. "Pharmaceutically acceptable carrier" refers to ingredients other than the active ingredient 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" (and grammatical variations thereof, such as "treat" or "treating") as used within the context of the recitations of Disclosure C, refers to clinical interventions to alter the natural course of the individual to be treated. Such clinical interventions may be prophylactic or performed during clinical lesions. Desired effects of treatment include, but are not limited to, prevention of disease occurrence or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological outcome of the disease, prevention of metastasis, slowing of disease progression, improvement or alleviation of disease state, and amelioration or improvement of prognosis. In one embodiment, C-disclosing antibodies can be used to slow the progression of a disease or disorder.

Within the scope of the recitations of Disclosure C, an "effective amount" of an antibody or pharmaceutical composition refers to an amount that is effective if used at the dosage and for the time period required to achieve the desired therapeutic or prophylactic result.

II. COMPOSITIONS AND METHODS In one embodiment, C is disclosed based on the use of an anti-IL-8 antibody with pH-dependent affinity for IL-8 as a pharmaceutical composition. Antibodies to C are disclosed to be useful, for example, in the diagnosis or treatment of diseases in which IL-8 is present in large quantities.

A. Anti-IL-8 Antibody Example In one embodiment, Disclosure C provides a primary anti-IL-8 antibody that has pH-dependent affinity for IL-8.

In one embodiment, Disclosure C provides an anti-IL-8 antibody having a pH-dependent affinity for IL-8 comprising at least 1, 2, 3, 4, 5, 6 in the following amino acid sequence , 7 or 8 amino acid substitution 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 71; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO:72.

In another embodiment, it is disclosed that C provides an anti-IL-8 antibody with pH-dependent affinity for IL-8 comprising at least one 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, comprising the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3, comprising SEQ ID The amino acid sequence of NO: 72.

Unless otherwise specified, the amino acid can be substituted with any other amino acid. In one embodiment, the anti-IL-8 antibody that discloses C comprises 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) at position 3 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) the sequence 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) aspartic acid 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) 9 of the sequence of SEQ ID NO:68 (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) 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) 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) at SEQ ID NO:68 Glutamic acid at position 1 of the sequence of ID NO:69; (z) asparagine at position 2 of the sequence of SEQ ID NO:69; (aa) 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) at SEQ ID NO: Aspartic acid at position 6 of the sequence of 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; (a g) 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) at position 11 of the sequence of SEQ ID NO:69 (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) at position 2 of the sequence of SEQ ID NO:70 Serine at position 3 of the sequence of 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 the sequence of SEQ ID NO:70; (ap) tyrosine at position 7 of the sequence of SEQ ID NO:70; (aq) the sequence of SEQ ID NO:70 (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) at Alanine at position 11 of the sequence of SEQ ID NO:70; (au) Aspartic acid at position 1 of the sequence of SEQ ID NO:71; (av) at 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) at 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; (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) at position 3 of the sequence of SEQ ID NO:72 (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) at position 5 of the sequence of SEQ ID NO:72 Phenylalanine at position 6 of the sequence of SEQ ID 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 that discloses C comprises one or more amino acid substitutions at a position selected from the group consisting of: (a) a propylamine 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) 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, an anti-IL-8 antibody that discloses C comprises a combination of amino acid substitutions at positions 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) 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 for C includes amino acid substitutions at: (a) tyrosine at position 9 of the sequence of SEQ ID NO: 68; (b) at SEQ ID NO: Arginine at position 11 of the sequence of 68; and (c) tyrosine at position 3 of the sequence of SEQ ID NO:69.

In one embodiment, the anti-IL-8 antibody disclosed for C includes amino acid substitutions at: (a) alanine at position 6 of the sequence of SEQ ID NO:68; (b) at 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 that discloses C comprises: (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 substituted for proline; and (c) at position 3 of the sequence of SEQ ID NO:69, tyrosine is substituted for histidine.

In one embodiment, an anti-IL-8 antibody that discloses C comprises: (a) at position 8 of the sequence of SEQ ID NO:68, replacing glycine with tyrosine; and (b) at SEQ ID NO:68 At position 9 of the sequence, tyrosine is substituted for histidine.

In one embodiment, the anti-IL-8 antibody that discloses C comprises: (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 In the 8th position of the sequence, glycine is replaced by tyrosine; (c) in the 9th position of the sequence of SEQ ID NO:68, tyrosine is replaced by histidine; (d) in the sequence of SEQ ID NO:68 At position 11, arginine is substituted for proline; and (e) at position 3 of the sequence of SEQ ID NO:69, tyrosine is substituted for histidine.

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

In one embodiment, the anti-IL-8 antibody disclosed for C comprises HVR-H3, comprising the amino acid sequence of SEQ ID NO:74.

In one embodiment, the anti-IL-8 antibody that discloses C comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 73, and HVR-H2 comprising 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 that discloses C comprises one or more amino acid substitutions at a position selected from the group consisting of: (a) the filament at position 8 of the sequence of SEQ ID NO:70 (b) aspartic acid 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) 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, an anti-IL-8 antibody that discloses C comprises a combination of amino acid substitutions at positions selected from the group consisting of: (a) serine at position 8 of the sequence of SEQ ID NO:70 (b) aspartic acid 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) at position 5 of the sequence of SEQ ID NO:71 : Glutamate at position 1 of the sequence of 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 disclosed for C includes amino acid substitutions at: (a) aspartic acid at position 1 of the sequence of SEQ ID NO:71; (b) at SEQ ID 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 disclosed for C includes amino acid substitutions at: (a) serine at position 8 of the sequence of SEQ ID NO: 70; (b) at SEQ ID NO: Aspartic acid at position 1 of the sequence of 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 acid.

In one embodiment, an anti-IL-8 antibody that discloses C comprises: (a) at position 1 of the sequence of SEQ ID NO:71, replacing aspartic acid with lysine; (b) at position 1 of SEQ ID NO:71; At position 5 of the sequence of 71, leucine is substituted for histidine; and (c) at position 1 of the sequence of SEQ ID NO: 72, glutamic acid is substituted for lysine.

In one embodiment, an anti-IL-8 antibody that discloses C comprises: (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 Position 1 of the sequence, replacing aspartic acid with lysine; (c) at position 5 of the sequence of SEQ ID NO:71, replacing leucine with histidine; and (d) in SEQ ID NO:72 The 1st position of the sequence replaces glutamic acid with lysine.

In one embodiment, the anti-IL-8 antibody that discloses C comprises HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75.

In one embodiment, the anti-IL-8 antibody disclosed for C comprises HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76.

In one embodiment, the anti-IL-8 antibody that discloses C comprises 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. The amino acid sequence of NO: HVR-L3 of 76.

In one embodiment, the anti-IL-8 antibody disclosed for C includes amino acid substitutions at: (a) alanine at position 55 of the sequence of SEQ ID NO:77; (b) at 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) ) glutamic acid 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) at position 87 of the sequence of SEQ ID NO:77 Tyrosine at position 103.

In one embodiment, an anti-IL-8 antibody that discloses C comprises: (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; (c) at position 58 of the sequence of SEQ ID NO:77, tyrosine is replaced by histidine; (d) at position 58 of the sequence of SEQ ID NO:77 60, substituted arginine to proline; (e) substituted glutamic acid to threonine at position 84 of the sequence of SEQ ID NO:77; (f) 87 of the sequence of SEQ ID NO:77 (g) At position 103 of the sequence of SEQ ID NO: 77, substituted tyrosine is histidine.

In one embodiment, the anti-IL-8 antibody disclosed for C comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78.

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

In one embodiment, an anti-IL-8 antibody of C is disclosed comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:78; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:79 acid sequence. This anti-IL-8 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 can be pH-dependent Anti-IL-8 antibodies that bind sexually to IL-8. This anti-IL-8 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 can be stably in the Anti-IL-8 antibodies that maintain IL-8 neutralizing activity in vivo (eg, in plasma). This anti-IL-8 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 may be immunocompromised original antibody.

In an alternative aspect, anti-IL-8 antibodies that disclose C also include those that have pH-dependent affinity for IL-8 and contain at least one 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 SEQ ID NO (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, anti-IL-8 antibodies that disclose C also include those that have pH-dependent affinity for IL-8 and contain at least one 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 SEQ ID NO (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2, containing the amino acid sequence of SEQ ID NO: 112; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO:113.

In an alternative aspect, anti-IL-8 antibodies that disclose C also include those that have pH-dependent affinity for IL-8 and contain at least one 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 SEQ ID NO (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, anti-IL-8 antibodies that disclose C also include those that have pH-dependent affinity for IL-8 and contain at least one 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 SEQ ID NO (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, anti-IL-8 antibodies that disclose C also include those that have pH-dependent affinity for IL-8 and contain at least one 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 SEQ ID NO (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, anti-IL-8 antibodies that disclose C also include those that have pH-dependent affinity for IL-8 and contain at least one 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 SEQ ID NO (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, anti-IL-8 antibodies that disclose C also include those that have pH-dependent affinity for IL-8 and contain at least one 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 SEQ ID NO (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, the anti-IL-8 antibody of C is disclosed to have IL-8 neutralizing activity. IL-8 neutralizing activity refers to activity that inhibits the biological activity of IL-8, or may refer to activity that inhibits receptor binding of IL-8.

In an alternative aspect, it was revealed that the anti-IL-8 antibody of C was an anti-IL-8 antibody that bound to IL-8 in a pH-dependent manner. In the context of Reveal C, an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner refers to an antibody that has a binding affinity for IL-8 at acidic pH compared to 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 revealed that the anti-IL-8 antibody of C has at least 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 15-fold greater affinity for IL-8 at neutral pH compared to 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, 1000 times, 10000 times or more. Binding affinity can be measured using, but not limited to, a surface plasmon resonance method (such as BIACORE (registered trademark)). Association rate constants (kon) and dissociation rate constants (koff) can be calculated using BIACORE (registered trademark) T200 Evaluation Software (GE Healthcare) by simultaneously fitting association and dissociation sensorgrams according to a simple one-to-one Langmuir binding model. Equilibrium dissociation constant (KD) is calculated as the ratio of koff/kon. In order to screen for an antibody whose binding affinity is changed depending on pH, it is not particularly limited, and ELISA, kinetic exclusion assay (KinExA ^™ ), etc., and surface plasmon resonance method (such as BIACORE (registered trademark)) can be used. pH-dependent IL-8 binding capacity refers to the property of binding to IL-8 in a pH-dependent manner. Meanwhile, whether an antibody can bind to IL-8 multiple times can be assessed according to the method described in WO2009/125825.

In one embodiment, it is desirable to reveal that the anti-IL-8 antibody of C has a small dissociation constant (KD) for IL-8 at neutral pH. In one embodiment, the dissociation constant of the antibody revealing C against IL-8 at neutral pH is, for example, but not limited to, 0.3 nM or less. In one embodiment, the dissociation constant of the antibody revealing C against IL-8 at neutral pH is, for example, but not limited to, 0.1 nM or less. In one embodiment, the dissociation constant of the antibody revealing C against IL-8 at neutral pH is, for example, but not limited to, 0.03 nM or less.

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

In one embodiment, anti-IL-8 antibodies that reveal C have a large dissociation constant (KD) for IL-8 at acidic pH. In one embodiment, the dissociation constant of the antibody revealing C against IL-8 at acidic pH is, for example, but not limited to, 3 nM or more. In one embodiment, the dissociation constant of the antibody revealing C against IL-8 at acidic pH is, for example, but not limited to, 10 nM or more. In one embodiment, the dissociation constant of the antibody revealing C against IL-8 at acidic pH is, for example, but not limited to, 30 nM or more.

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

In one embodiment, it is desirable to reveal that the binding affinity of the anti-IL-8 antibody of C for IL-8 is greater at neutral pH than at acidic pH.

In one embodiment, the ratio of dissociation constants between acidic pH and neutral pH, i.e., [KD (acid pH)/KD (neutral pH)], of anti-IL-8 antibodies of C is disclosed, for example, 30 or more But not limited to this. In one embodiment, the ratio of dissociation constants between acidic pH and neutral pH, i.e., [KD (acid pH)/KD (neutral pH)], of anti-IL-8 antibodies of C is disclosed, 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, it is disclosed that the anti-IL-8 antibody of C has a dissociation constant ratio between pH 5.8 and pH 7.4, that is, [KD (pH 5.8)/KD (pH 7.4)] is 30 or more but is not limited to this. In one embodiment, the ratio of dissociation constants between pH 5.8 and pH 7.4, i.e., [KD (pH 5.8)/KD (pH 7.4)], of the anti-IL-8 antibody of C is disclosed to be, for example, 100 or more, such as 200 the But not limited to this.

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

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

In one embodiment, it is desirable to reveal that anti-IL-8 antibodies to C stably maintain IL-8 neutralizing activity in solution (eg, PBS). Whether the activity is stably maintained in solution can be assessed 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, but not limited to, 1, 2, 3, or 4 weeks. In one embodiment, the storage temperature is, for example, but not limited to, 25°C, 30°C, 35°C, 40°C, or 50°C. In one embodiment, the storage temperature is, for example, but not limited to, 40° C.; and the storage period is, for example, but not limited to, 2 weeks. In one embodiment, the storage temperature is, for example, but not limited to, 50° C.; and the storage period is, for example, but not limited to, 1 week.

In one embodiment, it is desirable to reveal that anti-IL-8 antibodies to C stably maintain IL-8 neutralizing activity in vivo (eg, plasma). Whether the activity is stably maintained in vivo can be assessed by measuring the IL-8 neutralizing activity of the C-revealing antibody added to animal (eg, mouse) or human plasma before and after storage at a specific temperature for a specific period. In one embodiment, the storage period is, for example, but not limited to, 1, 2, 3, or 4 weeks. In one embodiment, the storage temperature is, for example, but not limited to, 25°C, 30°C, 35°C, or 40°C. In one embodiment, the storage temperature is, for example, but not limited to, 40° C.; and the storage period is, for example, but not limited to, 2 weeks.

In one embodiment, the cellular uptake rate of the anti-IL-8 antibody of C is revealed, where the antibody and IL-8 form a complex extracellular (eg, plasma) than the antibody alone. IL-8 revealed that the antibody of C, when complexed with IL-8, is more likely to enter cells from when it is complexed than when it is not complexed with IL-8.

In one embodiment, the immunogenicity of the anti-IL-8 antibody to reveal C, ie, predicted in a human host, is preferably reduced. "Low immunogenicity" can mean, but is not limited to, for example, when a sufficient amount of anti-IL-8 antibody is administered for a sufficient period of time to achieve a therapeutic effect in at least half or more of the subject in vivo, without eliciting an immune response. Inducing an immune response can include the production of anti-drug antibodies. "Low anti-drug antibody production" and "low immunogenicity" are used interchangeably. Human immunogenicity levels were assessed using a T cell epitope prediction program. Such T cell epitope prediction programs include Epibase (Lonza), iTope/TCED (Antitope), EpiMatrix (EpiVax), and the like. EpiMatrix is a system for predicting the immunogenicity of protein sequences of interest. It uses the amino acid sequence of the protein to be analyzed for immunogenicity to be cleaved to automatically design the sequence of peptide fragments, and then calculates its binding to 8 major MHC classes. Ability of II paired 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 T cell epitope prediction program described above to design sequences with reduced immunogenicity. Preferred sites for amino acid modifications to reduce the immunogenicity of the anti-IL-8 antibody of Reveal 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 Amino acid at position 81 and/or 82b of numbering.

In one embodiment, Disclosure C provides a method for enhancing the elimination of IL-8 from an individual as compared to using a reference antibody, comprising administering to the individual an anti-IL-8 antibody of Disclosure C. In one embodiment, the use of an anti-IL-8 antibody of C is disclosed for enhancing the elimination of IL-8 from an individual as compared to the use of a reference antibody. In one embodiment, disclosure of C relates to the use of an anti-IL-8 antibody of disclosure C for enhanced elimination of IL-8 from an individual as compared to the use of a reference antibody. In one embodiment, disclosure of C relates to the use of an anti-IL-8 antibody of disclosure C in the manufacture of a pharmaceutical composition for enhancing the elimination of IL-8 from a living body as compared to the 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 the elimination of IL-8 from a living body as compared to the use of a reference antibody. In one embodiment, Disclosure C relates to a method for enhancing the elimination of IL-8 from a living body as compared to using 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 to obtain the antibody of disclosure C, or the antibody with strong IL-8 binding affinity at both acidic and neutral pH. The reference antibody can be an antibody comprising the amino acid sequences of SEQ ID NOs: 83 and 84 or SEQ ID NOs: 89 and 87.

In one embodiment, Disclosure C provides a pharmaceutical composition comprising an anti-IL-8 antibody of Disclosure C, wherein the anti-IL-8 antibody of Disclosure C binds to IL-8 and then to the extracellular matrix. In one embodiment, the disclosure of C relates to the use of the anti-IL-8 antibody of disclosure C in the manufacture of a pharmaceutical composition, wherein the anti-IL-8 antibody of disclosure C is bound to IL-8 and then 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 this disclosure C comprises 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 this 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 their sequences.

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

1. Chimeric and Humanized Antibodies In certain embodiments, the antibody provided in Disclosure C can be a chimeric antibody. Some chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984). In one example, a chimeric antibody can include variable regions that are not human (eg, from a mouse, rat, hamster, rabbit, or non-human primate, such as monkey), and human constant regions.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody includes one or more variable regions, wherein the HVRs, eg, CDRs (or portions thereof), are derived from non-human antibodies, 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 can be substituted with residues of the corresponding non-human antibody (eg, the antibody from which its HVR residues are derived), eg, to maintain or improve antibody specificity or affinity.

Humanized antibodies and methods of making are reviewed, for example, in Almagro et al., Front. Biosci. 13:1619-1633 (2008) and described 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) (record-specific) Sex Determining Region (SDR) Transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (described "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (recording "FR dragging"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (recording "Guidelines for FR dragging") choice" method).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using "best-fit" methods (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); or framework regions derived from the consensus sequences of human antibodies of a particular subgroup of heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. , J. Immunol., 151:2623 (1993)); and 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 can be antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments and others described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review 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 US Patent Nos. 5,571,894 and 5,587,458.

Diabodies are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific. See, eg, 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 ). Tri- and tetra-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 an antibody. In certain embodiments, the single domain antibody can be a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516 B1). Antibody fragments can be made using a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or bacteriophage), as described within the scope of Disclosure C's description.

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 by 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 transgenic animals to produce fully human antibodies or whole antibodies bearing human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci, which are replaced by animal (non-human) immunoglobulin loci, either extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the animal (non-human) immunoglobulin loci are 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 using combinations of different human constant regions. Human antibodies can also be prepared using fusion tumor-based methods. Human myeloma and mouse-human heteromyeloma 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, for example, methods for making monoclonal human IgM antibodies from fusion tumor cell lines, such as described in U.S. Patent No. 7,189,826, and methods for making monoclonal human IgM antibodies, such as 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 described 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 colony variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences can be combined with desired human invariant domains. Techniques for selecting human antibodies from antibody libraries are described below.

4. Antibodies Derived from the Library Antibodies revealing C can be isolated by screening combinatorial libraries against the antibody for the desired activity. For example, various methods exist for generating phage display libraries and screening libraries for antibodies possessing desired binding properties. Such methods are described in Hoogenboom et al., in Meth. Mol. Biol. 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and e.g. 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. , J. Immunol. Meth. 284(1-2):119-132 (2004).

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

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

Finally, synthetic constructs can also be unaltered by cloning unrearranged V-gene segments from stem cells, using PCR primers containing random sequences to encode the hypervariable CDR3 regions, and rearranging in vitro (see below with 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. Herein, antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments.

5. Multispecific Antibodies In certain embodiments, the antibody provided in accordance with Disclosure C can be, for example, a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.

In certain embodiments, one of the binding specificities is for IL-8 and the other is for any other antigen.

In certain embodiments, bispecific antibodies can bind to 2 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 making multispecific antibodies include, but are not limited to, recombination of 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 US Pat. No. 5,731,168). Multispecific antibodies can be prepared by cross-linking 2 or more antibodies or fragments, using electrostatic steering effects to produce Fc-heterodimeric molecules (WO 2009/089004A1) (US Pat. No. 4,676,980; and Brennan et al. ., Science 229:81 (1985)), by using leucine zippers to make bispecific antibodies (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)), by using "Diabody" technology to make 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 3 or more functional antigen binding sites, including "Octopus antibodies", are also included herein (see eg US 2006/0025576).

Within the scope of the recitation of Disclosure C, this antibody or antibody fragment also includes a "dual-acting Fab" or "DAF", including an antigen binding site that binds to IL-8 as well as to a different antigen (see eg 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, deletions, and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. The final construct can include any combination of deletions, insertions, and substitutions, with the principle that the final construct is an antibody with the desired properties disclosed in the C context.

In one embodiment, Disclosure C provides an antibody variant having one or more amino acid substitutions. Such a substitution site can be any position in the antibody. Conservatively substituted amino acids are shown in Table 10, titled "Conservative Substitutions". Commonly substituted amino acids that would result in more substantial changes are shown in Table 10, titled "General Substitutions," and are further described in reference to amino acid side chain classes.

[Table 10]

Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: n-leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain alignment: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions can cause one member of one of these classes to become 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 methionine residue. Insertional variants of other antibody molecules include those derived from N- or C-terminal fusion of the antibody to an enzyme (such as ADEPT) or a polypeptide that increases the plasma half-life of the antibody.

7. Glycated variants In one embodiment, the antibody provided in accordance with Disclosure C may be a glycosylated antibody. Glycation sites can be created or removed by changing the amino acid sequence by addition or deletion from the antibody.

When the antibody includes an Fc region, the additional sugar chains can be varied. Unaltered antibodies produced by animal cells typically contain branched, bibranched oligosaccharides that are N-linked to Asn297 of the CH2 domain of the Fc region (see Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharides include, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose, the GlcNAc attached to the "stem" of the bibranched oligosaccharide structure. In one embodiment, it is disclosed that the oligosaccharides of the antibodies of 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 those with 1, 2, 3, or more amino acid modifications (eg, substitutions) in the native human Fc region sequence (eg, the Fc region of human IgGl, IgG2, IgG3, or IgG4).

Anti-IL-8 antibodies revealing C may include an Fc region having at least one of the following 5 properties, but are not limited: (a) Binding affinity to FcRn relative to the native Fc region at acidic pH. Increased binding affinity to FcRn of the Fc region; (b) decreased binding affinity of the Fc region to existing ADA relative to the binding affinity of the native Fc region to existing ADA; (c) relative to the native Fc region the plasma half-life of the Fc region is increased; (d) the plasma clearance of the Fc region is decreased relative to the plasma clearance of the native Fc region; and (e) relative to the native Fc region for effector receptors The binding affinity of the Fc region is reduced for effector receptors. 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, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold increase in FcRn binding affinity compared to the FcRn binding affinity of an antibody containing the native IgG Fc region 15-fold, 20-fold, 30-fold, 50-fold or 100-fold Fc region variants.

In one embodiment, Fc region variants include safe and favorable 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 an epitope on a therapeutic antibody. As used in the context of Disclosure C, the term "existing ADA" refers to the presence of detectable anti-drug antibodies in a patient's blood prior to administration of the therapeutic antibody to the patient. Pre-existing ADA includes rheumatoid factors. Fc region variants with low binding affinity to existing ADA include, but are not limited to, ADA binding affinities reduced to 1/10 or less, 1/10 compared to the ADA binding affinity of an antibody containing a native IgG Fc region 50 or less or 1/100 or less Fc region variants.

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 reduced binding affinity for complement proteins by 1/10 or less compared to the complement protein binding affinity of an antibody containing a native IgG Fc region , 1/50 or less or 1/100 or less.

In one embodiment, Fc region variants include Fc region variants with low or no binding affinity for an effector receptor. Effector receptors include, but are not limited to, FcyRI, FcyRII, and FcyRIII. FcyRI includes, but is not limited to, FcyRIa, FcyRIb, and FcyRIc and subtypes thereof. FcyRII includes, but is not limited to, FcyRIIa (there are 2 subtypes: R131 and H131) and FcyRIIb. 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 to effector receptors include, but are not limited to, those that have reduced binding affinity to effector receptors to at least 1/2 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 comprises an Fc region having one or more amino acid substitutions at any one of the following positions according to EU numbering compared to the native Fc region: 235, 236, 239 , 327, 330, 331, 428, 434, 436, 438, and 440 bits.

In one embodiment, the Fc region variant comprises 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 region.

In one embodiment, the Fc region variant comprises one or more of 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 comprises 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 disclosed for C comprises the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82. This 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 be an anti-IL-8 that binds to IL-8 in a pH-dependent manner Antibody. This 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 can stably maintain IL-8 neutralizing activity in vivo (eg, plasma). 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 be a low immunogenic antibody. 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 , FcRn at acidic pH (eg pH 5.8) binds Fc regions with increased affinity. This 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 binding affinity compared to the native Fc region of existing ADA, Fc region with reduced binding affinity for existing ADA. 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 an Fc region that has an extended half-life in plasma compared to a 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 effector receptors compared to a native Fc region Reduced Fc region. In yet another embodiment, the anti-IL-8 antibody comprises all 7 properties in combination of any 2, 3, 4, 5, 6 of the properties listed above.

In one embodiment, the anti-IL-8 antibody disclosed for C comprises the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82. This 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 be an anti-IL-8 that binds to IL-8 in a pH-dependent manner Antibody. This 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 can stably maintain IL-8 neutralizing activity in vivo (eg, in plasma) . This 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 be an antibody with low immunogenicity. This 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 FcRn binding affinity compared to the native Fc region at acidic pH FcRn binds to an Fc region with increased affinity. This 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 binding affinity compared to the native Fc region of existing ADA, Fc region with reduced binding affinity for existing ADA. Such an 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 a native Fc region. This 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 effector receptors compared to a native Fc region Reduced Fc region. In yet another embodiment, the anti-IL-8 antibody comprises all 7 properties in combination of any 2, 3, 4, 5, 6 of the properties listed above.

In certain embodiments, Disclosure C includes an antibody variant having 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 the reduction/total loss of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to identify antibodies that lack Fc[gamma]R binding (and thus ADCC activity) ability but retain FcRn binding ability.

Primary cultured cells for intermediary ADCC and NK cells express FcγRIII only, while monocytes express FcγRI, FcγRII and FcγRIII. FcRs expressed on heme-producing cells are listed in Table 3 on page 464 of Ravetch et al., Annu. Rev. Immunol. 9:457-492 (1991). A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest is described in US Pat. No. 5,500,362 (see, eg, 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, non-radioactive isotope assays can be employed 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). Useful effector cells in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.

Alternatively or additionally, 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 this 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, a CDC assay can be performed (see, eg, 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 (US Pat. 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 with alanine substitutions at positions 265 and 297, called "DANA". "Fc region variants (US 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 blood half-life and improved FcRn binding at acidic pH are described in. US2005/0014934. The antibodies described include one or more substituted Fc regions that improve binding of the Fc region to FcRn. Such Fc region variants are included 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 , 413, 424 or 434 at one or more positions, such as substitution at position 434 of the Fc region (US Pat. No. 7,371,826).

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

9. Antibody Derivatives In certain embodiments, the antibodies provided by Disclosure C can be further modified to contain additional non-proteinaceous structures known in the art and readily available. 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 , 3-dioxolane, poly-1,3,6-tri[a] alkane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), poly(n- vinylpyrrolidone) polyethylene glycol, propyl propylene glycol homopolymer, polypropylene oxide (prolylpropylene oxide)/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and their mixture. Polyethylene glycol propionaldehyde has advantages in industrialization because it is stable in water.

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

In another embodiment, conjugates of C-revealing anti-IL-8 antibodies to non-proteinaceous 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, eg, Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including, but not limited to, being harmless to humans but heating the non-protein structure to a temperature that would kill cells proximate the antibody-non-protein structure.

B. Recombinant Methods and Compositions Antibodies to IL-8 Rev. C can be made 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 presented at Disclosure C. Such nucleic acids can encode amino acid sequences comprising the VL of the antibody and/or amino acid sequences comprising the VH of the antibody (eg, the light and/or heavy chains of the antibody). In yet another embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In one embodiment, host cells comprising such nucleic acids are provided. In one embodiment, the host cell comprises (eg, has been transformed): (1) a vector comprising nucleic acids encoding the VL of this antibody and the VH of the antibody, or (2) a first vector comprising the VL encoding an antibody The nucleic acid; and the second vector, including the nucleic acid encoding the VH of this antibody.

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

In one embodiment, a method of making an anti-IL-8 antibody of Reveal C is provided, the method comprising: culturing a host cell comprising a nucleic acid encoding such an 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).

For recombinant production of anti-IL-8 antibodies, eg, as described above, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further colonization 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 nucleic acids encoding the heavy and light chains of the antibody).

Host cells suitable for colonizing or expressing vectors encoded as antibodies include prokaryotic or eukaryotic cells described within the scope of disclosure C. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For bacterial expression of antibody fragments and polypeptides, see, e.g., 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 expressing antibody fragments in E. coli). After expression, the antibody can be isolated from the soluble fraction of bacterial cell paste and further refined.

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable hosts for the colonization or expression of vectors encoded as antibodies, including fungal and yeast strains in which the glycation pathway has been "humanized" and capable of producing partially or Fully human glycosylated antibody. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Host cells suitable for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Without particular limitation, a combination of baculovirus and insect cells can be used to transfect Spodoptera frugiperda cells, and most baculovirus strains have been identified.

Plant cell cultures can be utilized as hosts. See US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (described PL Antibody™ technology for the production of antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension are useful. Other useful mammalian host cells are the monkey kidney CV1 strain, which is transformed by SV40 (COS-7); the human embryonic kidney cell line (293 cells, described in Graham et al., J. Gen Virol. 36:59 (1977) )); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, described 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 mouse hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); Mouse mammary tumor (MMT 060562); TRI cells, 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, NSO and Sp20, but not limited. For review of other mammalian host cell lines suitable for antibody production, 8AD6, 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 nucleic acid encoding the antibody as described above under conditions suitable for antibody expression can be isolated from within or outside the host cell (medium, milk) and refined to substantially Pure homogeneous antibody. The isolation/purification method generally used for the purification of polypeptides can be appropriately used to isolate and purify the antibody; however, the method is not limited to the above-mentioned examples. Antibodies can be obtained by, for example, appropriate selection and combination of column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization to appropriately separate and purify, but not limited. Chromatography includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. Such chromatography can be carried out 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), and the like.

For the properties of anti-IL-8 antibodies, such as the aforementioned increased extracellular matrix binding activity and enhanced cellular uptake complexes, Disclosure C provides a method for selecting antibodies with increased extracellular matrix binding activity and antibodies with enhanced cellular uptake complexes. In one embodiment, Disclosure C provides a method of producing an anti-IL-8 antibody comprising a variable region whose binding activity to IL-8 is pH-dependent, comprising the steps of: (a) assessing 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 comprising a vector carrying a nucleic acid encoding the antibody; and (d) removing the from this antibody.

The binding to the extracellular matrix can be performed by any method without limitation, as long as it is known to those of ordinary skill in the art. For example, the binding of the antibody to the extracellular matrix can be detected using an ELISA system in which the antibody system is applied to an extracellular matrix immobilized plate and a labeled antibody against the antibody is added. Alternatively, for example, the assay can use an electrochemiluminescence (ECL) method, wherein a mixture of the antibody and ruthenium antibody is applied 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 contacted 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, when evaluating extracellular matrix binding, based on the - the binding of IL-8 antibody is higher than the value representing the binding of the extracellular matrix to the reference antibody such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 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 the anti-IL-8 antibody to the extracellular matrix. The control anti-IL-8 antibody used in comparing several modified anti-IL-8 antibodies can be an unmodified anti-IL-8 antibody. In this case, the conditions should be the same except for the presence of IL-8. Specifically, in one embodiment, revealing C includes selecting an antibody representing a higher value of extracellular matrix binding from among several anti-IL-8 antibodies that have not been contacted with IL-8. In another embodiment, revealing C includes selecting an antibody representing a higher value of extracellular matrix binding from among several anti-IL-8 antibodies contacted with IL-8. In an alternative embodiment, step (b) of "selecting an anti-IL-8 antibody that binds strongly to the extracellular matrix" means that when assessing extracellular matrix binding, it may be based on the presence of IL-8 depending on, Antibodies are selected for differences in their binding to the extracellular matrix. A value representing the ratio of extracellular matrix binding of anti-IL-8 antibodies contacted with IL-8 to extracellular matrix binding of anti-IL-8 antibodies not contacted with IL-8, eg, 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 the disclosure C can be identified, screened or characterized for their physical/chemical properties or biological activity using various methods known in the art.

1. Combining assays and other assays in one aspect, this reveals that antibodies to C can utilize known methods for their antigen-binding activity, such as ELISA, Western blotting, kinetic exclusion assay (KinExA ^™ ), and surface plasmon Resonance is assessed using eg a BIACORE (GE Healthcare) apparatus.

In one embodiment, binding affinity can be assessed using a BIACORE T200 (GE Healthcare) as follows. An appropriate amount of capture protein (eg, Protein A/G (PIERCE)) was immobilized on a sensor chip CM4 (GE Healthcare) by amine coupling, so that the antibody of interest was captured. The diluted antigen solution is then injected with running buffer (as a reference solution: e.g. 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 sensing chip. The sensing chip was regenerated using 10 mM glycine HCl solution (pH 1.5). The measurement is carried out 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), both kinetic parameters, were calculated from the obtained sensorgrams. The KD (M) of an antibody against this antigen is calculated based on these constants. Parameters were calculated using BIACORE T200 Evaluation Software (GE Healthcare).

In one embodiment, IL-8 can be quantified as described below. An anti-human IL-8 antibody comprising a mouse IgG constant region was immobilized on the plate, and a solution comprising IL-8 bound to a humanized anti-IL-8 antibody that did not compete with the above-mentioned anti-human IL-8 antibody, added to the immobilization plate. After stirring, biotinylated anti-human Igκ light chain antibody was added and allowed to react for a period of time. SULFO-Tag-labeled streptavidin was then added and allowed to react for a period of time. Assay buffer was then added and assayed immediately with a SECTOR Imager 2400 (Meso Scale Discovery).

2. Activity Assay In one aspect, assays are provided to identify biologically active anti-IL-8 antibodies. Biological activities include, for example, IL-8 neutralizing activity and activity that blocks IL-8 signaling. Disclosure C also provides antibodies having such biological activity in vivo and/or in vitro.

In one embodiment, the method for determining the neutralization level of IL-8 is not particularly limited, and can also be determined by the following method. PathHunter ^™ CHO-K1 CXCR2 Beta-Arrestin Cell Line (DiscoveRx, Cat.# 93-0202C2) is an artificial cell line created to express human CXCR2, a known human IL-8 receptor, and expressed by human IL-8 It emits chemiluminescence when it receives a signal. When human IL-8 was added to the culture medium of cells, chemiluminescence was emitted from the cells in a manner dependent on the concentration of added human IL-8. When a combination of human IL-8 and anti-human IL-8 antibody was added to the medium, the chemiluminescence of the cells was reduced or undetectable compared to when the antibody was not added, because the anti-human IL-8 antibody could block the IL-8 signaling. Specifically, the stronger the neutralizing activity of human IL-8 of this antibody, the weaker the level of chemiluminescence; and the weaker the neutralizing activity of human IL-8 of this antibody, the stronger the level of chemiluminescence. Therefore, the human IL-8 neutralizing activity of the anti-human IL-8 antibody can be evaluated by examining the above differences.

D. Pharmaceutical formulations Pharmaceutical formulations comprising anti-IL-8 antibodies within the scope of disclosure C can be frozen by admixing anti-IL-8 antibodies of the desired degree of purity with one or more optionally pharmaceutically acceptable carriers It is prepared by mixing in the form of dry or aqueous formulations (see, eg, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages 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 octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butylphenol or benzyl alcohol) ; phenylparabens, such as methylparaben or propylparaben; 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, glutamic acid, amino acids Paragamic, 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 counter ions, such as sodium; metal complexes (eg, Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG).

Pharmaceutically acceptable carriers, including enteric drug dispersions, such as soluble hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX ^™ , Baxter International, Inc. .).) Examples of certain sHASEGPs, and methods of use, including rHuPH20, are described in US Application Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is in combination with one or more glycosaminoglycanase enzymes, eg, chondroitinase.

Examples of lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous Antibody Formulations US Patent No. 6,171,586 and WO2006/044908 described; WO2006/044908 formulations include histidine-acetic acid buffer.

Formulations within the scope of disclosure C may also include more than one active ingredient necessary for the treatment of a particular indication, preferably with complementary activities without detrimental to each other. Such active ingredients are suitably present in combination in an amount effective for the purpose.

Active ingredients can be encapsulated in microcapsules, for example, prepared by coacervation techniques or interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (such as microlipid) globules, 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 semipermeable matrices of solid hydrophobic polymers containing anti-IL8 antibodies of the disclosure C, wherein the matrices are in the form of shaped articles such as films or microcapsules.

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

E. METHODS AND COMPOSITIONS OF TREATMENT 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 are provided for use as pharmaceutical compositions. In an alternative aspect, anti-IL-8 antibodies are provided for the treatment of diseases in which IL-8 is present in excess. In one embodiment, anti-IL-8 antibodies are provided for use in a method of treating a disease in which IL-8 is present in excess. In one embodiment, Disclosure C provides a method for treating an individual with a disease in which IL-8 is present in excess, such as a disease caused by the presence of excess IL-8, comprising administering to the individual an effective amount of anti-IL-8 Antibody. In another embodiment, Disclosure C provides an anti-IL-8 antibody for use in such a method. In one embodiment, line C is disclosed for a pharmaceutical composition comprising an effective amount of an anti-IL-8 antibody for treating a disease in which IL-8 is present in excess. In one embodiment, line C is disclosed for the use of an anti-IL-8 antibody in the manufacture of a pharmaceutical composition for treating a disease in which IL-8 is present in excess. In one embodiment, C is disclosed for the use of an effective amount of an anti-IL-8 antibody to treat a disease in which IL-8 is present in excess. Diseases in which IL-8 is present in excess include, but are not limited to, inflammatory skin diseases such as inflammatory 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, Alcoholic Hepatitis, 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; malignancies such as ovarian, lung, prostate, gastric, breast, melanoma, head and neck, and kidney cancers; sepsis caused by infection; cystic fibrosis; pulmonary fibrosis; and acute lung injury.

In an alternative embodiment, Disclosure C provides an anti-IL-8 antibody for inhibiting the accumulation of biologically active IL-8. "Inhibiting the accumulation of IL-8" can be achieved by preventing an increase in the amount of IL-8 present in the body or by reducing the amount of IL-8 present 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. As used herein, "IL-8 present in vivo" may refer to IL-8 complexed with an anti-IL-8 antibody or free IL-8; or, total IL-8. As used herein, "existing in vivo" may refer to "extracellular secretion in vivo". 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 an individual. In one embodiment, Relation C is disclosed for a pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, comprising an effective amount of an anti-IL-8 antibody. In one embodiment, disclosure C relates to the use of anti-IL-8 antibodies to produce pharmaceutical compositions for inhibiting the accumulation of biologically active IL-8. In one embodiment, it is disclosed that C relates to the use of an effective amount of an anti-IL-8 antibody to inhibit the accumulation of biologically active IL-8. In one embodiment, it is revealed that the anti-IL-8 antibody of C inhibits IL-8 accumulation compared to the anti-IL-8 antibody without pH-dependent binding activity. In the above-described embodiments, the "individual" is preferably a human.

In an alternative embodiment, Disclosure C provides 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 Line C relates to a pharmaceutical composition for inhibiting angiogenesis, comprising an effective amount of an anti-IL-8 antibody. In one embodiment, line C is disclosed regarding the use of anti-IL-8 antibodies for the production of pharmaceutical compositions for inhibiting angiogenesis. In one embodiment, it is disclosed that line C relates to the use of an effective amount of an anti-IL-8 antibody to inhibit angiogenesis. In the above embodiment, the "individual" is preferably a human.

In an alternative aspect, it is revealed that C provides an anti-IL-8 antibody for inhibiting promotion of neutrophil migration. In one embodiment, Disclosure C provides a method of inhibiting neutrophil migration promotion in an individual comprising administering to the antibody an effective amount of an anti-IL-8 antibody; and providing an anti-IL for use in the method -8 antibody. In one embodiment, Line C is disclosed for a pharmaceutical composition for inhibiting neutrophil migration in an individual. In one embodiment, line C is disclosed regarding the use of anti-IL-8 antibodies to manufacture pharmaceutical compositions that inhibit neutrophil migration promotion in an individual. In one embodiment, it is disclosed that C relates to the use of an effective amount of an anti-IL-8 antibody to inhibit neutrophil migration promotion in an individual. In the above-described 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, eg, for use in any method of treatment. In one embodiment, the pharmaceutical composition includes the anti-IL-8 antibody provided in Disclosure C and a pharmaceutically acceptable carrier.

C-disclosed antibodies can be used alone or in combination with other agents in therapy. For example, it is disclosed that an antibody to C can be co-administered with at least one additional therapeutic agent.

The C-disclosed antibody (and any additional therapy) can be administered using any suitable method, including parenteral, intrapulmonary, and intranasal, and can be administered focal if local treatment is desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, eg, injection, eg, intravenous or subcutaneous, depending in part on whether the administration is brief or chronic. Various administration schedules include, but are not limited to, single or multiple administrations at various time points, bolus administrations, and intermittent infusions.

It is revealed that the antibody of C should be formulated, administered and administered in accordance with good medical practice. Factors considered in this context include the particular condition 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 medical prescribers have know factor. The antibody is not necessarily, but may alternatively be formulated with one or more agents currently used in the prevention or treatment of the disorder of interest.

The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. Usually administered at the same dose, via the same route, within the scope of the disclosure for C, or 1 to 99% of the dose within the scope of the disclosure for C, or any dose deemed appropriate experimentally/clinically and any path.

For the prevention or treatment of a disease, it is revealed that the appropriate dose of antibody to C (when alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether this Antibody variants are administered for prophylactic or therapeutic use, previous treatment, the patient's clinical history and response to this antibody, and the judgment of the attending physician. Such antibodies are suitable for administration to a patient once or in a series of treatments. Depending on the type and severity of the disease, the initial dose to the patient may be about 1 μg/kg to 15 mg/kg (eg, 0.1 mg/kg to 10 mg/kg) of the antibody, eg, a single dose or several separate doses. given or continuous infusion. The usual daily dose is about 1 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. When administration is repeated over several days or longer, depending on the condition, treatment generally continues until the desired inhibitory effect of disease symptoms is exhibited. Typical doses of the antibody range, for example, from about 0.05 mg/kg to about 10 mg/kg. Thus, eg, one or more doses of about 0.5 mg/kg, eg, 2.0 mg/kg, eg, 4.0 mg/kg, or eg 10 mg/kg (or any combination) may be administered to a patient. Such administration may be intermittent, eg, every week or every three weeks (eg, in such a manner that the patient receives from about 2 to about 20 doses or about 6 doses of the antibody). A higher loading dose can be administered initially, followed by one or more lower doses; but other dosing regimens are useful. The progress of therapy can be easily monitored by conventional techniques and analytical methods.

F. Manufactured Items In another aspect of Disclosure C, the present disclosure provides articles of manufacture, including materials useful in treating, preventing, and/or diagnosing the aforementioned disorders. Such articles of manufacture include containers, labels, or drug labels on or associated with containers. Suitable containers include, for example, bottles, vials, and bags of intravenous solutions. Containers can be made of various materials, such as glass or plastic. Such containers hold compositions, by themselves or with other compositions useful for treating, preventing and/or diagnosing conditions, and may also have sterile access ports (e.g., the container may be a bag of intravenous solutions or may be pierced by a hypodermic needle). small glass vial with an overcapped cap). At least one active ingredient in the composition is a C-revealing antibody. The label or drug label indicates that the composition is used for the treatment of the selected condition.

Also, an article of manufacture can include: (a) a first container comprising a composition comprising an antibody of disclosure C; and (b) a second container comprising a composition comprising an additional cytotoxic agent or a different therapeutic agent. The article of manufacture of the embodiment of Disclosure C may further include a pharmaceutical replica indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture further includes, for example, a second (or third) container comprising a pharmaceutically acceptable buffer, such as Bacteriostatic Water for Injection (BWFI), Phosphate Buffered Saline, Ringer's solution and glucose solution. The article of manufacture may further include other materials desirable for sale or user perspective, including other buffers, filters, needles, and syringes.

Those of ordinary skill in the art will know according to the ordinary skill in the art that the present disclosure C includes all combinations of part or all 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 herein by reference.

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

Although various elements described herein are denoted as, 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 disclosure of A, B, and C, for example, a first element may be referred to as a second element, and similarly, a second element may be referred to as a first element.

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

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

As used herein, unless the context clearly dictates otherwise, "comprising" is intended to indicate the presence of the recited item (member, step, element, quantity, etc.); and the term does not exclude the presence of other items (member, step, element, quantity, etc.) .

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

Unless the context is inconsistent, numerical systems used herein are to be understood as specific ranges understood by those of ordinary skill in the art. For example, the expression "1 mg" is understood as "about 1 mg", with some deviations. For example the expression "1 to 5" is to be understood specifically and individually as "1, 2, 3, 4, 5" unless inconsistent with the context.

Hereinafter, A, B, and C will be specifically described using Embodiments 1 to 4 and 21 to 23, Embodiments 5 to 7, 19 and 20, and Embodiments 8 to 19, respectively, but it is not intended to be limited thereto. It should 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 Fv4-IgG1 disclosed in WO2009/125825 is an antibody that binds to the human IL-6 receptor in a pH-dependent manner, comprising VH3-IgG1 (SEQ ID NO:24) as a heavy chain and VL3-CK (SEQ ID NO:32) as a light chain. 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 number of negatively charged amino acids The number of positively charged amino acids such as arginine and lysine. Specifically, VH3(High_pI)-IgG1 (SEQ ID NO: 25) can be generated by substituting glutamic acid at position 16 of the heavy chain VH3-IgG1 according to the Kabat numbering with glutamic acid, position 43 The glutamic acid at position 64 is substituted with arginine, the glutamic acid at position 64 is substituted with lysine, and the glutamic acid at position 105 is substituted with glutamic acid, so as to increase the isoelectric point value of the heavy chain. Likewise, VL3(High_pl)-CK (SEQ ID NO: 33) can be generated by substituting serine at position 18 with arginine, gluten at position 24 in light chain VL3-CK according to Kabat numbering Arginine is replaced by arginine, glutamic acid at position 45 is replaced by lysine, glutamic acid at position 79 is replaced by glutamic acid, and glutamic acid at position 107 is replaced by lysine. The isoelectric point value of the chain increases. When a substitution was introduced at position 79 of VL3-CK, modifications involving substitution of alanine at position 80 to proline and alanine at position 83 to isoleucine were introduced simultaneously, even if not to increase the isoelectric point value.

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

Then using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) for each The theoretical isoelectric point value of the generated antibody was calculated. The cysteine side chains of this antibody molecule were all assumed to be used to form disulfide bonds, and the contribution of the cysteine side chains to the pKa was excluded from the calculations.

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

WO2011/122011 discloses 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 substitution to Fv4-IgG1 It enhances the Fc region and confers FcRn binding ability under neutral pH conditions. WO2013/125667 also discloses that the FcγR-mediated uptake of Fv4-IgG1-F1180 (hereinafter referred to as Low_pI-F1180) into cells is enhanced by introducing amino acid substitutions into the Fc region of Fv4-IgG1 to enhance neutral pH conditions FcγR binding ability. At the same time, an amino acid modification that increases plasma retention of this antibody by increasing FcRn binding by acidic pH conditions in the endosome was introduced into Fv4-IgG1-F1180. The antibody systems shown below were made by increasing the isoelectric point values 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: 26) of WO2013/125667 28) According to the Kabat numbering method, the glutamic acid at the 16th position is replaced by glutamic acid, the glutamic acid at the 43rd position is replaced by arginine, the glutamic acid at the 64th position is replaced by lysine, and the 105th position is replaced by lysine. glutamic acid was substituted with glutamic acid to make VH3(High_pI)-F11 (SEQ ID NO:31), VH3(High_pl)-F939 (SEQ ID NO:27), and VH3(High_pl)-F1180 (SEQ ID NO:27), respectively ID NO: 29), as the heavy chain with increasing isoelectric point value.

The following antibodies were made using these heavy chains according to the method of Reference Example 2: (1) Low_pI-F939, including VH3-IgG1-F939 as heavy chain and VL3-CK as 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, Include VH3-IgG1-F1180 as heavy chain and VL3-CK as light chain; (5) Middle_pI-F1180, including VH3-IgG1-F1180 as heavy chain and VL3(High_pI)-CK as light chain; (6) High_pI-F1180 , including VH3(High_pI)-F1180 as heavy chain and VL3(High_pI)-CK as light chain; (7) Low_pI-F11, including VH3-IgG1-F11 as heavy chain and VL3-CK as light chain; and (8) High_pI-F11, including VH3(High_pI)-F11 as heavy chain and VL3(High_pI)-CK as light chain.

Then, the theoretical isoelectric point values of the produced antibodies were each calculated by a method similar to that described above using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION). The calculated theoretical isoelectric point values are shown in Table 3. In all novel Fc region variant-containing antibodies, the theoretical isoelectric point values increase in steps in the order of Low_pl, Middle_pl, and High_pl.

[實施例2] [table 3]

[Example 2]

Antigen elimination effect of antibodies showing increased isoelectric point value for pH-dependent binding (2-1) In vivo assay of pH-dependent human IL-6 receptor-binding antibody with adjusted isoelectric point In vivo assays were performed using various pH-dependent human IL-6 receptor-binding antibodies produced in Example 1 as follows: 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 referred to as "hsIL-6R") prepared according to the method of Reference Example 3, anti-human IL-6 receptor antibody and human immunoglobulin preparation Sanglopor were simultaneously administered to human FcRn 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 receptors. 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) were added to 10 mL via the tail vein. /kg administered once. Since the amount of anti-human IL-6 receptor antibody is in sufficient excess over soluble human IL-6 receptor, it is assumed that almost all 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 minutes to obtain plasma. Separated plasma was cryopreserved at -20°C or lower until measurement.

(2-2) Measurement of soluble human IL-6 receptor concentration in plasma by electrochemiluminescence Soluble human IL-6 receptor concentrations in mouse plasma were measured by electrochemiluminescence. 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 50-fold or more dilutions were prepared for mouse plasma assays, respectively sample. The samples were mixed with Ru-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 human IL-6 Tocilizumab (CAS number: 375823-41-9) of the receptor-binding antibody was mixed and then reacted at 37°C overnight. The final concentration of Tocilizumab was adjusted to 333 μg/mL. The reaction solution was then dispensed onto Streptavidin Gold Multi-ARRAY plates (Meso Scale Discovery). After an additional 1 hour reaction at room temperature, the reaction solution was washed. Read Buffer T(x4) (Meso Scale Discovery) was then immediately dispensed into the plate and measurements were performed using a SECTOR Imager 2400 (Meso Scale Discovery). Soluble human IL-6 receptor concentrations were calculated from responses from calibration curves using the analytical software SOFTmax PRO (Molecular Devices).

Changes in plasma concentrations of soluble human IL-6 receptor in human FcRn gene-transfected mice following intravenous administration are shown in Figures 1, 2, and 3. Fig. 1 shows that in the case of the native IgG1 constant region, the isoelectric point value of the variable region is increased, and the antigen elimination effect is enhanced. Fig. 2 shows that the antibody (F939), which has been imparted with the ability to bind to FcRn under neutral pH conditions, has an increased isoelectric point value of the variable region and enhanced antigen elimination effect. Fig. 3 shows that the antibody (F1180) with enhanced FcγR-binding ability under neutral pH conditions increases the isoelectric point value of the variable region and enhances the antigen elimination effect.

In all cases, it was shown that by increasing the isoelectric point value of the antibody, the rate of antigen elimination by the pH-dependently bound antibody was accelerated. It was also shown that by further conferring an increase in the binding ability to FcRn or FcγR under neutral pH conditions, the rate of antigen elimination could be further accelerated compared to the increase in the isoelectric point of the pH-dependent binding antibody (compare Figure 1). and Figures 2 and 3).

(2-3) In vivo analysis method of pH-dependent human IL-6 receptor-binding antibody with adjusted isoelectric point value The following in vivo assays 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 subcutaneously transplanted into human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice, Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) model animals maintained constant plasma concentrations of soluble human IL-6 receptor on the back. Anti-human IL-6 receptor antibodies were administered to model animals, and the in vivo kinetics of the antibodies were evaluated after administration.

Specifically, a monoclonal anti-mouse CD4 antibody obtained by a known method was injected into the tail vein as a single injection at 20 mg/kg to inhibit the neutralization of the anti-soluble human IL-6 receptor possibly produced by the mouse itself. Antibody. An infusion pump containing 92.8 μg/ml of 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 in a single dose at 1 mg/kg into the tail vein. 15 minutes, 7 hours, 1 day, 2 days, 3 days or 4 days, 6 days or 7 days, 13 or 14 days, 20 or 21 days, and 27 or 28 days after administration of the anti-human IL-6 receptor antibody day, blood was collected from the mice. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to obtain plasma. Separated plasma was cryopreserved at -20°C or lower until measurement.

(2-4) Measurement of plasma hsIL-6R concentration by electrochemiluminescence The concentration of hsIL-6R in mouse plasma was measured by electrochemiluminescence. Samples of hsIL-6R 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 50-fold or more dilutions of mouse plasma assay samples were prepared, respectively. Samples were 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 before React overnight at 37°C. The final concentration of Tocilizumab was adjusted to 333 μg/mL. The reaction solution was then dispensed onto Streptavidin Gold Multi-ARRAY plates (Meso Scale Discovery). After an additional 1 hour reaction at room temperature, the reaction solution was washed. Read Buffer T(x4) (Meso Scale Discovery) was then immediately dispensed onto the plate and measurements were performed using a SECTOR Imager 2400 (Meso Scale Discovery). hsIL-6R concentrations were calculated from the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

The change in the measured human IL-6 receptor concentration is shown in FIG. 4 . For both an antibody whose Fc region belongs to native IgG1 (High_pI-IgG1), and this antibody containing a novel Fc region variant (High_pI-F11) with enhanced binding to FcRn at neutral pH conditions, compared to the administration of Low isoelectric point antibodies (also referred to as "Low_pI"), plasma concentrations of soluble human IL-6 receptor decreased in the case of administration of higher isoelectric point antibodies (also referred to as "High_pI").

Without being bound by a particular theory, the results obtained from these experiments can also be explained as follows: when the Fc region of the administered antibody belongs to a native IgG antibody, the uptake into cells is thought to be mainly due to non-specific uptake (pinocytosis). occur. 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 whole molecule is biased towards positive charge), the more accessible the complex is to the cell membrane, and the more likely it is that non-specific uptake occurs. When an antibody with an increased isoelectric point value forms a complex with an antigen, 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 the antibody exhibiting pH-dependent antigen binding, the rate 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 low level.

In these embodiments, the isoelectric point of the antibody is increased by introducing amino acid substitutions that may be exposed on the surface of the antibody molecule that reduce the number of negatively charged amino acids and/or increase the number of positively charged amino acids, in the variable region of the antibody. Those of ordinary skill in the art will appreciate that the effect obtained due to such an increase in isoelectricity is not primarily (or substantially) dependent on the type of target antigen or the amino acid sequence that makes up the antibody, but may be expected to depend 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 that their primary metabolic pathway does not involve renal excretion. Knowing that IgG antibodies with Fc are recovered in a salvage pathway via FcRn expressed by cells including endothelial cells of blood vessels, and thus have long half-lives, IgG antibodies are thought to be primarily metabolized in endothelial cells. Specifically, it is believed that IgG that enters endothelial cells non-specifically is recovered by binding to FcRn, while molecules that cannot bind FcRn are metabolized. IgGs with reduced FcRn binding capacity have shorter half-lives, conversely, blood half-lives can be extended by increasing their binding capacity to FcRn. In this way, the aforementioned method for controlling the kinetics of IgG in blood involves modifying Fc to change the binding ability to FcRn; however, the working examples of WO2007/114319 (mainly the technique of substituting amino acids in the FR region) and WO2009/041643 (mainly the technique of substituting amino acids in the CDR region) shows that regardless of the antigen type, 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 believed to depend on physiochemical Coulomb interactions between the negatively charged cell surface and IgG antibodies. Therefore, reducing (increasing) the isoelectric point value of IgG antibodies and thus reducing (increasing) the Coulomb interaction is believed to reduce (increase) its non-specific uptake into endothelial cells, thereby reducing (increasing) its metabolism in endothelial cells, thereby reducing (increasing) its metabolism in endothelial cells. Can control plasma pharmacokinetics. Because the Coulombic interaction between endothelial cells and the negative charge on the cell surface is a physiochemical interaction, it is believed that this interaction is not primarily dependent on the antibody constitutive amino acid sequence itself. Therefore, the method for controlling plasma pharmacokinetics provided here can be applied not only to specific antibodies, but also to any polypeptides containing variable regions of antibodies. A decrease (increase) in Coulomb interaction herein refers to a decrease (increase) in Coulomb force, expressed as attraction, and/or an increase (decrease) in 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 for introducing a single amino acid substitution or a combination of amino acid substitutions into positions exposed on the surface of the antibody molecule. Alternatively, the plurality of amino acid substitutions introduced may be close to each other in configuration. The inventors have discovered 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 the antibody molecule, or when a preexisting positively charged amino acid (preferably lysine) is used, arginine or lysine), it is advisable to further replace those amino acids in conformation with positively charged amino acids (in some cases, even one or more amino acids buried in the antibody molecule) one or more amino acids in order to obtain a state with locally positively charged clusters at positions close in configuration. The definition of "configurationally close position" here is not particularly limited, but may, for example, refer to the introduction of a single amino acid substitution or multiple amino acid substitutions within 20 angstroms, preferably 15 angstroms or more preferably within 10 angstroms of each other. . Whether the amino acid substitution of interest is at a position exposed on the surface of the antibody molecule, or whether the amino acid substitution is at a nearby 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 charge buried in the antibody molecule and the charge on the surface of the antibody molecule are treated indiscriminately. Considering the effect of charge to make antibody molecules, not only the isoelectric point but also the surface charge and the local clustering of charges on the antibody molecule, can further accelerate the elimination of antigens from plasma, and even maintain the concentration of this antigen in plasma at lower level.

Receptors such as FcRn or FcγR are expressed on the cell membrane, 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 is shifted to the positive charge), the administered antibody-antigen complex is more accessible to the cell membrane, and uptake via Fc receptors is more likely to occur. Therefore, antibodies with increased affinity for FcRn or FcγR and increased isoelectric point value under neutral pH conditions also show increased uptake into cells via Fc receptors when they form complexes with antigens. Thus, for antibodies that bind to antigen in a pH-dependent manner and have enhanced affinity for FcRn or FcγR at neutral pH, the rate of elimination of antigen from plasma can be enhanced by increasing the isoelectric point, and plasma antigen concentration can be increased. maintained at low levels. [Example 3]

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

Similar to the method of Example 1, three types of antibodies with different isoelectric points were prepared as antibodies showing pH-dependent binding to the IL-6 receptor: Low_pI-IgG1, Middle_pI-IgG1 and High_pI-IgG1. According to the method of Reference Example 2, they were respectively produced: Low_pI(NPH)-IgG1, including H54 (SEQ ID NO:34) and L28 (SEQ ID NO:35); and High_pI(NPH)-IgG1 described 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, 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 bound pH-dependently.

[Table 4]

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

The results are shown in FIG. 5 . Both antibodies showing pH-dependent binding as well as antibodies not showing pH-dependent binding increased binding to the extracellular matrix by increasing their isoelectric points. Furthermore, unexpectedly, in antibodies that bind to 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 by a particular theory, the results obtained by such experiments can also be explained as follows. Introduction of histidine modifications into antibody variable regions is known to be one method of conferring pH-dependent antigen binding to antibodies (see eg WO2009/125825). Histidine has an imidazole group on the side chain and is uncharged at neutral to alkaline pH, but is known to be positively charged at acidic pH. Using this property of histidine, by introducing histidine into the variable region of the antibody, especially the CDRs close to the site of interaction with the antigen, the charge environment at the site of interaction 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. Those of ordinary skill in the art will appreciate that the effect obtained by such introduction of histidine does not depend primarily (or substantially) on the type of target antigen or the amino acid sequence that makes up the antibody, but on histamine The acid introduction site or the number of histidine residues introduced.

WO2009/041643 is described in general terms as follows: protein-protein interactions are composed of hydrophobic interactions, electrostatic interactions and hydrogen bonds, and the strength of such binding can usually be determined using binding constants (affinity) or apparent Binding constant (avidity) was expressed. pH-dependent binding, the change in binding strength between neutral pH conditions (eg, pH 7.4) and acidic pH conditions (eg, pH 5.5 to pH 6.0), depends on naturally occurring protein-protein interactions. For example, the binding between the aforementioned IgG molecule and FcRn, which is known to be a salvage receptor for IgG molecules, shows strong binding under acidic pH conditions and very weak binding under neutral pH conditions. Histidine residues are involved in many protein-protein interactions that are altered in a pH-dependent manner. The pKa of histidine residues is close to 6.0 to 6.5, so the proton dissociation state of histidine residues varies between neutral and acidic pH conditions. More specifically, histidine residues are uncharged and neutral under neutral pH conditions and act as hydrogen atom acceptors; and are positively charged and act as hydrogen atom donors under acidic pH conditions. Also, as in the IgG molecule-FcRn interaction described above, the 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, substitution of amino acid residues involved in protein-protein interactions with histidine residues or introduction of histidine at the interaction site can confer pH dependence on protein-protein interactions. A similar approach has been used for protein-protein interactions between antibodies and antigens; antibody mutations with reduced antigen affinity at acidic pH have been successfully obtained by introducing histidine into the CDR sequences of anti-lysozyme antibodies body (Ito et al., FEBS Lett. 309(1):85-88 (1992)). In addition, it has been reported that an antibody specifically binds to an antigen at low pH in cancer tissues and weakly binds to the corresponding antigen at neutral pH due to the introduction of histidine in the CDR sequence (WO2003/105757).

The amino acid residues introduced to increase the isoelectric point value are preferably lysine, arginine or histidine with positively charged side chains. Standard pKa of these amino acid side chains: 10.5 for lysine, 12.5 for arginine, and 6.0 for histidine (Skoog et al., Trends Anal. Chem. 5(4):82-83 (1986) ). According to the acid-base balance theory known in the art, these pKa values refer to a solution at pH 10.5 in which 50% of the lysine 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, and becomes about 9% in a pH 11.5 solution with a pH higher than the pKa of 1 lysine. On the other hand, as the pH of the solution decreases, the positively charged ratio increases, and in a solution of pH 9.5 with a pH lower than the pKa value of 1 lysine, the positively charged ratio becomes about 91%. The same theory holds true for arginine and histidine. More specifically, in a neutral pH solution (eg, pH 7.0), almost 100% of lysine or arginine is positively charged, while about 9% of histidine is positively charged. Therefore, although histidine under neutral pH condition is positively charged, its degree is lower than that of lysine or arginine. Therefore, lysine and arginine are considered to be more suitable to be introduced to increase the isoelectric point. value of amino acids. Also 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 thought to be effective for increasing extracellular matrix binding, for the reasons described above, Substitution to introduce lysine or arginine is considered more suitable than substitution to introduce histidine.

As previously revealed, by combining the introduction of histidine to confer pH-dependent antigen binding and the modification of the antibody to increase the 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 the isoelectric point 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 for extracellular matrix was significantly higher. This showed that the affinity for extracellular matrix is not necessarily explained by the isoelectric point level alone, and it can be said that the synergistic effect is shown by the introduction of a combination of isoelectric point-increasing modification and histidine modification. This result is unexpected and unexpected.

However, it should be noted that the pKa of the amino acid side chain of a protein is greatly affected by the surrounding environment, but does not always conform to the above theoretical pKa value. More specifically, the above records are presented here based on general scientific theory; but one can easily speculate that there will be many exceptions to actual proteins. For example, Hayes et al., J. Biol. Chem. 250(18):7461-7472 (1975) stated that when the pKa of histidine contained in muscle protein was determined experimentally, the value was concentrated at about 6.0, but it was reported at 5.37 ~8.05 change. Naturally, histidines with high pKa are mostly positively charged at neutral pH. Therefore, the above theory does not deny the effect of increasing the isoelectric point value of the introduction of histidine and the true three-dimensional structure of the protein. Like the introduction of lysine or arginine modification, the amino acid modification of histidine has a sufficient possibility to exert the effect of increasing the isoelectric point. [Example 4]

Utilize 1 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 an antigen in a pH-dependent manner by introducing amino acid substitutions into the variable region of the antibody. In addition, methods of increasing the isoelectric point value of an antibody can also be performed by making as few as 1 amino acid substitution in the constant region of the antibody.

The method of adding 1 amino acid 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 variable regions, amino acid substitutions introduced into the constant region are preferably to reduce the number of negatively charged amino acids (such as aspartic acid or glutamic acid) and increase the number of positively charged amino acids (such as arginine or lysine).

Without limitation, the position at which the amino acid substitution is introduced into the constant region is preferably a position where the side chain of the amino acid may be exposed on the surface of the antibody molecule. Desirable examples include methods for introducing combinations of amino acid substitutions at positions that can be exposed on the surface of the antibody molecule. Alternatively, the multiple amino acid substitutions are preferably introduced at positions such that they are conformationally close to each other. Furthermore, without limitation, the plurality of amino acid substitutions introduced are preferably substituted into positively charged amino acids such that in some cases they result in states where the plurality of positive charges are in configurationally close positions. The "conformationally proximity site" is not particularly limited, but may, for example, refer to the introduction of one or more amino acid substitutions into each other, eg, within 20 angstroms, preferably within 15 angstroms, 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 positions of amino acid substitution are close together can be assessed using known methods such as X-ray crystallography.

In addition, the method of imparting a plurality of positively charged positions close to the configuration includes, in addition to the above-mentioned methods, a method of using an amino acid originally positively charged in the IgG constant region. Examples of such positively charged amino acid positions include: (a) arginine at positions 255, 292, 301, 344, 355 or 416 according to EU numbering; and (b) arginine at 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 into positively charged amino acids at sites that are conformationally close to these positively charged amino acids, it is possible to impart multiple positive charges at conformationally close positions.

(4-1) Production of pH-dependent anti-IgE-binding antibody The following 3 antibodies were produced as pH-dependent anti-human IgE antibodies according to the method of Reference Example 2: (1) Ab1, a conventional antibody comprising Ab1H (SEQ ID NO: 38) as the heavy chain and Ab1L (SEQ ID NO: 38) NO:39) as a light chain; (2) Ab2, a conventional antibody comprising Ab2H (SEQ ID NO:40) as a heavy chain and Ab2L (SEQ ID NO:41) as a light chain; and (3) Ab3, as A conventional antibody, comprising Ab3H (SEQ ID NO:42) as the heavy chain and Ab3L (SEQ ID NO:43) as the light chain.

(4-2) Evaluation of pH dependence of human IgE binding The affinity of Ab1 for human IgE at pH 7.4 and pH 5.8 was evaluated as follows. Kinetic analysis of human IgE and Ab1 was performed using a BIACORE T100 (GE Healthcare). The measurement system used 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) by amine coupling to capture the antibody of interest. Human IgE was then interacted with the antibody captured on the sensor chip by injecting diluted IgE solution with running buffer (as a control solution). For the running buffer, either of the above buffers (1) and (2) was used, and human IgE was diluted with each buffer. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were carried out at 25°C. The KD (M) of human IgE of each antibody was calculated from the kinetic parameters calculated from the sensorgram obtained by the 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 assessed as follows. The binding activity of the anti-hIgE antibody to hIgE (dissociation constant KD (M)) was carried out using BIACORE T200 (GE Healthcare). The measurement system used 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.

A peptide derived from the human glypican 3 (also known as GPC3) protein present in chemical synthesis (the amino acid sequence is (VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO: 44))) ("biotinylated GPC3 peptide") will be utilized by attaching biotin to A suitable amount of peptide prepared by Lys at the C-terminus of ) was added to Sensor chip SA (GE Healthcare), and was immobilized on the chip using the affinity between streptavidin and biotin. The appropriate concentration was injected. and immobilized on the wafer by capturing the biotinylated GPC3 peptide. An appropriate concentration of anti-hIgE antibody was injected as the analyte and interacted with hIgE on the sensor wafer. The sensor wafer was then regenerated and injected 10 mM Glycine-HCl, pH 1.5. All measurements were performed at 37°C. Association rate constants ka (1/Ms) and dissociation rate constants 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-mentioned values.

The results are presented in Table 5. All the antibodies, Ab1, Ab2 and Ab3, showed pH-dependent binding to human IgE, and their affinity at acidic pH (pH 5.8) was significantly higher than that at neutral pH (pH 7.4). weak. Therefore, administration of these antibodies to live animals is expected to accelerate the elimination of human IgE as an 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 by single amino acid modification in the constant region Ab1 prepared in Example (4-1) is an antibody having natural human IgG1 as a constant region. Ab1H-P600 is made by modifying the Fc region of Ab1H of the heavy chain of Ab1 by substituting proline at position 238 according to EU numbering with aspartic acid, and replacing serine at position 298 according to EU numbering Acid substitution to alanine to prepare. Further various Fc variant systems were prepared according to the method of Reference Example 2, by introducing various single amino acid substitutions indicated in Tables 7-1 and 7-2 into the Fc region of Ab1H-P600, respectively. For all Fc variants, Ab1L (SEQ ID NO:39) was used as the light chain. The affinity of these antibodies for hFcyRII2b was comparable to that of the P600 variant (data not shown).

[Table 7-1]

[Table 7-2]

(4-4) Human FcγRIIb Binding Assay Using Novel Fc Region Variant-Containing Antibodies Using BIACORE The binding assay was performed using BIACORE (registered trademark) T200 (GE Healthcare). Soluble hFcyRIIb in His-tagged molecular form is produced using methods known in the art. An appropriate amount of anti-His antibody was immobilized on Sensor chip CM5 (GE Healthcare) by amine coupling using His capture kit (GE Healthcare) to capture hFcyRIIb. Antibody-antigen complexes were then injected with running buffer (as a reference solution) to interact with hFcyRIIb captured on the sensor wafer. 20 mM N-(2-acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, _1.2 mM CaCl, and 0.05% (w/v) Tween 20 at pH 7.4 were used as running buffers, respectively buffer to dilute soluble hFcyRIIb. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were carried out at 25°C. The analysis is based on the calculated binding (RU) of the sensorgram obtained by the measurement and shows the relative value when the binding amount of P600 is defined as 1.00. To calculate the 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. There are several Fc variants showing improved affinity for hFcyRIIb immobilized on a BIACORE (registered trademark) sensor wafer.

While not being bound by 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 wafer is presumed to be similar to the binding of this antigen-antibody complex to hFcγRIIb present on the negatively charged cell membrane surface.

An antibody prepared by introducing an isoelectric point-increasing modification into the Fc region has a more positively charged Fc region (constant region) than an antibody before the modification was introduced. Therefore, it is believed that the Coulomb interaction between the Fc region (positively charged) and the sensing chip surface (negatively charged) is enhanced by the modification of the increased isoelectric point. This effect is also expected to occur on the surface of negatively charged cell membranes as well; it is also expected to show the effect of accelerating the rate or rate of uptake into cells in vivo.

From the above results, it is considered that the ratio of hFcyRIIb bound to the variant is about 1.2 times or more compared to the hFcyRIIb bound to Ab1H-P600, indicating that the antibody has a strong charge effect on the binding of hFcyRIIb on the sensing chip. Modifications that are expected to produce a charge effect include, for example, in accordance with EU numbering at 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 modifications. 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 preferred amino acid substitutions introduced at position 430 are positively charged arginine or lysine, or glycine or threonine in uncharged residues.

(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 produced novel Fc region variant-containing antibodies, the following assays were performed.

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

The results are shown in Tables 7-1 and 7-2 (see the "Contrast" 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. This antigen-antibody complex binds to hFcyRIIb expressed on the cell membrane via the Fc region of the antibody, 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; thus the antibody can dissociate from this antigen. Because the dissociated antigen was labeled with pHrodoRed as described earlier, the endosomes fluoresced. Therefore, if the intracellular fluorescence intensity is stronger than that of the control group, it is considered that the uptake of the antigen-antibody complex into the cells occurs faster or more frequently.

Here, compared with the fluorescence intensity of Ab1H-P600, the fluorescence intensity of antigen entering cells of this variant is about 1.05 times or more, which is considered to have a charge effect on antigen entry into cells. Compared with the fluorescence intensity of Ab1H-P600, the fluorescence intensity of antigen entering cells of this variant was about 1.5 times or more, which is considered to have a strong charging effect on antigen entry into cells. Therefore, the above results show that by introducing a modification with an increased isoelectric point at an appropriate position in the Fc region, the uptake into cells can be accelerated compared to before the introduction of the modification. Amino acid modification positions showing such effects are, for example, according to EU numbering 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 positions 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. Without limitation, 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 residues at position 399 according to EU numbering. [Example 5]

Fc variants with enhanced FcRn binding made in acidic pH conditions to improve retention in plasma At acidic pH conditions in endosomes, IgG antibodies that have entered cells are known to return to plasma by binding to FcRn. Therefore, IgG antibodies generally have longer plasma half-lives than proteins that are not bound to FcRn. Taking advantage of this property, methods for enhancing plasma retention of antibodies by introducing amino acid modifications in the Fc region of antibodies to increase FcRn affinity under acidic pH conditions are known. Specifically, methods for improving the plasma retention of antibodies by increasing the FcRn affinity under acidic pH conditions by amino acid modification are known, for example, 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 described above, Fc variants known to have increased FcRn affinity under acidic pH conditions will show undesired affinity for rheumatoid factor (RF) (WO2013/046704). The following assays were therefore performed to create Fc variants that reduce or substantially do not bind to rheumatoid factors that improve plasma retention.

(5-1) Production of Novel Fc Region Variant-Containing Antibodies Fc variants with increased FcRn affinity under acidic pH conditions, for example including 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 having amino acid modifications introduced into the Fc region of the heavy chain (VH3-IgG1m) of Fv4-IgG1, which is an anti-human IL-6 receptor, was produced Antibody. These heavy chains were used to make the following antibodies according to the method of Reference Example 2: (a) Fv4-IgG1, including VH3-IgG1m (SEQ ID NO: 46) as heavy chain and VL3-CK as light chain; (b) Fv4- YTE, including VH3-YTE (SEQ ID NO:47) as heavy chain and VL3-CK as light chain; (c) Fv4-LS, including VH3-LS (SEQ ID NO:48) as heavy chain and VL3-CK as 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 heavy chain and VL3-CK as light chain; (h) Fv4-F1889m, including VH3-F1889m (SEQ ID NO:53) as heavy chain and VL3-CK as light chain; (i) ) Fv4-F1927m, including VH3-F1927m (SEQ ID NO:54) as a heavy chain and VL3-CK as a light chain; and (j) Fv4-F1168m, including VH3-F1168m (SEQ ID NO:55) as a heavy chain and VL3-CK as light chain.

(5-2) Kinetic analysis of binding to human FcRn An antibody containing VH3-IgG1m or the above variant as a heavy chain and L(WT) (SEQ ID NO: 37) as a 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 a BIACORE T100 (GE Healthcare). An appropriate amount of Protein L (ACTIGEN) was immobilized on Sensor chip CM4 (GE Healthcare) by amine coupling to capture the antibody of interest. Human FcRn and the antibody captured on the sensor wafer were then allowed to interact by injecting diluted FcRn solution with running buffer (as a reference solution). For running buffers, 50 mM sodium phosphate, 150 mM NaCl, and 0.05% (w/v) Tween 20 at pH 6.0 were used, each buffer was also used to dilute FcRn. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were carried out at 25°C. The KD (M) of human FcRn for each antibody was calculated based on the kinetic parameters calculated from the sensorgrams obtained from the measurements, the on-rate constant ka (1/Ms) and the dissociation rate constant kd (1/s). Parameters were calculated using BIACORE T100 Evaluation Software (GE Healthcare).

The results are shown in Table 8.

[實施例6] [Table 8]

[Example 6]

Assessing the affinity of Fc region variant-containing antibodies for rheumatoid factor with enhanced FcRn binding under acidic pH conditions Anti-drug antibodies (ADAs) affect the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes cause severe side effects; therefore, the clinical utility and efficacy of therapeutic antibodies may be limited by the production of ADA. 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 (also referred to as "pre-existing ADA") present in the patient prior to administration of the therapeutic antibody may present similar problems. Specifically, for therapeutic antibodies directed against patients with autoimmune diseases such as rheumatoid arthritis (RA), rheumatoid factor (RF), which is an autoantibody against human IgG, may cause " Pre-existing ADA" problem. Recently, a humanized anti-CD4 IgG1 antibody with an N434H (Asn434His) mutation was 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 the rheumatoid factor in the Fc region to the antibody compared to the parent human IgG1.

Rheumatoid factor is a polyclonal autoantibody against human IgG, its epitope on human IgG is different depending on the colony, and it seems to be located in the CH2/CH3 junction region, which may bind to FcRn in the CH3 domain. base overlap. Therefore, mutations that increase the binding activity (binding affinity) to FcRn may increase the binding activity (binding affinity) to a specific colony of rheumatoid factor.

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

On the other hand, in WO2013/046704, some amino acid modifications that selectively inhibit the affinity for rheumatoid factor without affecting the affinity for FcRn are disclosed, wherein, a combination of 2 amino acid mutations is indicated, Namely Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D. Therefore, it was revealed for the first time that the introduction of Q438R/S440E had a new increased affinity under acidic pH conditions to examine whether the binding to rheumatoid factor would be reduced.

(6-1) Assay for rheumatoid factor binding of antibodies containing Fc region variants Binding analysis to rheumatoid factor was performed using electrochemiluminescence (ECL) at pH 7.4 using individual sera (Proteogenex) from 30 RA patients. Each mixed 50-fold diluted serum sample, biotinylated test antibody (1 μg/mL), and SULFO-TAG NHS Ester (Meso Scale Discovery)-labeled test antibody (1 μg/mL), and incubated at room temperature Cultivate hours. Thereafter, this mixture was added to streptavidin-coated MULTI-ARRAY 96-well plates (Meso Scale Discovery), which were 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 in a SECTOR imager 2400 Reader (Meso Scale Discovery) and chemiluminescence was measured.

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

Figure 18 shows the mean values of rheumatoid factor binding affinity in the serum of 30 RA patients for each variant. All 6 new variants showed lower affinity than 3 pre-existing variants (YTE, LS, and N434H), and they also showed lower affinity for rheumatoid factor than native human IgGl. Thus, when considering the clinical development of therapeutic antibodies with improved affinity for FcRn for autoimmune diseases such as rheumatoid arthritis, the Fc variants disclosed for the first time here may inhibit the concerns of pre-existing Fc variants. risk associated with rheumatoid factor, it can be used more safely than the currently known Fc variants. [Example 7]

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

(7-1) Production of novel antibodies containing Fc region variants The following anti-human IgE antibodies were made: (a) OHB-IgG1, including OHBH-IgG1 (SEQ ID NO:56) as heavy chain and OHBL-CK (SEQ ID NO:57) as light chain; (b) OHB-LS, Include OHBH-LS (SEQ ID NO:58) as heavy chain and OHBL-CK as light chain; (c) OHB-N434A, including OHBH-N434A (SEQ ID NO:59) as heavy chain and OHBL-CK as light chain (d) OHB-F1847m including OHBH-F1847m (SEQ ID NO:60) as heavy chain and OHBL-CK as light chain; (e) OHB-F1848m including OHBH-F1848m (SEQ ID NO:61) as heavy chain chain and OHBL-CK as light chain; (f) OHB-F1886m including OHBH-F1886m (SEQ ID NO:62) as heavy chain and OHBL-CK as 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, including OHBH-F1927m (SEQ ID NO: 64) as the heavy chain and OHBL-CK as the light chain.

(7-2) PK analysis of novel antibodies containing Fc region variants in monkeys The in vivo kinetics of anti-human IgE antibodies in plasma were assessed following administration of anti-human IgE antibodies to Malay monkeys. The anti-human IgE antibody solution was administered once intravenously at 2 mg/kg. 5 minutes after administration, (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 minutes to obtain plasma. Separated plasma was cryopreserved 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 by ELISA Anti-human IgE antibody concentrations in Malay monkey plasma were measured by ELISA. Anti-human IgG kappa chain antibody (antibody solution) was first dispensed on Nunc-ImmunoPlate, MaxiSorp (Nalge Nunc International) and allowed to stand at 4°C overnight to make anti-human IgG kappa chain antibody immobilized plate. Calibration curve samples with plasma concentrations of 640, 320, 160, 80, 40, 20 or 10 ng/mL and Malay monkey plasma measurement samples diluted 100-fold or more were prepared. These calibration curve samples and plasma measurement samples were prepared so that the monkey IgE (a product prepared in-house) was spiked at a concentration of 1 μg/mL. Then, this sample was dispensed onto an anti-human IgG kappa chain antibody immobilized plate and left at room temperature for 2 hours. HRP-anti-human IgG gamma chain antibody (Southern Biotech) was then dispensed and left to stand at room temperature for 1 hour. The color-forming reaction was then performed using TMB Chromogen Solution (Life Technologies) as a substrate, and after the reaction was stopped by adding 1N sulfuric acid (Wako), the absorbance at 450 nm was measured with a microplate reader. Anti-human IgE antibody concentrations in monkey plasma were calculated from the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). Changes in the concentrations of anti-human IgE antibodies measured in monkey plasma are shown in FIG. 19 . Changes in anti-human IgE antibody concentrations were measured from 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. In calculating the change in anti-human IgE antibody concentration and the clearance rate in monkey plasma, samples from individuals who were positive for antibodies in plasma to the anti-sample-administered samples were excluded.

[Table 9]

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

Results All novel Fc variants were confirmed to show greatly improved plasma retention compared to the Fc region of native IgGl.

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

The 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 a heavy chain and VL3-CK as a light chain; and (b) Fv4-F1718, including VH3- F1718 (SEQ ID NO: 65) as the heavy chain and VL3-CK as the light chain.

1 mg/kg in the tail vein of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice; Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010) 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 cryopreserved at -20°C or lower until use.

(7-6) The concentration of anti-human IL-6 receptor antibody in plasma was measured by ELISA The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA. Anti-human IgG (gamma chain specific) F(ab') ₂ fragment of antibody (SIGMA) was first distributed to Nunc-ImmunoPlate, MaxiSorp (Nalge nunc International) and left at 4°C overnight to make anti-human IgG immobilized plates . 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. 200 μL of 20 ng/mL soluble human IL-6 receptor was added to 100 μL of the calibration curve sample or the plasma measurement sample, and then the mixed solution was allowed to stand at room temperature for 1 hour. This mixed solution was then dispensed into the wells of an anti-human IgG immobilized plate, and the plate was left to stand at room temperature for 1 hour. Then, biotinylated anti-human IL-6R antibody (R&D) was added, and the reaction was carried out at room temperature for 1 hour. Then, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added and reacted at room temperature for 1 hour, using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate to carry out the color-forming reaction of the reaction solution. After stopping the reaction by adding 1 N sulfuric acid (Showa Chemical), the absorbance at 450 nm of the reaction solution in each well was measured on a microplate reader. Antibody concentrations in mouse plasma were calculated from the absorbance values of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

The results are shown in Figure 20. F1718, an Fc variant described in WO2013/046704 that has increased binding to FcRn at acidic pH, did not show any effect in prolonging antibody PK, but showed plasma retention equivalent to that of native IgG1.

F1718 has 4 mutations, namely N434Y/Y436V/Q438R/S440E introduced in the Fc region. In contrast, F1848m, which was first disclosed here, also introduced four mutations, namely N434A/Y436V/Q438R/S440E. The amino acid mutation introduced by these two types of Fc differs only in that F1718 is mutated into Y (tyrosine) at position 434 according to EU numbering, and A (alanine) in F1848m. In Example (7-2), F1848m showed improved plasma retention compared to native IgG1, whereas F1718 did not show any improvement in plasma retention. Therefore, without limitation, this suggests that it is desirable to introduce A (alanine) as an amino acid mutation at position 434 to improve plasma retention.

(7-7) Predicted immunogenicity scores of Fc variants The production of anti-drug antibodies (ADA) affects the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes leads to serious side effects; therefore, the production of ADA may limit the clinical utility and drug efficacy of therapeutic antibodies. The immunogenicity of therapeutic antibodies is known to be influenced by a number of factors, and in particular the importance of effector T cell epitopes carried by therapeutic antibodies has been reported many times.

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

EpiMatrix was used to calculate the immunogenicity scores of the Fc variants to be evaluated. EpiMatrix is a system for predicting the immunogenicity of a protein of interest. It automatically designs the sequence of peptide fragments by cutting the amino acid sequence of the protein to be predicted immunogenicity by 9 amino acids, and then calculates them. Binding capacity to 8 major MHC class II dual genes (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, F1718, F1848m, F1756 and F1847m, calculated as above, are shown in Table 31 in the "EpiMatrix Score" column. Also, regarding the EpiMatrix score, the immunogenicity score corrected for Tregitope content is shown in the column "tReg Adjusted Epx Score". Tregitope is a peptide fragment sequence predominantly found in naturally occurring antibody sequences and is thought to inhibit immunogenicity by activating regulatory T cells (Treg).

[Table 31]

In accordance with these results, both the "EpiMatrix Score" and the "tReg Adjusted Epx Score" showed a decrease in the immunogenicity scores of the N434A variants F1848m and F1847m compared to the N434Y variant. This indicates that A (alanine) was ideally introduced as an amino acid mutation at position 434 to obtain a lower immunogenicity score. [Example 8]

Production of Humanized Anti-Human IL-8 Antibody (8-1) Production of humanized anti-human IL-8 antibody hWS-4 US Patent No. 6,245,894 discloses humanized anti-IL-8 antibodies that bind to human IL-8 (hIL-8) and block its physiological function. 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 nearly 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, and the native human IgG1 sequence or native human IgG4 sequence can be used as the heavy chain constant region sequence, and the native human Kappa sequence can be used as the light chain constant region sequence.

From the humanized IL-8 antibodies disclosed in US Pat. No. 6,245,894, the coding sequence of hWS4H-IgG1 (SEQ ID NO: 83), which combines the heavy chain variable region RVHg with the native human anti-IgG1 sequence for the heavy chain The constant region was fabricated 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 as a humanized WS-4 antibody (hereinafter referred to as hWS-4).

(8-2) Production of humanized anti-human IL-8 antibody Hr9 Human common framework sequences with different FRs used in hWS-4 were used to make new humanized antibodies.

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, excluding the sequence according to the Kabat numbering method) 82a), using the sequence found in JH1 as FR4. These sequences were linked to the CDR sequences of the hWS-4 heavy chain to create Hr9-IgG1 (SEQ ID NO: 85), a novel humanized antibody heavy chain.

Two types of antibodies were then made, namely: hWS-4, with hWS4H-IgG1 as heavy chain and hWS4L-kOMT as light chain; and Hr9, with Hr9-IgG1 as heavy chain and hWS4L-kOMT as light chain. In the context of revealing C, when specifying a 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 in the amounts of antibodies 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 for human IL-8 was determined using a BIACORE T200 (GE Healthcare) in the following manner.

A running buffer with the following composition was used: 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) by amine coupling and the antibody of interest was captured. Human IL-8 was then interacted with the antibody captured on the sensor chip by injecting diluted human IL-8 solution with running buffer (as a reference solution). For the running buffer, use the composition as above and also dilute human IL-8 with this buffer. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were performed at 37°C. The KD (M) of human IL-8 for each antibody was calculated from the kinetic parameters calculated from the sensorgram obtained by the measurement, the association rate constant kon (1/Ms) and the dissociation rate constant koff (1/s). Parameters were calculated using BIACORE T200 Evaluation Software (GE Healthcare).

The results are shown in Table 12. It was confirmed that hWS-4 and Hr9 have equivalent 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 that, from the above tests, more appropriate human common framework derived sequences were selected to combine with the HVR sequences of hWS-4 and produce Hr9 with improved production levels and maintaining binding affinity for human IL-8. [Example 9]

Production of antibodies with pH-dependent IL-8 affinity (9-1) Production of Hr9-modified antibody to confer pH dependence The purpose of the study was to impart pH-dependent IL-8 affinity to the Hr9 antibody obtained in Example 8.

While not being bound by a particular theory, antibodies with pH-dependent affinity for IL-8 may exhibit the following behavior in vivo. This antibody, administered to a living organism, strongly binds to IL-8 and blocks its function in an environment that maintains neutral pH (eg, in plasma). A portion of such IL-8/antibody complexes are taken up into cells by nonspecific interactions with cell membranes (pinocytosis) (hereinafter referred to as nonspecific uptake). Under acidic pH conditions in endosomes, the binding affinity of the above-mentioned antibodies to IL-8 becomes weak, and thus the antibodies dissociate from IL-8. Antibodies dissociated from IL-8 can then be re-entered extracellularly via FcRn. The above-mentioned antibodies returned to the extracellular (intraplasmic) in this way can re-bind to other IL-8 and block its function. Antibodies with pH-dependent affinity for IL-8 are thought to bind to IL-8 multiple times by the mechanism described above.

Conversely, in the case that the antibody does not have the properties possessed by the above-mentioned antibodies, the antibody molecule can only neutralize the antigen once, and cannot neutralize the antigen multiple times. Generally, since IgG antibodies have 2 Fabs, a single antibody molecule is capable of neutralizing 2 molecules of IL-8. On the other hand, an antibody that can bind to IL-8 multiple times can bind to IL-8 any number of times as long as it stays in the living body. For example, a single molecule of a pH-dependent IL-8 binding antibody, when administered 10 times taken up into cells, can neutralize up to 20 molecules of IL-8 until eliminated. Therefore, antibodies that can bind IL-8 multiple times have the advantage of neutralizing several IL-8 molecules even in small amounts. From another viewpoint, an antibody capable of multiple binding to IL-8 has the advantage of being able to maintain a state capable of neutralizing IL-8 for a longer period of time than when an antibody of the same amount without this property is administered. From another point of view, an antibody that binds IL-8 multiple times has the advantage of being able to block the biological activity of IL-8 more strongly than an equivalent antibody that does not have this property when administered.

To achieve these advantages, amino acid modifications, mainly histidine, were introduced into the variable regions of Hr9-IgG1 and WS4L-k0MT to create antibodies that could bind to IL-8 multiple times. Specifically, the variants shown in Table 13 were fabricated according to the methods of Reference Examples 1 and 2.

Labels such as "Y97H" in Table 13 show the mutation introduction positions according to the Kabat numbering method, the amino acid before the introduction of the mutation and the amino acid after the introduction of the mutation. Specifically, when "Y97H" is indicated, the amino acid residue at position 97 of the Kabat numbering is shown to be substituted from Y (tyrosine) to H (histidine). In addition, when a plurality of amino acid substitutions are introduced in combination, it is described in the form of "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) as follows. The following 2 running buffers were used: (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 and the antibody of interest was captured. Human IL-8 was then interacted with the antibody captured on the sensor chip by injecting diluted human IL-8 solution with running buffer (as a reference solution). For the running buffer, any of the above solutions were used, and human IL-8 was diluted with each buffer. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were performed at 37°C. The KD (M) of human IL-8 for each antibody was calculated from the kinetic parameters calculated from the sensorgram obtained by the measurement, the association rate constant kon (1/Ms) and the dissociation rate constant koff (1/s). Parameters were calculated using BIACORE T200 Evaluation Software (GE Healthcare).

The results are shown in Table 14-1. First, compared to Hr9, Hr9/L16 with L54H modification in the light chain slightly increased the binding affinity of human IL-8 at neutral pH (pH 7.4), but decreased 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) made by combining various light chains and H89 containing Y97H modifications to the heavy chain all showed reduced binding affinity for human IL-8 at acidic pH and reduced binding affinity of human IL-8 at neutral pH.

[Table 14-1]

(9-3) Production and Evaluation of Modified Antibodies to Confer pH Dependence Combinations of promising modifications found in 9-2 and new amino acid mutations were evaluated and the following combinations were found.

[Table 14-2]

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

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

Surprisingly, H89/L118 with H89-IgG1 as the heavy chain and L118-kOMT (SEQ ID NO: 88) as the light chain was found to have enhanced human IL-8 binding at neutral pH compared to Hr9 affinity (KD), but weaker binding affinity (KD) for human IL-8 at acidic pH compared to Hr9. 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 a strong binding affinity (small KD) so that it can be strongly neutralized under neutral pH conditions. the antigen (eg in plasma). On the other hand, the antibody should preferably have a large dissociation rate constant (koff) and/or a weak binding affinity (large KD) in order to rapidly release the antigen under acidic pH conditions (eg, in endosomes) . Compared to Hr9, H89/L118 has achieved these favorable properties under both neutral pH and acidic pH conditions.

Thus 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 to this, it was shown that pH-dependent IL-8 binding antibodies that are advantageous as medicines can be generated by introducing a single or a combination of multiple amino acid modifications selected from these modifications.

Without being bound by a particular theory, it is thought that an important factor in the use of pH-dependent antigen-binding antibodies as medicines is whether or not the administered antibody can release the antigen in the endosome. In this regard, a sufficiently weak binding (large dissociation constant (KD)) or sufficiently fast dissociation rate (large dissociation rate constant (koff)) under acidic pH conditions is believed to be important. Therefore, the following experiments were performed to examine whether the obtained KD or koff of H89/L118 was sufficient for dissociation of this antigen in vivo in vivo with BIACORE. [Example 10]

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

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

Human IL-8 binding affinity was assessed using H998/L63 in a similar manner to Example 9-2. The obtained induction map is shown in Fig. 21.

Unexpectedly, H998/L63 showed a slow dissociation rate at both neutral pH and acidic pH, and had a stronger binding affinity for IL-8 than Hr9. However, it is known that analytical values such as dissociation rate constant (koff) and dissociation constant (KD) cannot be calculated correctly in the case of protein-protein interactions with low dissociation rates due to mechanical limitations of BIACORE. Since the correct analytical value cannot be obtained for H998/L63, its analytical value is not shown here. However, the experimental results confirm that H998/L63 has very strong binding affinity at both neutral pH and acidic pH, and is suitable as an antibody for comparison in 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 The elimination rate of human IL-8 in vivo was evaluated using H89/L118 produced in Example 9 and H998/L63 produced in Example 10.

The pharmacokinetics of human IL-8 was evaluated after simultaneous administration of human IL-8 and anti-human IL-8 antibody to 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, respectively) was administered in a single dose of 10 mL/kg into the tail vein. At this time, there is a sufficient excess of anti-human IL-8 antibody relative to human IL-8, so it is considered that almost all of the 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 minutes to obtain plasma. Separated plasma was cryopreserved at -20°C or lower until measurement.

(11-2) Measurement of human IL-8 concentration in plasma Human IL-8 concentrations in mouse plasma were determined by electrochemiluminescence. Anti-human IL-8 antibody (prepared in-house) including mouse IgG constant region was first dispensed into MULTI-ARRAY 96-well plates (Meso Scale Discovery) and left to stand at room temperature for 1 hour. Anti-human IL-8 antibody immobilized plates were then prepared by blocking with 5% BSA (w/v) in PBS-Tween for 2 hours at room temperature. Calibration curve samples were prepared containing human IL-8 at 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-fold or more. The sample and hWS-4 were mixed and allowed to react overnight at 37°C. Then, 50 μL of the mixed solution was dispensed 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, and then react with SULFO-TAG-labeled streptavidin (Meso Scale Discovery) for 1 hour at room temperature, distribute Read Buffer T (x1 ) (Meso Scale Discovery), measurements were performed immediately with a SECTOR Imager 2400 (Meso Scale Discovery). Human IL-8 concentrations were calculated from the response of the calibration curve using the analytical software SOFT Max PRO (Molecular Devices).

Data for human IL-8 concentrations 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 can be seen from Figure 22, co-administration of human IL-8 and H89/L118 was unexpectedly faster from mouse plasma than co-administration of human IL-8 and H998/L63. The CL values, which also quantitatively represent the rate of elimination of human IL-8 from mouse plasma, indicate that the rate of elimination of human IL-8 for H89/L118 is approximately 19-fold increased compared to H998/L63.

Without being bound by a particular theory, it can be speculated from the data obtained as follows. Most of the human IL-8 administered at the same time as the antibody binds to the antibody in plasma and exists as a complex. Human IL-8, which has been bound to H998/L63, still exists in the state bound to the antibody even in the endosome under acidic pH conditions due to the strong affinity of the antibody. After that, H998/L63 is still in the form that binds human IL-8 and can be returned to plasma by FcRn; so when this happens, human IL-8 also returns to plasma at the same time. Therefore, most of the human IL-8 entering the cells will be returned to the plasma. That is, when H998/L63 was administered concurrently, the rate of elimination of human IL-8 from plasma was significantly reduced. On the other hand, as described above, human IL-8 entering cells becomes a complex with H89/L118 (pH-dependent IL-8-binding antibody), and this antibody may be dissociated under acidic pH conditions in the endosome. Human IL-8 dissociated from the antibody may be degraded after delivery to the lysosome. Therefore, pH-dependent IL-8-binding antibody can significantly accelerate the elimination of human IL-8, compared with IL-8-binding antibody such as H998/L63, which has strong binding affinity at both acidic pH and neutral pH.

(11-3) Mouse PK assay for increasing doses of H89/L118 Experiments to confirm the effect of dose changes of H89/L118 were then carried out as follows. Pharmacokinetics of human IL-8 was assessed following 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 at a single dose of 10 mL/kg. At this time, since there is a sufficient excess of anti-human IL-8 antibody compared to human IL-8, it is 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 minutes to obtain plasma. Separated plasma was cryopreserved at -20°C or lower until measurement.

Human IL-8 concentrations in mouse plasma were measured in a similar manner to Example 11-2. The results of human IL-8 concentrations 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 the antibody had an approximately 2-fold slower elimination rate of human IL-8 compared to the group administered with 2 mg/kg of H89/L118.

The following inventors will describe the possible factors that may lead to the above results based on scientific background, but the content of revealing C is not limited to the following discussion.

Among the antibodies returned to plasma from the endosome via FcRn, the proportion of antibodies that bind to human IL-8 is preferably low. Focusing on the presence of human IL-8 in endosomes, a high proportion of free rather than bound antibody is expected. When human IL-8 was administered with an antibody without pH-dependent IL-8-affinity, the majority (almost 100%) of human IL-8 endosomes were thought to be present in antibody-complexed form, with a small amount of (close to 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 be present in the free form in the endosome. Hypothetically, the free ratio 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 proportion of free human IL-8 in the endosome understood by the above formula is ideally high, such as 20% is better than 0%, 40% is better than 20%, 60% is better than 40%, 80% is better than 60%. %, and 100% are better than 80%.

Thus, there is a correlation between the above-mentioned ratio 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 the endosome. However, in the case of pH-dependent IL-8-binding antibodies, which can make the ratio of free human IL-8 approach 100% in endosomes, further weakening binding affinity and/or increasing dissociation rate at acidic pH may not necessarily be effective Increase the proportion of free human IL-8. We can readily understand that, for example, even if the proportion of free human IL-8 improves from 99.9% to 99.99%, the magnitude of the improvement may not be significant.

According to the general chemical equilibrium theory, when anti-IL-8 antibody coexists with human IL-8, and its 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 ratio of free human IL-8 decreases. On the other hand, when the concentration of the antibody is low, when the concentration of the antigen is low or when the dissociation constant (KD) is large, the complex is not easily formed, and the proportion of free human IL-8 increases.

In this experiment, when the dose of H89/L118 was 8 mg/kg, the rate of elimination of human IL-8 was slower than when the dose of this antibody was 2 mg/kg. This therefore implies that in endosomes, the proportion of free human IL-8 will be lower when the antibody is administered at 8 mg/kg than when the antibody is administered at 2 mg/kg. The reason for the decrease may be that increasing the antibody dose in endosomes by a factor of 4, thus promoting the formation of IL-8-antibody complexes in endosomes. That is, in the group in which the dose of antibody was increased, the ratio of free human IL-8 in the endosome decreased, and thus the rate of elimination of human IL-8 decreased. It also suggested that the dissociation constant (KD) of H89/L118 at acidic pH was not sufficient to achieve almost 100% free human IL-8 when the antibody was administered at a dose of 8 mg/kg. More specifically, in the case of an antibody with a larger dissociation constant (KD) (weaker binding) in acidic pH conditions, almost 100% free IL-8 can be achieved even when the antibody is administered at a dose of 8 mg/kg. In the state of IL-8, the elimination rate of human IL-8 was equivalent to that when the antibody dose was 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 the endosome, it is not particularly limited, and we can verify whether there is room to increase the elimination of this antigen in vivo. degree of effect. For example, a method comparing the rate of elimination of human IL-8 using a novel pH-dependent IL-8 binding antibody with H89/L118, wherein the novel antibody has a weaker binding affinity at acidic pH than H89/L118 and /or an increased dissociation rate at acidic pH. If the novel pH-dependent IL-8 binding antibody described above shows an equivalent elimination rate of human IL-8 to H89/L118, this implies that at acidic pH, the binding affinity and/or dissociation rate of H89/L118 is sufficient to achieve at The endosome level is almost 100% free human IL-8 ratio. On the other hand, when the novel pH-dependent IL-8 binding antibody described above shows a higher elimination rate of human IL-8, it is suggested that the binding affinity and/or dissociation rate of H89/L118 at acidic pH has room for improvement. [Example 12]

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

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

Some results are shown in Table 17. Antibodies H553/L118 including H553-IgG1 (SEQ ID NO:90) as heavy chain and L118-kOMT as light chain, and antibodies including H496-IgG1 (SEQ ID NO:101) as heavy chain and L118-kOMT as 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 was introduced into the heavy chain of H89/L118, had improved binding affinity for human IL-8 at neutral pH compared to H89/L118, but at acidic pH, human IL-8 8 Binding affinity was little changed. More specifically, the R57P modification introduced into H89/L118 is a modification that increases the binding affinity of human IL-8 only at neutral pH, but does not change the binding affinity at acidic pH. Also, H553/L118, generated by introducing Y55H modification into the heavy chain of H496/L118, had maintained or slightly enhanced binding affinity at neutral pH, but on the other hand, compared to H89/L118, at acidic pH The binding affinity is reduced. That is, when a combination of two amino acid modifications, ie, Y55H and R57P to H89/L118, was introduced, the reduced binding affinity at acidic pH was further enhanced while the binding affinity at neutral pH was maintained or slightly enhanced.

(12-2) Mouse PK assay using H553/L118 H553/L118 was used to assess the rate of human IL-8 elimination in mice by a method similar to that of Example 11-2. The resulting data on human IL-8 concentrations 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, no major differences were observed between H553/L118 and H89/L118 when comparing data from mice administered 2 mg/kg antibody; but when comparing data from mice administered 8 mg/kg antibody, It was demonstrated that H553/L118 accelerated the elimination of human IL-8 by a factor of 2.5 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 antigen elimination rate was observed with increasing antibody dose for H89/L118 .

Without being particularly limited, the reasons for obtaining such results can be discussed as follows. When this antibody was administered at 2 mg/kg and 8 mg/kg, H533/L118 showed equivalent elimination rates of human IL-8. The proportion of free IL-8 shown in endosomes could reach levels close to 100% because IL-8 binding by H553/L118 at acidic pH was sufficiently weak even at 8 mg/kg administration. 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 attenuated at a high dose of about 8 mg/kg. On the other hand, even at a high dose of 8 mg/kg, H553/L118 still achieved the maximum effect of eliminating human IL-8.

(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 to 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, it is stably maintained (the IL-8 neutralization of this antibody) 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 methods were used to evaluate the stability of these antibodies in mouse plasma.

Mouse plasma was collected from blood of C57BL/6J (Charles River) and 200 μL of 200 mM PBS (Sigma, P4417) was added to 800 μL of mouse plasma to make 1 mL according to methods known in the art. Moreover, sodium azide (sodium azide) was added as an antibacterial agent at a final concentration of 0.1%. 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 was collected as a starting sample. The remaining samples were stored at 40°C. After storage for 1 and 2 weeks, a part of each sample was collected and used as the sample stored for 1 week and the sample stored for 2 weeks. All samples were frozen at -80°C and stored until each analysis was performed.

Their human IL-8 neutralizing activity was then assessed with 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, an artificial cell line to transmit signals in human IL-8- to emit chemical electricity luminescence. Although not particularly limited, the neutralizing activity of human IL-8 possessed by anti-human IL-8 antibodies can be assessed using such cells. When human IL-8 was added to the cell culture, a certain amount of chemiluminescence was visualized in a manner dependent on the concentration of added human IL-8. When human IL-8 and anti-human IL-8 antibody are added to the culture medium together, when the anti-human IL-8 antibody binds to human IL-8, human IL-8 signaling can be blocked. Therefore, the chemiluminescence caused by the addition of human IL-8 can be inhibited by the anti-human IL-8 antibody, and the chemiluminescence will be weaker than without the addition of the 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 neutralization activity of human IL-8 carried by the antibody decreases, the degree of chemiluminescence increases.

The same is true for an antibody that has been added to mouse plasma and stored for a certain period of time. If the neutralizing activity of this antibody is not changed due to storage in mouse plasma, the degree of the above-mentioned chemiluminescence before and after storage will not be the same. should be changed. On the other hand, if the neutralizing activity of the antibody is reduced by preservation in mouse plasma, the degree of chemiluminescence using the preserved antibody will increase compared to before preservation.

Next, it was examined whether the antibody stored in the mouse plasma maintains the neutralizing activity using the above-mentioned cell line. Cell lines were first suspended in AssayComplete(tm) Cell Plating 0 Reagent, and then seeded in a 384-well plate at 5000 cells/well. One day after the start of cell culture, the following experiments were performed to determine the concentration of human IL-8 to be added. Serial dilutions of human IL-8 solutions at 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 the detection reagent according to the experimental procedure of the product, and use the chemiluminescence detector to detect the relative chemiluminescence level. From the results, the reactivity of cells to human IL-8 was confirmed, and the human IL-8 concentration 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.

The neutralizing activity of the antibody contained therein was then evaluated using the above-mentioned mouse plasma to which the anti-human IL-8 antibody had been added. Human IL-8 at a concentration determined as above and mouse plasma containing the anti-human IL-8 antibody described above were added to the cell culture. The amount of mouse plasma to be added was determined to contain gradient concentrations of anti-human IL-8 antibody ranging from 2 μg/mL (13.3 nM) to 0.016 μg/mL (0.1 nM). The detection reagent is then added according to the product experimental procedure, and the relative chemiluminescence level is detected using a chemiluminescence detector.

Here, the relative value of the relative chemiluminescence level of each antibody concentration is defined as follows: the average relative chemiluminescence level when human IL-8 and antibody are not added to the well is 0%, and only human IL-8 is added but The average relative chemiluminescence level with no 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 25 A shows the results from the starting sample (mouse plasma without preservative treatment), and Figure 25 B shows preservation at 40 Results for samples that were stored at 40°C for 1 week, and Figure 25C shows the results for samples stored at 40°C for 2 weeks.

Results For Hr9 and H89/L118, no difference in 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 the neutralizing activity of human IL-8 after storage for 2 weeks. Therefore, the human IL-8 neutralizing activity of H553/L118 in mouse plasma is more easily reduced 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]

Production of antibodies with reduced predicted immunogenicity scores using an in silico system (13-1) Predicted immunogenicity scores of various IL-8-binding antibodies Antidrug antibody (ADA) production affects the potency and pharmacokinetics of therapeutic antibodies, and sometimes causes severe side effects; therefore, the clinical utility and drug efficacy of therapeutic antibodies may be limited by the production of ADA. The immunogenicity of therapeutic antibodies is known to be influenced by a number of factors, and there have been numerous reports describing the importance of effector T cell epitopes in therapeutic antibodies.

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

Here EpiMatrix was used to calculate the immunogenicity score of each anti-IL-8 antibody. EpiMatrix is a system for predicting the immunogenicity of a protein of interest. It automatically designs the sequence of peptide fragments by cutting the amino acid sequence of the protein to be predicted immunogenicity by 9 amino acids, and then calculates their corresponding 8 Binding capacity of major MHC class II dual 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 for the heavy and light chains of each anti-IL-8 antibody, calculated in the manner described above, 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 shown in the "tReg Adjusted Epx score" column. Tregitope is a peptide fragment sequence that occurs primarily in native antibody sequences and is thought 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]

According to 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 to reduce.

Furthermore, using EpiMatrix, the frequency of ADA production of predicted antibody molecules can be compared as a whole by considering the heavy and light chain fractions of the actual frequency of ADA production by various commercially available antibodies. The results of performing such an analysis are shown in FIG. 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 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 was 10.42% for hWS-4, a known humanized anti-human IL-8 antibody, but the newly identified H89/L118 (5.52%), H496 The frequencies of /L118 (4.67%) or H553/L118 (3.45%) were significantly lower compared to hWS-4.

(13-2) Manufacture of modified antibodies with lower predicted immunogenicity scores As mentioned above, compared with hWS-4, H89/L118, H496/L118, and H553/L118 have lower immunogenicity scores; but from Table 19, it can be seen that the immunogenicity scores of heavy chains are higher than those of light chains, 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 for amino acid modifications that could reduce the immunogenicity fraction. As a result of hard search, 3 variants were found, namely H496v1, in which the alanine at position 52c according to the Kabat numbering method was substituted with aspartic acid, and H496v2, in which the glutamic acid at position 81 according to the Kabat numbering method was substituted with Threonine, H496v3, in which serine at position 82b according to the Kabat numbering is substituted with aspartic acid. Furthermore, H1004 containing all these 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]

The three heavy chains, H496v1, H496v2 and H496v3, all contained a single modification, showing a smaller immunogenicity fraction than H496. Also 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 in combination with H1004. Therefore, when calculating the immunogenicity score, both the L118 combination and the L395 combination were used. As shown in Table 20, H1004/L118 and H1004/L395, both heavy and light chains, also showed very low immunogenicity scores.

The frequency of ADA production by these combinations was then predicted in a manner similar to that of 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 frequency of ADA production, with a predicted value of 0%.

(13-3) Measurement of IL-8 binding affinity of H1004/L395 H1004/L395, an antibody comprising H1004-IgG1m (SEQ ID NO:91) as the heavy chain and L395-kOMT (SEQ ID NO:82) as the light chain, was produced. The binding affinity of H1004/L395 for human IL-8 was measured using a BIACORE T200 (GE Healthcare) as described below.

The following 2 running buffers were used and measurements were performed at 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) by amine coupling method, and the antibody of interest was captured. Human IL-8 was then interacted with the antibody captured on the sensor chip by injecting diluted human IL-8 solution or running buffer (as a reference solution). For the running buffer, either of the above buffers was used, and human IL-8 was diluted with each buffer. To regenerate the sensor wafer, 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) were used. The KD (M) of human IL-8 for each antibody was calculated from the kinetic parameters calculated from the sensorgram obtained by the measurement, the association 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 an equivalent KD for human IL-8 at neutral pH, but has a larger KD and koff at acidic pH; The nature of IL-8 dissociation.

[實施例14] [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 From the evaluation of Example 13, H1004/L395 was obtained, which has pH-dependent IL-8 binding capacity and a lower immunogenicity score. Elaborate investigations were then carried out to generate variants with these favorable 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]

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

Human IL-8 binding affinity at neutral and acidic pH conditions was measured in a manner similar to that of 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 the antibody in terms of IL-8 binding when stored in PBS was then assessed as follows.

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

Then, BIACORE measurement of IL-8 binding affinity was carried out as follows using the starting sample and the sample for the heating accelerated test.

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

An appropriate amount of Protein A/G (PIERCE) was immobilized on Sensor chip CM4 (GE Healthcare) by amine coupling and the antibody of interest was captured. Human IL-8 was then interacted with the antibody captured on the sensor chip by injecting 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 wafer, 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) were used. 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 as well as the sample for the heating accelerated test. Also, the ratio of the binding level of human IL-8 in the starting sample relative to the sample in the heat-accelerated test was calculated.

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

[Table 22]

Using the above test, H1009/L395 was obtained, which is an antibody comprising 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 had slightly improved binding affinity for human IL-8 at neutral pH, but decreased binding affinity at acidic pH, i.e., pH-dependent further strengthen. Also when exposed to severe conditions, such as 50°C in PBS, H1009/L395 showed slightly improved stability in IL-8 binding 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 assessment of H1009/L395 It was then assessed whether the IL-8 neutralizing activity of H1009/L395 was stably maintained in mouse plasma in a manner similar to Example 12-3. Here, H1009/L395-F1886s, which will be detailed in Example 19 below, was 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 native human IgG1. The variable region of H1009/L395, especially the region surrounding the HVR, is responsible for human IL-8 binding and the IL-8 neutralizing activity of the antibody, and the introduction of modifications to the constant region is not thought to affect these properties.

Stability assessment in mouse plasma was performed as follows. 150 μL of 200 mM phosphate buffer (pH 6.7) was added to 585 μL of mouse plasma. Sodium azide was then added as an antibacterial agent at a final concentration of 0.1%. Each antibody (Hr9, H89/L118 or H1009/L395-F1886s) was added to the above mouse plasma at a final concentration of 0.4 mg/mL. At this point, a portion of the sample was collected as a starting sample. Store the rest of the samples at 40°C. After storage for 1 and 2 weeks, a part of each sample was collected and used as the sample stored for 1 week and the sample stored for 2 weeks. All samples were frozen at -80°C and stored until each analysis was performed.

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

The results of the 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 storage in mouse plasma), and Figure 28B shows protection against storage at 40°C Results for 1 week samples, Figure 28C shows results for samples stored at 40°C for 2 weeks.

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

(14-3) Mouse PK assay using H1009/L395 The rate of H1009/H395 elimination of human IL-8 in mice was assessed as follows. H1009/L395, H553/L118 and H998/L63 were used as antibodies. Administration to mice, collection of blood and measurement of human IL-8 concentration in mouse plasma were carried out by the method shown in Example 11.

The results of human IL-8 concentration 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 dose 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. The clearance (CL) value, which quantitatively represents the rate of elimination of human IL-8 from mouse plasma, was shown to be approximately 30-fold higher than that of 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 antigen is maintained at a nearly constant concentration, and the production rate and elimination rate of the antigen will also be maintained at an almost constant value. When the antibody is administered under such conditions, even though the rate of antigen production is unaffected, the rate of antigen elimination may be altered by the formation of a complex between the antigen and the antibody. In general, since the rate of elimination of the antigen is greater than the rate of elimination of the antibody, in this case, the rate of elimination of the antigen that has complexed with the antibody is reduced. As this rate of antigen elimination decreases, the concentration of antigen in plasma increases, but the extent of the increase in this case can also be defined by the ratio of the rate of elimination when the antigen is present alone relative to that when the antigen forms a complex. That is, if the elimination rate when the complex is formed is reduced by a factor of 10 compared to the elimination rate when the antigen is present alone, the antigen concentration 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 administration to the organism can be defined as the antigen CL before and after antibody administration under each condition.

Here, there was an approximately 30-fold difference in the CL of human IL-8 when H998/L63 and H1009/L395 were administered, suggesting that when these antibodies were administered to humans, the increased level of human IL-8 concentration in plasma would There is about a 30-fold difference. In addition, the 30-fold difference in plasma human IL-8 concentration means that the amount of antibody required to completely block the biological activity of human IL-8 also differs by about 30-fold under each condition. That is, compared with H998/L63, H1009/L395 can block the biological activity of IL-8 in plasma by about 1/30, and the amount of this antibody is very small. When H1009/L395 and H998/L63 were administered to humans individually at the same dose, H1009/L395 could 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 having 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 excellent effect of H1009/L395 shown in Example 14 in eliminating human IL-8 by a factor of 30 is a surprising 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 antibody-antigen complexes into cells. That is, if the rate of pH-dependent antigen-binding antibody uptake into cells is increased, the antigen-eliminating effect of pH-dependent antibodies increases when antigen-antibody complexes are formed, compared to when no complexes are formed. Methods for increasing the rate of antibody uptake into cells are known, including: conferring FcRn-binding ability of antibodies at neutral pH conditions (WO 2011/122011), methods for enhancing the binding ability of antibodies to FcγRs (WO 2013/047752) , and methods for facilitating the formation of complexes comprising multivalent antibodies and multivalent antigens (WO 2013/081143).

However, the above technique is not used in the constant region of H1009/L395. Also, although IL-8 is known to form a homodimer, the human IL-8 bound by H1009/L395 is found to exist in a unitary form, because H1009/L395 recognizes the homobinary of human IL-8 The body forms the surface. Thus the antibody does not form multivalent complexes.

More specifically, although the above techniques were 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 are only possible speculations of the inventor based on the technical background, and the content of the disclosure of C is not limited to the following discussion content.

Human IL-8 is a protein with a high electric point (pI), and the theoretical isoelectric point calculated by known methods is about 10. That is, under neutral pH conditions, human IL-8 is a protein whose charge is biased toward the positive side. The pH-dependent IL-8-binding antibody represented by H1009/L395 is also a protein whose charge is biased to the positive side, 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 electrical point and is originally rich in positive electricity) with human IL-8 (has a high electrical point) is higher than that of H1009/L395 alone.

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

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

Evaluation of the amount of antibody bound to extracellular matrix by ECL (electroluminescence) method 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 at 5 μL per well into a MULTI-ARRAY 96-well plate, high binding, bare plate (manufactured by Meso Scale Discovery: MSD), and immobilized overnight at 4°C. It was then blocked with 150 mM NaCl, 0.05% Tween20, 0.5% BSA, 0.01% NaN ₃ in 20 mM ACES buffer (pH 7.4).

Antibodies to be evaluated were prepared as follows. Separately added antibody samples were 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), followed by buffer 2 (20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20, 0.1% BSA and 0.01% NaN3, pH 7.4) was prepared by diluting to a final concentration of ₃ μg/mL.

On the other hand, an antibody sample to be added as a complex with human IL-8 was prepared by adding human IL-8 at a molar concentration 10 times that of the antibody to the antibody sample, and then diluting each antibody with buffer-1 , so that the antibody concentration was 9 μg/mL, respectively, and then further diluted with Buffer-2 to a final antibody concentration of 3 μg/mL. At this point, the human IL-8 concentration was about 0.6 μg/mL. It was shaken for 1 hour at room temperature for complex formation.

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

The results are shown in Figure 30. Interestingly, all anti-IL-8 antibodies such as H1009/L395 showed hardly any binding to the extracellular matrix when the antibody was alone (-IL8), but when complexed with human IL-8 ((+hlL8 )), binds to the extracellular matrix.

As mentioned above, the properties of anti-IL-8 antibodies that acquire affinity for extracellular matrix by binding to human IL-8 have not been elucidated. Again, without limitation, combining such properties with pH-dependent IL-8 binding antibodies 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 determine whether a complex of human IL-8 and pH-dependent IL-8-binding antibody was formed, and whether the uptake of the complex into cells was increased in mice.

An antibody variant comprising the variable region of H1009/L395 and an Fc region lacking binding affinity for various Fc receptors was first made. Specifically, as a modification to delete the binding ability to human FcRn under acidic pH conditions, the isoleucine at position 253 of the heavy chain H1009-IgG1 was substituted with alanine and serine at position 254 according to EU numbering. for aspartic acid. Also, as a modification to delete binding to mouse FcγR(s), leucine at position 235 was substituted with arginine, glycine at position 236 was substituted with arginine, and serine at position 239 was substituted with lysine. acid. H1009-F1942m (SEQ ID NO: 93) was made as a heavy chain containing 4 of these modifications. Also, H1009/L395-F1942m was produced, which includes H1009-F1942m as a heavy chain and L395-kOMT as a light chain.

Antibodies with this Fc region lack FcRn binding affinity under acidic pH conditions and do not transfer from endosomes to plasma. Therefore, such antibodies are eliminated from plasma in vivo faster than antibodies comprising the native Fc region. In this case, when the antibody including the native Fc region is taken up into the cell, only part of it that is not salvaged by FcRn will be degraded after transfer to the lysosome, but if the antibody includes an Fc that does not have FcRn binding affinity region, all antibodies taken up into the cell will be degraded in the lysosome. More specifically, antibodies comprising such modified Fc regions may be eliminated from plasma at a rate that is equivalent to the rate of entry into cells. That is, the rate of intracellular uptake of an antibody whose binding affinity for FcRn has been deleted can also be confirmed by measuring the rate of elimination of this antibody from plasma.

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

For when anti-human IL-8 antibody alone was administered to human FcRn gene-transfected mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice; Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)), and the respective 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 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 individually administered to the tail vein at 10 mL/mL. kg is administered once. In this case, since there is a sufficient excess of anti-human IL-8 antibody to human IL-8, it is believed that almost all human IL-8 binds to this antibody. Blood was collected 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 minutes to obtain plasma. Separated plasma was cryopreserved at -20°C or lower until measurement.

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

Anti-human IL-8 antibody (prepared in-house) containing mouse IgG constant region was then added to the plate and allowed to react for 1 hour at room temperature. Anti-mouse IgG (BECKMAN COULTER) labeled with Ruthenium-SULFO-TAG NHS Ester (Meso Scale Discovery) was again added to the plate and allowed to react for 1 hour. Measurements were then performed using a SECTOR Imager 2400 (Meso Scale Discovery) immediately after dispensing Read Buffer T(x1) (Meso Scale Discovery) to the plate. Anti-human IL-8 antibody concentrations were calculated from the responses in the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

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

[Table 24]

Compared to the intracellular uptake rate of H1009/L395-F1942m, the intracellular uptake rate of the complex of H1009/L395-F1942m and human IL-8 showed at least a 2.2-fold increase. It is marked here as "at least 2.2 times" for the following reasons: one is to include the possibility of 5 times, 10 times or 30 times the actual value. Since the rate of elimination of human IL-8 was relatively fast 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 plasma decreased rapidly after administration. More specifically, even if human IL-8 was administered at the same time, not all H1009/L395-F1942m in the plasma were present in the human IL-8-bound form, in fact, most of them were already in the free form after about 7 hours of administration. exist. Since the uptake rate was evaluated under such conditions, the intracellular uptake rate of the complex of H1009/L395-F1942m and human IL-8 was actually increased by 5-fold, 10-fold or 30-fold even compared to the uptake rate of H1009/L395-F1942m times, but the results are only partially reflected in the experimental system; so the effect presented may be 2.2 times and so on. Therefore, from the obtained results, 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 the 2.2-fold increase.

It is not particularly limited, and the following inferences can be obtained from the findings so far. When H1009/L395, which is a pH-dependent IL-8-binding antibody, forms a complex with human IL-8, the complex has a higher isoelectric point and is more positively charged than the antibody alone. At the same time, the affinity of the complex to the extracellular matrix increases more than the affinity of the antibody alone. For example, raising the isoelectric point and enhancing extracellular matrix binding are thought to be factors that promote the uptake of antibodies into living cells. Also from the mouse experiment, the intracellular uptake of the complex of H1009/L395 and human IL-8 was increased by 2.2 times or more than the uptake rate of H1009/L395. From the above, theoretical explanations, as well as in vitro properties and in vivo phenomena consistently support the hypothesis that H1009/L395 forms a complex with human IL-8 to facilitate uptake of the complex into cells and lead to a marked increase in the elimination of human IL-8.

Some antibodies against IL-8 have been reported so far, but there is no report on the increased binding affinity to extracellular matrix and increased uptake of the complex into cells after complexing with IL-8.

In addition, based on the observation that when the antibody and IL-8 form a complex, the intracellular uptake of the anti-IL-8 antibody increases, it is considered that the anti-IL-8 antibody that has formed a complex with IL-8 in the plasma quickly enters the cell , while the free antibody that is not complexed with IL-8 tends 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 the IL-8 molecule in the cell and return to the outside of the cell, which can then bind to another IL-8 8 molecules; therefore, the increased intracellular uptake after the formation of the complex will have a stronger effect of eliminating IL-8. That is, selecting an anti-IL-8 antibody with increased binding to the extracellular matrix or an anti-IL-8 antibody with increased uptake into cells can also be another embodiment of Disclosure C. [Example 17]

Immunogenicity prediction of pH-dependent IL-8 binding antibody H1009/L395 using an in silico system The immunogenicity fraction and ADA-producing frequency of H1009/L395 were then predicted in a similar manner to Example 13-1. The results are shown in Table 25 and FIG. 32 . In Figure 32, H1009/L395 is labeled "H1009L395".

[Table 25]

The results in Table 25 show that H1009/L395 and H1004/L395 had the same degree of low immunogenicity scores. Also, in Figure 32, the predicted ADA generation frequency for H1009/L395 was 0%, and the same for H1004/L395.

Therefore, the predicted immunogenicity of H1009/L395 is greatly 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 variant with enhanced FcRn binding in acidic pH conditions As described in the previous examples, the pH-dependent IL-8 binding antibody H1009/L395 is an antibody with superior properties among antibodies with native IgG1 as a constant region. Such antibodies can also be used as antibodies containing amino acid substitutions in the constant region, as shown in Example 5, comprising an Fc region with enhanced 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) Production of H89/L118 Fc region-modified antibody with enhanced FcRn binding at acidic pH Various modifications for enhancing FcRn binding described in Example 5-1 were introduced into the Fc region of H89/L118. Specifically, the following variants were made by introducing the modifications used in F1847m, F1848m, F1886m, F1889m, F1927m, and F1168m into the Fc region of H89-IgG1: (a) H89/L118-IgG1, including H89-IgG1m (SEQ ID NO:94) as heavy chain and L118-KOMT as light chain; (b) H89/L118-F1168m includes H89-F1168m (SEQ ID NO:95) as heavy chain and L118-KOMT as light chain; (c) ) H89/L118-F1847m, including H89-F1847m (SEQ ID NO:96) as a heavy chain and L118-KOMT as a light chain; (d) H89/L118-F1848m, including H89-F1848m (SEQ ID NO:97) as a Heavy chain and L118-KOMT as light chain; (e) H89/L118-F1886m, including H89-F1886m (SEQ ID NO:98) as heavy chain and L118-KOMT as light chain; (f) H89/L118-F1889m, including H89-F1889m (SEQ ID NO:99) as heavy chain and L118-KOMT as light chain; and (g) H89/L118-F1927m including H89-F1927m (SEQ ID NO:100) as heavy chain and L118-KOMT as a light chain. The Malay monkey PK assay using these antibodies was carried out according to the following formula.

(18-2) Novel Malay monkey PK assay for antibodies containing Fc region variants The biokinetics of the anti-human IL-8 antibody was assessed after administration of the anti-human IL-8 antibody to Malay monkeys. The anti-human IL-8 antibody solution was administered once intravenously 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 minutes to obtain plasma. Separated plasma was cryopreserved at -60°C or lower until measurement.

Anti-human IL-8 antibody concentrations in Malay monkey plasma were measured by electrochemiluminescence. Anti-hKappa Capture Ab (Antibody Solutions) was first dispensed into MULTI-ARRAY 96-well plates (Meso Scale Discovery) and stirred for 1 hour at room temperature. Anti-human antibody immobilized plates were then prepared by blocking with 5% BSA (w/v) in PBS-Tween for 2 hours at room temperature. Samples for calibration curves were prepared, which contained anti-human IL-8 antibody concentrations in plasma at 40.0, 13.3, 4.44, 1.48, 0.494, 0.165, and 0.0549 μg/mL, and samples for measurement in Malay monkey plasma were prepared. Dilute 500-fold or more. 50 μL of the solution was dispensed into each well of the anti-human antibody immobilized plate, and the solution was stirred at room temperature for 1 hour. Anti-hKappa Reporter Ab, Biotin conjugate (Antibody Solutions) was then added to the aforementioned plate and allowed to react for 1 hour at room temperature. 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 dispensed to the plate, using a SECTOR Imager 2400 (Meso Scale Discovery) ) to measure immediately. Anti-human IL-8 antibody concentrations were calculated from the responses in the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

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

[Table 26]

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

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

On the other hand, IL-8 is a soluble interleukin, and the use of an anti-IL-8 antibody as a medicine is mainly expected to show a pharmacological action by neutralizing the function of IL-8 at a site where IL-8 is excessively present. The site where IL-8 is present in excess is not particularly limited, and may be, for example, an inflamed site. It is known that various immune cells are generally aggregated and activated at such an inflamed site. It is not always beneficial to transmit unexpected activation signals to these cells via Fc receptors and to induce activities such as ADCC and ADCP in the unintended cells. Therefore, it is not particularly limited, but from the viewpoint of safety, it is preferable that the anti-IL-8 antibody administered in vivo has a 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 male monkey FcγRs, amino acid modifications were further introduced into the Fc region of H1009/L395-F1886m. Specifically, H1009-F1886s (SEQ ID NO:81) was made by implementing the following substitutions to the H1009-F1886m heavy chain: according to EU numbering, L at position 235 was substituted with R, G at position 236 was substituted with R, S at position 239 is substituted with K. Likewise, H1009-F1886m was replaced by the following to make H1009-F1974m (SEQ ID NO:80): According to EU numbering, the L at position 235 was replaced with R, and the G at 236 was replaced with R, which would be according to EU numbering, The region from positions 327 to 331 was substituted with the native human IgG4 sequence. H1009/L395-F1886s and H1009/L395-F1974m were produced as antibodies with these heavy chains and with L395-kOMT as the light chain.

(19-2) Confirmation of affinity for various human FcγRs The affinity of H1009/L395-F1886s or H1009/L395-F1974m for soluble FcγRIa or FcγRIIIa was then confirmed in humans or Malay monkeys as follows.

Assays were performed using a BIACORE T200 (GE Healthcare) for binding of H1009/L395-F1886s or H1009/L395-F1974m to FcyRIa or the soluble form of FcyRIIIa in humans or Malay monkeys. Soluble FcγRIa and FcγRIIIa in both human and Malay monkeys were prepared as His-tagged molecules according to methods known to those of ordinary skill in the art. An appropriate amount of rProtein L (BioVision) was immobilized on a sensor chip CM4 (GE Healthcare) by amine coupling and the antibody of interest was captured. Soluble FcyRIa or FcyRIIIa was then injected with running buffer (as a reference solution) to interact with the antibody captured on the sensor wafer. Soluble FcyRIa or FcyRIIIa was also diluted with HBS-EP+ (GE Healthcare) as running buffer. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were carried out at 20°C.

The results are shown in Figure 34. Here, the labels of human FcyRIa, human FcyRIIIa, Malay monkey FcyRIa and Malay monkey FcyRIIIa are hFcyRIa, hFcyRIIIa, cynoFcyRIa and cynoFcyRIIIa in this 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 of the FcγRs.

(19-3) Mouse IL-8 Elimination Assay for Fc Variants Then, for H1009/L395-F1886s and H1009/L395-F1974m, the elimination rate of human IL-8 and the retention of antibody in mouse plasma were confirmed according to the following experiments. Three doses of H1009/L395-F1886s, 2 mg/kg, 5 mg/kg, and 10 mg/kg were used for evaluation here, so the effect of increasing antibody doses on H1009/L395-F1886s can also be assessed.

Simultaneous administration of human IL-8 to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mice; Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) and anti-human IL-8 antibody, the biokinetics of human IL-8 was assessed. A mixed solution of 10 mL/kg 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 in a single dose via the tail vein. In this case, since the anti-human IL-8 antibody is present in sufficient excess over human IL-8, almost all of the human IL-8 is considered to have 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 minutes to obtain plasma. Separated plasma was cryopreserved at -20°C or lower until measurement.

Human IL-8 concentrations in mouse plasma were measured similarly to Example 11. The results of human IL-8 concentration 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 groups administered at a dose of 2 mg/kg, H1009/L395 containing the Fc region of native IgG1 and H1009/L395-F1886s containing the modified Fc region showed the same effect of eliminating human IL-8.

Then, when the dose of H1009/L395-F1886s antibody was changed, although the IL-8 concentration in plasma was slightly different one day after administration, there was no significant difference in the clearance rate of human IL-8 between 2 mg/kg and 10 mg/kg doses. difference. This strongly suggests that the antibody comprising the variable region of H1009/L395 showed sufficient IL-8 depletion even though the antibody was administered at high doses.

[Table 27]

(19-4) Malay monkey PK assay of Fc variants Plasma retention of antibodies in Malay monkeys was then validated using H1009/L395-F1886s or H1009/L395-F1974m as follows.

The biokinetics of the anti-human IL-8 antibody was evaluated in the case of administration of the anti-human IL-8 antibody to Malay monkeys alone, and in the case of the simultaneous administration of the human IL-8 and the anti-human IL-8 antibody. The anti-human IL-8 antibody solution (2 mg/mL) or the mixed solution of human IL-8 (100 μg/kg) and anti-human IL-8 antibody (2 mg/kg) was administered intravenously at a single dose of 1 mL/kg. Inland 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 minutes to obtain plasma. Separated plasma was cryopreserved at -60°C or lower until measurement.

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

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

Also, when H1009/L395-F1886s and human IL-8 were administered simultaneously, the changes in plasma concentrations were equivalent to those of the antibody administered alone. Without being particularly limited, the following discussion can be derived from the above findings. As described 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 then transfer to lysosomes, where they are degraded by various degrading 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 the plasma through FcRn in the endosome; as long as there is sufficient FcRn salvage function, even if the intracellular uptake rate is accelerated, the plasma retention will not be affected. Here, even if H1009/L395-F1886s and human IL-8 were administered simultaneously to Malay monkeys, plasma retention was not affected. This suggests the possibility that even with an increased rate of antibody uptake into cells against H1009/L395-F1886s, it is still sufficiently rescued by FcRn to allow it to return to plasma.

Yet another Fc variant, H1009/L395-F1974m, also showed equivalent plasma retention relative to H1009/L395-F1886s. These Fc variants have been introduced with various modifications as above that reduce binding capacity for various Fc[gamma]Rs, but have not been shown to have an effect on plasma retention of the antibody itself. From the above, the plasma retention of H1009/L395-F1886s and H1009/L395-F1974m in Malay monkeys was significantly prolonged, which was extremely satisfactory compared to the antibody with the native IgG1 Fc region.

[Table 28]

As demonstrated in the above examples, H1009/L395 is the first to significantly increase the rate of elimination of human IL-8 in vivo by including pH-dependent IL-8 binding capacity and complex formation with IL-8 for rapid uptake into cells of antibodies. Furthermore, the binding affinity of this antibody to IL-8 at neutral pH is also increased compared to the known hWS-4 antibody, and this antibody can more strongly neutralize human IL-8 in 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 constructed based on Hr9 has a significantly improved production level compared to hWS-4, and is an antibody suitable for production from the viewpoint of production level. Furthermore, this antibody showed a very low score for immunogenicity in computer-simulated immunogenicity predictions, which is a significantly lower score compared to the known hWS-4 antibody and several other commercially available antibodies. It can be expected that H1009/L395 is not easy to generate ADA in humans and can be used safely for a long time. Therefore, compared with the known anti-human IL-8 antibody, H1009/L395 shows improvement in various aspects, and is very useful as a pharmaceutical.

As described above, H1009/L395 having the Fc region of native IgG is sufficiently useful; however, variants of H1009/L395 including a functionally improved Fc region can also be suitably used as antibodies for enhanced use. Specifically, FcRn binding can be increased under acidic pH conditions to prolong plasma retention and maintain the effect for a long time. Variants including the introduction of modifications to the Fc region to reduce the binding ability to FcyRs can also be used as highly safe therapeutic antibodies, avoiding unwanted immune cell activation and producing cytotoxic activity upon administration to an organism. As such Fc variants, the use of F1886s or F1974m utilized herein is particularly advantageous, but not limited to these Fc variants; therapeutics including other modified Fc regions may be used as long as the Fc variants have similar functions Antibodies as an embodiment of Disclosure C.

Therefore, the revealing C antibodies including H1009/L395-F1886s and H1009/L395-F1974m generated by the careful research of the present invention can maintain the condition that the biological activity of human IL-8 is safely and strongly inhibited for a long time. Here, it is understood that the level of known anti-IL-8 antibodies cannot be achieved, 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. Humanized anti-factor IXa/factor X bispecific antibody F8M (Q499-z121/J327-z119/L404-k: H chain (SEQ ID NO: 330)/H chain (SEQ ID NO) described in WO2012/067176 was used :331)/common L chain (SEQ ID NO:332)) In this example, 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 as anti-factor IXa/factor X bispecific antibodies according to the method of Reference Example 2: (a) F8M-F1847mv, a conventional antibody, including F8M-F1847mv1 (SEQ ID NO: 323) with F8M-F1847mv2 (SEQ ID NO: 324) as a heavy chain and F8ML (SEQ ID NO: 325) as a light chain; (b) F8M-F1868mv, a conventional antibody, including F8M-F1868mv1 (SEQ ID NO: 323) : 326) with F8M-F1868mv2 (SEQ ID NO: 327) as a heavy chain and F8ML (SEQ ID NO: 325) as a light chain; and (c) F8M-F1927mv, a conventional antibody including F8M-F1927mv1 (SEQ ID NO: 326) 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 sequences mentioned in Example 5 with respect to enhancing FcRn binding and reducing 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 the monoclonal antibodies, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were evaluated after intravenous administration of a single bolus at a dose of 0.6 mg/kg to male Malay monkeys, respectively. Plasma concentrations of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were determined by sandwich ELISA. Pharmacokinetic parameters were calculated using WinNonlin ver 6.4 software. As shown in Table 30, the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were 29.3 days, 54.5 days, and 35.0 days, respectively. A PK study using F8M in Malay monkeys was performed at a dose of 6 mg/kg on different days and showed a half-life of 19.4 days. It is known that F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv have longer half-lives than F8M. This suggests that the half-life of the anti-Factor IXa/X bispecific antibody may be extended by the same modification of the Fc region sequence as described in Example 5.

[實施例21] [Table 30]

[Example 21]

Evaluation of IgE clearance from plasma using pi-increasing Fab variants In order to improve the clearance of human IgE, pH-dependent antigen binding antibodies were used in this example to evaluate the substitution of the Fab portion of the antibody to increase the pi. The method of adding amino acid substitution to this antibody variable region to increase pI is not particularly limited, but can be implemented using the method disclosed in WO2007/114319 or WO2009/041643. The amino acid substitutions introduced into the variable region are preferably those that reduce the number of negatively charged amino acids (such as aspartic and glutamic acids) and increase the number of positively charged amino acids (such as arginine and lysine). By. Also, 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 side chain of the amino acid may be exposed on the surface of the antibody molecule.

(21-1) Production of antibodies with increased isoelectric point values by modifying amino acids in variable regions The antibodies to be tested are listed in Table 32 and Table 33.

The heavy chain Ab1H003 (also referred to as H003, SEQ ID NO: 144) was obtained by introducing a pI-increasing substitution H32R into Ab1H (SEQ ID NO: 38). Other heavy chain variants were also prepared according to the method shown in Reference Example 1 by introducing each of the substitutions presented in Table 32 into Ab1H. All heavy chain variants were expressed 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 evaluated pi-increasing substitutions in the light chain.

The light chain Ab1L001T (also referred to as L001, SEQ ID NO: 164) was prepared by introducing a pI-increasing substitution G16K into Ab1L. Other light chain variants were also 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) Binding assay of human FcγRIIb using BIACORE using variants with increased pI As for the produced antibodies containing Fc region variants, binding assays using BIACORE T200 (GE Healthcare) were used to conduct binding assay for soluble human FcγRIIb and antigen - Binding between antibody complexes. Soluble human FcyRIIb (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 amine-coupled to a sensor chip CM5 (GE Healthcare) using a His capture kit (GE Healthcare) to capture human FcγRIIb. Antibody-antigen complexes were then injected with running buffer (as a reference solution) and interacted with human FcyRIIb captured on the sensor wafer. 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 Soluble human FcyRIIb was diluted in each buffer. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were performed at 25°C. The analysis was performed based on the binding (RU) calculated 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. To calculate parameters, BIACORE T100 Evaluation Software (GE Healthcare) was used.

The results of the SPR analysis are summarized in Tables 32 and 33. Some variants showed improved binding to human FcyRIIb immobilized on a BIACORE sensor wafer.

Antibodies prepared by introducing pi-increasing modifications into the variable regions of antibodies are those with more positive charges in the variable regions than before the introduction of the modifications. It is therefore believed that the Coulomb interaction between the variable region (positively charged) and the sensing wafer surface (negatively charged) is enhanced by the modification with increased pi. Furthermore, such an effect can be expected to occur also on the surface of the negatively charged cell membrane; therefore, an effect of accelerating uptake into a living cell can also be expected.

Here, this variant bound about 1.2-fold or more to hFcyRIIb than the original Ab1 to hFcyRIIb, which is believed to have a strong charge effect on binding the antibody to hFcyRIIb on the sensor wafer.

Among the heavy chain variants with increased pI, antibodies with substitutions (according to Kabat numbering) of Q13K, G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G or Q105R, alone or in combination, showed higher binding to hFcyRIIb. Single amino acid substitutions or combinations of these substitutions in the heavy chain are presumed to have a strong charge effect on hFcyRIIb bound to the sensor wafer. One or more positions that would therefore be expected to increase the rate or rate of uptake into a cell in vivo by introducing a pI-increasing modification into the variable region of the antibody heavy chain may include, for example, positions 13, 15, 64, 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.

Light chain variants with increased pi-increase, alone or in combination with G16K, Q24R, A25R, S26R, E27R, Q37R, G41R, Q42K, S52K, S52R, S56K, S56R, S65R, T69R, T74K, S76R, S77R, Q79K The substituted antibody (according to Kabat numbering) showed higher binding to human FcγRIIb. Single amino acid substitutions or combinations of these substitutions in the light chain are presumed to have a strong charge effect on human FcyRIIb bound to the sensor chip. One or more positions that would therefore be expected to increase the rate or rate of uptake into a cell in vivo by introducing a pI-increasing modification into the variable region of the antibody light chain may include, for example, positions 16, 24, 25, 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) Cellular uptake of pi-increased Fab region variant-containing antibodies To assess the rate of intracellular uptake into hFcγRIIb-expressing cell lines using the manufactured antibodies containing the Fab region variant, an assay similar to (4-5) above was performed, except that the amount of antigen taken up was expressed relative to Ab1H/Ab1L (original Ab1H/Ab1L). ) value (let be 1.00) relative value representative.

Quantitative results of cellular uptake are summarized in Tables 32 and 33. Intense fluorescence from this antigen in cells was observed in various Fc variants. Here, the antigen entry into the cell is considered to have a strong charge effect when the fluorescence intensity of the antigen taken up into cells of this variant is about 1.5 times or more than the fluorescence intensity of the original Ab1.

Among the heavy chain variants with increased pI, antibodies bearing P41R, G44R, T77R, D82aN, D82aG, D82aS, S82bR or E85G substitutions (according to Kabat numbering), alone or in combination, showed greater antigen uptake into cells. Single amino acid substitutions or combined substitutions in the heavy chain are presumed to have a strong charge effect on the uptake of the antigen-antibody complex into cells. It is therefore expected to result in one or more positions at which uptake of the antigen-antibody complex into the cell is faster or greater by introducing a pi-increasing modification into the variable region of the antibody heavy chain, which may include, for example, positions 41, 44 according to the 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 substitutions of S77R or Q79K (according to the Kabat numbering) showed stronger antigen uptake into cells. Single amino acid substitutions or combinations of these substitutions in the light chain are presumed to have a strong charge effect on 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 uptake of the antigen-antibody complex into the cell by introducing a pi-increasing modification into the variable region of the antibody light chain results 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 the 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 glutamic acid, arginine or lysine, preferably arginine or lysine.

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. This antigen-antibody complex binds to human FcγRIIb expressed on the cell membrane via the Fc region of the antibody, 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; thus the antibody can dissociate from the antigen in the intracellular endosome (acidic pH conditions). Because the dissociated antigen is transported to the lysosome and accumulates, it fluoresces intracellularly. Therefore, strong intracellular fluorescence intensity is thought to indicate that uptake of antigen-antibody complexes into cells occurs faster or more.

(21-4) Evaluation of the 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 co-injection model to assess their ability to accelerate clearance of IgE from plasma. For the co-injection 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 concentrations were determined by anti-IgE ELISA. Anti-human IgE (Clonite 107, MABTECH) was first dispensed into microwell plates (Nalge nunc International) and left at room temperature for 2 hours or at 4°C overnight to prepare anti-human IgE antibody immobilized plates. Samples against the standard curve and samples and excess anti-IgE antibody (manufactured in-house) were mixed to form a homogeneous structure of immune complexes. These samples were added to the anti-human IgE antibody immobilized plate and left at 4°C overnight. These samples were then sequentially reacted with human GPC3 nucleoprotein (manufactured in-house), biotinylated anti-GPC3 antibody (manufactured in-house), 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 the IgE plasma concentration time profile in C57BL6J mice.

After administration of the Fab variant with increased pi with pH-dependent antigen binding, the plasma total IgE concentration was lower than that of the original Ab1. These results show that this antigen-antibody immune complex with a high pi variant of pH-dependent antigen binding binds more strongly to plasma membrane receptors such as FcyR, increasing cellular uptake of the antigen-antibody immune complex. This antigen taken up into cells can be efficiently released from the antibody in the endosome, accelerating the elimination of IgE. The IgE concentration of Ab1H/Ab1L007-treated mice, which showed weak potency in in vitro experiments, was higher than that of other antibodies containing Fab variants with increased pi. These results also suggest that the sensitivity of the in-vitro system using the fluorescence intensity of the InCell Analyzer 6000 described above would be higher than the sensitivity of the in-vitro BIACORE system described above for estimation of in vivo antigen clearance in plasma. [Example 22]

Evaluation of C5 clearance from plasma using pi-increased Fab variants To improve clearance of human IgE, pH-dependent antigen-binding antibodies were used in this example to assess substitutions that increase the pi of the Fab portion of the antibody.

(22-1) Preparation of C5 [Expression and purification of recombinant human C5] Recombinant human C5 (NCBI GenBank access number: NP_001726.2, SEQ ID NO: 207) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). The adjusted medium expressing human C5 was diluted with an equal volume of milliQ water and then applied to a Q-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were pooled and then the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The resulting sample was applied to a SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were pooled and applied to CHT ceramic hydroxyapatite columns (Bio-Rad Laboratories, Hercules, CA, USA). The human C5 eluate was applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing human C5 were pooled and stored at -150°C. Recombinant human C5 prepared in-house or plasma from human C5 (CALBIOCHEM, Cat#204888) was used in this study.

Expression and purification of recombinant Malayan monkey C5 (NCBI GenBank access number: XP_005580972, SEQ ID NO: 208) was performed in exactly the same way as the human part.

(22-2) Preparation of synthetic calcium library (calcium libary) The gene repertoire of antibody heavy chain variable regions was used as a synthetic human heavy chain repertoire consisting of 10 heavy chain repertoires. Germline frameworks VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59, VH4-61 were selected based on germline frequency in human B cell inventory and biophysical properties of V gene families , VH4-b, VH5-51, and VH6-1 are libraries. The synthetic human heavy chain repertoire is diverse in antibody binding sites that mimic the human B cell antibody repertoire.

Gene repertoires of antibody light chain variable regions were designed with calcium binding motifs and diversity in positions likely to contribute to antigen recognition, with reference to the human B cell antibody repertoire. The design of a gene repertoire of antibody light chain variable regions exhibiting properties for calcium-dependent binding to antigens is described in WO 2012/073992.

The combination of the heavy chain variable region library and the light chain variable region library is inserted into the phagemid vector, and the phage library is constructed, refer to (de Heard et al., Meth. Mol. Biol. 178:87-100 (2002) )). The trypsin cleavage site was introduced into the linker region between the Fab and pill proteins in the phagemid vector. Phage presented as Fab was prepared using a modified M13KO7 helper phage with a trypsin cut between the N2 and CT domains of gene III.

(22-3) Isolated calcium-dependent anti-C5 antibody The phage display library was diluted in TBS supplemented with BSA and CaCl ₂ to final concentrations of 4% and 1.2 mM, respectively. As a panning method, a conventional magnetic bead selection method was used with reference to 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). Magnetic beads using NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin coated) or streptavidin coated beads (Dynabeads M-280 Streptavidin). Human C5 (CALBIOCHEM) , Cat#204888) were labeled with EZ-Link NHS-PEG4-Biotin (PIERCE, Cat No. 21329).

For the initial round of phage selection, the phage display pool was incubated with biotinylated human C5 (312.5 nM) for 60 minutes at room temperature. Phage presenting bound Fab variants were then captured using magnetic beads.

After incubating with magnetic beads for 15 minutes at room temperature, the magnetic beads were washed three times with 1 mL TBS containing 1.2 mM CaCl ₂ and 0.1% Tween 20, and then the magnetic beads were washed twice with 1 mL TBS containing 1.2 mM CaCl ₂ . Phage was eluted by suspending the magnetic 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 phage were precipitated with polyethylene glycol, supplemented with BSA and CaCl ₂ at final concentrations of 4% each and resuspended in 1.2 mM TBS for the next round of panning.

After the first round of panning, phages were selected on a calcium-dependent basis in which the antibody bound more strongly to C5 in the presence of calcium ions. Panning in rounds 2 and 3 was performed in the same manner as round 1, except that 50 nM (round 2) or 12.5 nM (round 3) biotinylated antigen was used, and finally 0.1 mL of elution buffer (50 mM MES, 2 mM EDTA, 150 mM NaCl, pH 5.5) was eluted and contacted with 1 μL of 100 mg/mL trypsin to select for its calcium dependence. After selection, the selected phage colonies were converted to IgG format.

Binding capacity of transformed IgG antibodies to human C5 was assessed under 2 different conditions: binding and dissociation at 1.2 mM _CaCl2 -pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM _CaCl2 ), binding at 1.2 mM CaCl2 ₂ - pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl ₂ ) and dissociated at 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. Sensorgrams of these antibodies are 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 ). Certain amino acid substitutions were introduced into the CFP0016 heavy chain variable region using methods known to those of ordinary skill 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 in parentheses represent abbreviated names.

[Table 34]

The full-length genes encoding the nucleotide sequences of the antibody heavy and light chains are synthesized and prepared according to general methods known to those of ordinary skill in the art. Heavy and light chain expression vectors are prepared by inserting the obtained plastid fragments into vectors for expression in mammalian cells. The expression vectors obtained are sequenced according to general methods known to those of ordinary skill in the art. For antibody expression, the prepared plastids were transiently transfected into FreeStyle293-F cell line (Thermo Fisher Scientific). Purification of the expression antibody from the adjusted medium was carried out using rProtein A Sepharose Fast Flow (GE Healthcare) by general methods known to those of ordinary skill in the art.

(22-5) Generation and characterization of pH-dependent anti-C5 bispecific antibodies A bispecific antibody that recognizes two different epitopes of C5, produced by the combination of CFP0020 and CFP0018. Bispecific antibodies are prepared in IgG format with 2 different colonies of Fab at each binding site of the antibody using general methods known to those of ordinary skill in the art. In this bispecific IgG antibody, the two heavy chains include heavy chain constant regions (G1dP1, SEQ ID NO: 227 and G1dN1, SEQ ID NO: 228) that are different from each other in order to efficiently form heterodimers of the two heavy chains Elemental body. An anti-C5 bispecific antibody comprising the binding site of anti-C5 MAb "X" and anti-C5 MAb "Y" is represented by "X//Y".

By introducing some amino acid substitutions into the heavy and light chain CDRs by general methods known to those of ordinary skill in the art, we obtained a 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 common light chain: CFP0020L233-k0, SEQ ID NO:231).

Kinetic parameters of optimisation 20//18 against recombinant human C5 were assessed using a BIACORE T200 instrument (GE Healthcare) under 2 different conditions at 37°C (eg (A) binding and dissociation at pH 7.4, (B) binding at pH 7.4 , dissociated at pH 5.8). Protein A/G (Pierce, Cat No. #21186) or anti-human IgG (Fc) antibody (in the Human Antibody Capture Kit; GE Healthcare, Cat No. BR-1008-39) was immobilized by amine coupling on the Series S CM4 (GE Healthcare, Cat No. BR-1005-34). Anti-C5 antibody was captured on the immobilized molecule and human C5 was injected. The running buffers used were ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM _CaCl2 , 0.05% Tween 20). Kinetic parameters for both pH conditions were determined by fitting sensorgrams to a 1:1 binding-RI (no bulk effect adjustment) model using BIACORE T200 Evaluation Software, Version 2.0 (GE Healthcare). 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 reported in Table 35 . Optimizing 20//18, the dissociation of human C5 was faster at pH 5.8 than the dissociation rate at pH 7.4.

[Table 35]

(22-6) Production of antibodies with increased isoelectric point values by modifying amino acids in the variable region 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 a P41R/G44R substitution with increased pi into 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 a pi-increased T77R/E85R 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 with two heavy chain variants of the CFP0018H0012-G1dN1 variant and CFP0020L233-k0 (SEQ ID NO: 231 ) as the light chain to obtain bispecific antibodies.

Likewise, we also assessed pi-increasing substitutions in 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 according to the method shown in Reference Example 1 by introducing each of the substitutions shown in Table 37 into CFP0020L233-k0. 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) Human FcγRIIb Binding Assay Using BIACORE Using the Variant with Increased pI For the manufactured antibody containing the Fc region variant, the binding assay of soluble hFcγRIIb to the antigen-antibody complex 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 a sensor chip CM5 (GE Healthcare) to capture hFcyRIIb using an amine coupling method using a His capture kit (GE Healthcare). The antibody-antigen complexes were then injected with running buffer (as a reference solution) to interact with hFcyRIIb captured on the sensor wafer. 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 were used as running buffers and each buffer to dilute soluble hFcyRIIb. To regenerate the sensor wafer, 10 mM glycine-HCl pH 1.5 was used. All measurements were performed at 25°C and the analysis was performed based on the calculated binding (RU) of the sensorgrams obtained from the measurements and shows the binding amount when CFP0020H0261-G1dP1/CFP0018H0012-G1dN1/CFP0020L233-k0 (original Ab2) binding was defined as 1.00 relative value. To calculate parameters, BIACORE T100 evaluation software (GE Healthcare) was used.

The results of the SPR analysis are summarized in Tables 36 and 37. There are variants showing improved binding to hFcyRIIb immobilized on BIACORE sensor wafers. The antibody is considered to have a strong charge effect on binding of hFcyRIIb on the sensor wafer when this variant binds to hFcyRIIb about 1.2-fold or more compared to the original Ab2 to hFcyRIIb.

Among the heavy chain variants with increased pI, antibodies with L63R, F63R, L82K or S82bR substitutions (according to Kabat numbering) showed higher binding to hFcyRIIb. Single amino acid substitution or a combination of these substitutions in the heavy chain is presumed to have a strong charge effect on the binding of hFcyRIIb on the sensor wafer. One or more positions that are expected to result in faster or greater uptake into cells in vivo by introducing pi-increasing modifications into the antibody heavy chain variable region may include, for example, positions 63, 82 or 82b according to the Kabat numbering. The amino acid substitution introduced into such a position may be arginine or lysine.

Among the light chain variants with increased pi, antibodies with G16K, Q27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (according to Kabat numbering) showed higher binding to hFcyRIIb. A single amino acid substitution in the light chain or a combination of these substitutions is believed to have a strong charge effect on binding of human FcγRIIb on the sensor chip. It is therefore expected that by introducing a pi-increasing modification into the variable region of the antibody light chain, one or more positions will result in faster or greater uptake into cells in vivo, which may include, for example, positions 16, 27, 41, The position of 52, 56, 65, 69, 74, 76, 77 or 79. 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 Fab region variant-containing antibodies To assess the rate of intracellular uptake into hFcyRIIb-expressing cell lines using the produced Fab region variant-containing antibodies, the following analysis was performed.

The MDCK (Madin-Darby canine kidney) cell line continuously expressing hFcyRIIb was produced according to a known method. Intracellular uptake of antigen-antibody complexes is assessed using these cells. Specifically, human C5 was labeled with Alexa555 (Life Technologies) according to established experimental procedures, and antigen-antibody complexes were formed in the culture medium with an antibody concentration of 10 mg/mL and an antigen concentration of 10 mg/mL. The culture solution containing the antigen-antibody complex was added to the culture plate of the above-mentioned MDCK cells expressing hFcγRIIb continuously, and incubated for 1 hour, and then the fluorescence intensity of the antigen taken up into the cells was quantified using an InCell Analyzer 6000 (GE healthcare). The amount of antigen taken up was expressed as a value relative to the original Ab2 value, which was taken as 1.00.

Quantitative results of cellular uptake are shown in Tables 36 and 37. Intense fluorescence from this antigen in cells was observed in some heavy and light chain variants. Here, when the fluorescence intensity of antigen uptake into cells of the variant is about 1.5 times or more compared to the fluorescence intensity of the original Ab2, it is considered that antigen uptake into cells has a strong charge effect.

Of the heavy chain variants with increased pI, any of the variants with a G8R, L18R, Q39K, P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN, S82bR, T83R, A85R or E85G substitution (according to Kabat numbering) Antibodies show stronger antigen uptake into cells. Single amino acid substitutions or combinations of these substitutions in the heavy chain are presumed to have a strong charge effect on uptake of the antigen-antibody complex into cells. It is therefore expected to result in faster or greater uptake of the antigen-antibody complex into the cell by introducing a pi-increasing modification into the variable region of the antibody heavy chain at one or more positions, which may include, for example, positions 8, 18 according to the 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 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 greater uptake of antigen into cells. Single amino acid substitutions or combinations of these substitutions in the light chain are presumed to have a strong charge effect on uptake of the antigen-antibody complex into cells. Variants with 4 or more amino acid substitutions tend to have stronger charge effects than those with fewer amino acid substitutions. As in Example 21, (21-3), the combination of 42K and 76R substitutions is effective in IgE antibody systems. In the case of the C5 antibody, the amino acid at position 42 of the Kabat numbering is already lysine, so we can observe the charge effect of 42K/76R by the single substitution of 76R. The fact that the variant with the 76R substitution has a strong charge effect shows that the C5 antibody also shows a strong charge effect of the 42K/76R combination regardless of the antigen. It is therefore expected to result in faster or greater uptake of the antigen-antibody complex into the cell by introducing a pi-increasing modification into the variable region of the antibody light chain at one or more positions, which may include, for example, positions 16, 27 according to the 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) Evaluation of the clearance rate of C5 in a mouse co-injection model Several anti-C5 bispecific antibodies (original Ab2, 20L233-005, 20L233-006 and 20L233-009) of the mouse co-injection model were tested to assess the ability to accelerate clearance of C5 from plasma. In the co-injection model, C5 premixed with anti-C5 bispecific antibody was administered as a single intravenous injection to C57BL6J mice (Jackson Laboratories). All groups received C5 at 0.1 mg/kg and anti-C5 bispecific antibodies at 1.0 mg/kg. Total C5 plasma concentrations were determined by anti-C5 ELISA. Anti-human C5 mouse IgG was first dispensed onto ECL plates, overnight at 5°C to prepare anti-human C5 mouse IgG immobilized plates. Samples and samples for standard curve were mixed with anti-human C5 rabbit IgG. These samples were added to an anti-human C5 mouse IgG immobilized plate and left at room temperature for 1 hour. These samples were then reacted with HRP-conjugated anti-rabbit IgG (Jackson Immuno Research). After incubating the plate for 1 hour at room temperature, conjugated thio-labeled anti-HRP antibody was added. ECL signals were read with a Sector Imager 2400 (Meso Scale discovery). Human C5 concentrations were calculated from the ECL signal in the standard curve using SOFTmax PRO (Molecular Devices). Figure 39 depicts the C5 plasma concentration time profile in C57BL6J mice.

All bispecific antibodies tested in this study with the pi-increasing substitution demonstrated rapid C5 clearance from plasma compared to the original Ab2. Therefore, it is suggested that the amino acid substitutions of T74K/S77R, S76R/Q79K and Q37R in the light chain 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 where fluorescence intensity is measured using the InCell Analyzer 6000 or in the in vitro BIACORE system described above seem unlikely to contribute to antigen clearance in plasma, the use of more sensitive in vivo The system finds contributors. These results also suggest that 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 in order to estimate the clearance rate of antigen from plasma in vivo. [Example 23]

Evaluation of IgE clearance in plasma using pi-increased Fc variants To improve clearance of human IgE or human C5, pH-dependent antibodies were used to assess pi-increasing substitutions in the Fc portion of antibodies. The method of adding amino acid to the constant region of the antibody to increase 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 a 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, and Ab1L as light chain.

[Table 38]

(23-2) Human FcγRIIb binding assay with BIACORE using an antibody containing an Fc region variant 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, (21-2). The analysis results are shown in Table 38. Here, when the variant binds about 1.2 times or more to hFcyRIIb than the original Abl binds to hFcyRIIb, it can be considered that the antibody binds to hFcyRIIb on the sensor chip with a strong charge effect.

Among the variants with increased pi by a single amino acid substitution 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 hFcyRIIb. The D413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K or G402K single amino acid substitutions are presumed to have a strong charge effect on the binding of hFcyRIIb on the sense chip. One or more positions that are expected to result in accelerated uptake into cells by introducing a pI-increasing modification into the antibody constant or Fc region may include, for example, positions 311, 343, 384, 401, 402, or 413 according to the Kabat numbering, According to the EU numbering method. The amino acid substitution introduced into such a position may be arginine or lysine.

(23-3) pi-increased cellular uptake of Fc region variant-containing antibodies In order to evaluate the intracellular uptake of the antigen-antibody complex formed by the antibodies described in Table 38, a cytometry analysis method similar to that described in Example 21, (21-3) was carried out. The analysis results are shown in Table 38. Here, compared to the fluorescence intensity of the original Ab1, the variant of antigen entering cells has about 1.5 times or more fluorescence intensity, Qi is considered to have a strong charge effect on antigen entry into cells.

Among the variants with increased pi by a single amino acid substitution 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. A single amino acid substitution at D413K, Q311R, N315K, N384R, Q342K, P343R, P343K, D401R, D401K or D413R is presumed to have a strong charge effect on antigen-antibody complex uptake into cells. Positions that are expected to result in faster or greater uptake of antigen-antibody complexes into cells by introducing pi-increasing modifications to the constant or Fc region of an antibody may include, for example, positions 311, 315, 342, 343, 384, 401 or 413. The amino acid substitution introduced into such a position may be arginine or lysine.

(23-4) Evaluation of 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) of the mouse co-injection model were tested to assess their ability to accelerate clearance of IgE from plasma. The assay was carried out in a manner analogous to Example 21, (21-4). Figure 40 depicts the plasma concentration time profile in C57BL6J mice.

Following administration of the high pi variants (only single amino acid substitutions) with pH-dependent antigen binding, plasma total IgE concentrations were lower than the original concentrations except for P1480m. P1480m, which showed weak potency in both in vitro studies, failed to accelerate IgE elimination, and in mice treated with the high pI variant without pH-dependent antigen binding, plasma total 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. Antigen complexed with pH-dependent antigen-binding antibodies taken up into cells can be efficiently released from antibodies in endosomes, accelerating the elimination of IgE. These results suggest that even though the substitution positions tested by the in vitro BIACORE system described above do not appear to contribute to in vivo clearance of the antigen from plasma, a more sensitive in vivo system can be used to find contributors. These results also suggest that the sensitivity of measuring fluorescence intensity in the in vitro system using the above-mentioned InCell Analyzer 6000 may be higher than that in the above-mentioned in-vitro BIACORE system in order to estimate the clearance rate of the antigen from plasma in vivo.

Reference Example 1 Construction of expression vector for amino acid substituted IgG antibody Mutants were prepared using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) using the method described in the accompanying instruction manual. The plastid fragment containing this mutant was inserted into an 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 the art.

Reference Example 2 Expression and purification of IgG antibody The antibody was expressed using the following method. HEK293H cell line (Invitrogen) derived from human embryonic kidney cancer cells was suspended in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Cells were seeded at 10 mL per dish against adherent cells (10 cm in diameter; CORNING) at a cell density of 5 to 6 x 10 ⁵ cells/mL and cultured in a CO ₂ incubator (37°C, 5% CO ₂ ) for 1 sky. The medium was then removed by aspiration and 6.9 mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plastids were introduced into cells by lipofection. The obtained culture supernatant was collected, centrifuged (about 2,000 g, 5 minutes, room temperature) to remove cells, and sterilized by filtration through a 0.22-μm filter MILLEX (registered trademark)-GV (Millipore) to obtain the supernatant. Antibodies were purified from the obtained culture supernatant using rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) using methods known in the art. To determine the concentration of purified antibody, 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 as follows. A CHO cell line that continuously expresses soluble human IL-6 receptor is constructed using methods known in the art, and the expression soluble human IL-6 receptor consists of the amino acid sequence of amino acids 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. The fraction eluting the main peak in the final step was used as the final purified product.

none.

Figure 1 shows changes in human IL-6 receptor concentration in plasma of human FcRn gene-transfected mice administered a human IL-6 receptor-binding antibody that binds to human in a pH-dependent manner IL-6 receptor, and its constant region is native IgG1 (Low_pI-IgG1), or an antibody is administered with an increased isoelectric point value of the variable region of the antibody (High_pI-IgG1). Figure 2 shows the change in the concentration of human IL-6 receptor in the plasma of human FcRn gene-transfected mice to which human FcRn gene-transfected mice were individually administered with a human IL-6 receptor-binding antibody, which was pH-dependent Binds sexually to the human IL-6 receptor and confers binding to FcRn at neutral pH (Low_pI-F939); and an antibody is administered that increases the isoelectric point value of the variable region of the antibody (Middle_pI-F939 , High_pI-F939). Figure 3 shows changes in the concentration of human IL-6 receptor in the plasma of human FcRn gene-transfected mice, which were individually administered with a human IL-6 receptor-binding antibody, which was pH-dependent Binds sexually to the human IL-6 receptor with increased FcγR binding at neutral pH (Low_pI-F1180), and upon administration of an antibody, the variable region of the antibody has an increased isoelectric point value (Middle_pI-F1180 , High_pI-F1180). Figure 4 shows the changes in the concentration of human IL-6 receptor in the plasma of the human FcRn gene-transfected mice, and the soluble human IL-6 receptor concentration in the plasma is maintained at a steady state. Administered a human IL-6 receptor-binding antibody, which binds to the human IL-6 receptor in a pH-dependent manner and whose constant region is native IgG1 (Low-pI-IgG1); and administered an antibody that binds to the human IL-6 receptor in a pH-dependent manner Include an Fc region variant, wherein the Fc region in the antibody has increased FcRn binding under neutral pH conditions (Low_pI-F11); and the antibody was administered with an isoelectric point value of the variable region of the antibody Increased (High_pI-IgG1, High_pI-F11). Figure 5 shows the degree of extracellular matrix binding of each of three types of antibodies with different isoelectric point values that bind to the human IL-6 receptor in a pH-dependent manner (Low_pI-IgG1, Middle_pI-IgG1, and High_pI-IgG1), and the degree of extracellular matrix binding (Low_pI(NPH)-IgG1 and High_pI(NPH)-IgG1) of each of two types of antibodies with different isoelectric point values that do not bind to the human IL-6 receptor in a pH-dependent manner. In the context of Disclosure A described herein, "NPH" means pH-independent. Figure 6 shows the range of relative values (measured as BIACORE (registered trademark)) for soluble human FcγRIIb binding of an antibody comprising an Fc region variant whose isoelectric point has been modified by 1 of the constant region of the Ab1H-P600 antibody amino acid residues, the antibody binds to IgE in a pH-dependent manner. Ab1H-P600 was set to 1.00. Figure 7 shows the relative rate of uptake into cells of hFcyRIIb-expressing cell lines of an antibody comprising an Fc region variant whose isoelectric point has been modified by 1 amino acid of the constant region of the Ab1H-P600 antibody residues increase. Ab1H-P600 was set to 1.00. Figure 8 shows the degree of binding of Fv4-IgG1 having 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 rheumatoid factors in serum from each RA patient. Figure 10 shows the extent of binding of Fv4-LS with an Fc region variant with increased FcRn binding to rheumatoid factor in the serum of each RA patient. Figure 11 shows the degree of binding of Fv4-N434H, an Fc region variant with increased FcRn binding, to rheumatoid factors in the serum of each RA patient. Figure 12 shows the extent of binding of Fv4-F1847m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the serum of each RA patient. Figure 13 shows the extent of binding of Fv4-F1848m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the serum of each RA patient. Figure 14 shows the extent of binding of Fv4-F1886m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the serum of each RA patient. Figure 15 shows the extent of binding of Fv4-F1889m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the serum of each RA patient. Figure 16 shows the extent of binding of Fv4-F1927m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the serum of each RA patient. Figure 17 shows the extent of binding of Fv4-F1168m, an Fc region variant with increased FcRn binding, to rheumatoid factors in the serum of each RA patient. Figure 18 shows the average binding of Fv4-IgG1 to rheumatoid factor in the serum of each RA patient, Fv4-IgG1 has the Fc region of native human IgG1, and each of the antibodies includes a novel Fc region variant, the Fc The regions have Fc region variants with increased binding to each FcRn. Figure 19 shows the concentration change of each anti-human IgE antibody in plasma of Malay monkeys administered OHB-IgG1, which is an anti-human IgE antibody and has the Fc region of native human IgG1, each of which has a novel Fc region Variants, each Fc region has Fc region variants with increased binding to FcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m and OHB-F1927m). Fig. 20 shows the change in the concentration of anti-human IL-6 receptor antibody in the plasma of human FcRn gene-transfected mice administered Fv4-IgG1, which is an anti-human IL-6 receptor antibody and has native human IgG1 Fc region, or administered Fv4-F1718, which increased binding to FcRn under acidic pH conditions. Figure 21 shows a sensogram of IL-8 binding to H998/L63 and Hr9 at pH 7.4 and pH 5.8 as measured by Biacore. Figure 22 shows the changes in human IL-8 concentration in mouse plasma when H998/L63 or H89/L118 was administered in a mixture at 2 mg/kg and human IL-8 to mice. Figure 23 shows the changes in human IL-8 concentration in mouse plasma when H89/L118 was administered in a mixture at 2 mg/kg or 8 mg/kg and human IL-8 to mice. Figure 24 shows changes in human IL-8 concentrations in mouse plasma when H89/L118 or H553/L118 were administered in a mixture at 2 mg/kg or 8 mg/kg and human IL-8 to mice. Figure 25A shows the relative value change of antibody concentration-dependent chemiluminescence with antibodies Hr9, H89/L118 or H553/L118 before storage in plasma. Figure 25B shows the relative value change of the antibody concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H553/L118 after storage in plasma for 1 week. Figure 25C shows the relative changes in the antibody concentration-dependent chemiluminescence of antibodies Hr9, H89/L118 or H553/L118 after storage in plasma for 2 weeks. Figure 26 shows the predicted frequency of ADA for each anti-IL-8 antibody (hWS4, Hr9, H89/L118, H496/L118 or H553/L118) predicted by EpiMatrix and for other pre-existing therapeutic antibodies The predicted frequency of occurrence of ADA. Figure 27 shows the predicted frequency of ADA occurrence for each anti-IL-8 antibody (H496/L118, H496v1/L118, H496v2/L118, H496v3/L118, H1004/L118 or H1004/L395) predicted by EpiMatrix, and Predicted frequency of occurrence of ADA for other pre-existing therapeutic antibodies. Figure 28A shows the relative value change of the antibody concentration-dependent chemiluminescence with antibody Hr9, H89/L118 or H1009/L395-F1886 before storage in plasma. Fig. 28B shows the relative value change of the antibody concentration-dependent chemiluminescence of antibody Hr9, H89/L118 or H1009/L395-F1886 after storage in plasma for 1 week. Figure 28C shows the relative value change of the antibody concentration-dependent chemiluminescence with antibody Hr9, H89/L118 or H1009/L395-F1886 after storage in plasma for 2 weeks. Fig. 29 shows changes in the concentration of human IL-8 in mouse plasma when each of H1009/L395, H553/L118, and H998/L63 and human IL-8 were administered in a mixture to mice. Figure 30 shows the extent of extracellular matrix binding when Hr9, H89/L118 or H1009/L395 were added to the extracellular matrix alone, and in admixture with human IL-8. Figure 31 shows that when the variable region of H1009/L395 and the antibody (F1942m) that does not bind to the Fc region of FcRn are administered alone or in a mixture with human IL-8 to human FcRn gene-transfected mice, little Changes in antibody concentrations in mouse plasma. Figure 32 shows the predicted frequency of ADA for H1009/L395 and H1004/L395 predicted by EpiMatrix, and the predicted frequency of ADA for other pre-existing therapeutic antibodies. Figure 33 shows the change in the concentration of each anti-human IL-8 antibody in the plasma of Malay monkeys administered with H89/L118-IgG1, which has the variable region of H89/L118 and the Fc region of native human IgG1, and each antibody has Fc region variants with increased binding to FcRn (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 antibodies that have the variable regions of H1009/L395 and whose Fc regions are variants against each FcyR (F1886m, F1886s or F1974m). Fig. 35 shows the changes in the concentration of human IL-8 in mouse plasma after administration of a mixture of anti-IL-8 antibody and human IL-8 to human FcRn gene-transfected mice. In this case, the anti-IL-8 antibody was 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 change in antibody concentration in plasma of Malay monkeys administered with Hr9-IgG1 or H89/L118-IgG1, both of which include the Fc region of native human IgG1, or administered with H1009/L395-F1886s or H1009/L395-F1974m, both including 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 (Panels 38A-38D) shows an Octet sensorgram of selected 25 [25] pH-dependent and/or calcium-dependent antigen-binding colonies. 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 (12)

An isolated anti-IL-8 antibody that binds to human IL-8, the anti-IL-8 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 67; (b) HVR- H2, including the amino acid sequence of SEQ ID NO:73; (c) HVR-H3, including the amino acid sequence of SEQ ID NO:74; (d) HVR-L1, including the amino acid sequence of SEQ ID NO:70 Sequences; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO:75; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO:76. The anti-IL-8 antibody according to claim 1, wherein the anti-IL-8 antibody is a humanized anti-IL-8 antibody. The anti-IL-8 antibody according to claim 1, wherein the anti-IL-8 antibody is a humanized anti-IL-8 antibody derived from a mouse antibody. An isolated nucleic acid encoding the anti-IL-8 antibody of any one of claims 1 to 3. A vector comprising the nucleic acid according to claim 4. A host cell comprising the vector of claim 5. A method of producing an anti-IL-8 antibody, comprising culturing the host cell of claim 6. The method of claim 7, further comprising isolating the antibody from the host cell culture medium. A pharmaceutical composition, comprising the anti-IL-8 antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. A use of the anti-IL-8 antibody according to any one of claims 1 to 3, for the manufacture of a pharmaceutical composition for treating a condition in which excess IL-8 exists. A use of the anti-IL-8 antibody according to any one of claims 1 to 3, for the manufacture of a pharmaceutical composition for inhibiting angiogenesis. A use of the anti-IL-8 antibody according to any one of claims 1 to 3, for the manufacture of a pharmaceutical composition for inhibiting the promotion of neutrophil migration.

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