TWI621628B - Il-8-binding antibodies and uses thereof - Google Patents

Abstract

A non-exclusive aspect provides a novel IL-8 antibody that is superior as a drug line.

Description

IL-8 binding antibody and use thereof

Cross-reference with related applications

This application is related to and claims the priority of Japanese Priority Patent Application 2015-185254 filed in Japan on September 18, 2015, the contents of which are incorporated by reference in their entirety.

In a non-exclusive aspect, the present disclosure provides an anti-IL-8 antibody, a pharmaceutical composition containing the antibody, a nucleic acid encoded by the antibody, and a host cell containing the nucleic acid. Methods and uses of manufacturing IL-8 antibodies and pharmaceutical compositions for treating, for example, IL-8 related disorders are also provided.

Antibodies have high stability in plasma and few side effects, so they are valued as pharmaceuticals. Among them, the majority of IgG-type therapeutic antibodies 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 technologies have been developed in terms of technologies applicable to second-generation therapeutic antibodies, including those for improving effector function, antigen-binding ability, pharmacokinetics or stability, and reducing the risk of immunogenicity. Technology (Kim et al., Mol. Cells. 20 (1): 17-29 (2005)). The dosage of therapeutic antibodies is usually very high, so the development of therapeutic antibodies The exhibition encountered difficulties such as making subcutaneous administration preparations and high manufacturing costs. Methods for improving the pharmacokinetics, pharmacodynamics, and antigen-binding properties of therapeutic antibodies provide a means by which the dosage and manufacturing costs associated with therapeutic antibodies can be reduced.

Substitution of amino acid residues in the constant region provides a method for improving antibody pharmacokinetics (Hinton et al., J. Immunol. 176 (1): 346-356 (2006); Ghetie et al., Nat. Biotechnol. 15 (7): 637-640 (1997)). Affinity maturation technology provides a method to enhance the antigen-neutralizing ability of antibodies (Rajpal et al., Proc. Natl. Acad. Sci. USA 102 (24): 8466-8471 (2005); Wu et al. , J. Mol. Biol. 368: 652 (2007)), and may increase 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 antibodies may improve the biological activity of the antibodies in vitro or reduce the dose, and may further improve the efficacy 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 the antigen; therefore, the antigen can be neutralized with a small amount of antibody by increasing the affinity. The affinity of an antibody against an antigen can be routinely increased using a variety of known methods (see, for example, Rajpal et al., Proc. Natl. Acad. Sci. USA 102 (24): 8466-8471 (2005)). Furthermore, if the antigens can be covalently bonded and the affinity can be infinite, theoretically, one antibody molecule can be used to neutralize one antigen molecule (when the antibody is divalent, two antigens can be neutralized). However, one limitation of the development of therapeutic antibodies so far is that 1 antibody molecule usually only binds and neutralizes 1 antigen molecule (2 molecules when the antibody is divalent). Antigen-binding antibody (hereinafter also referred to as "pH-dependent antibody" or "pH-dependent binding antibody"), which enables one antibody molecule to bind and neutralize multiple antigen molecules (see, for example, WO2009 / 125825; Igawa et al., Nat. Biotechnol. 28: 1203-1207 (2010)). The pH-dependent antibody strongly binds to the antigen under neutral conditions in plasma and dissociates from the antigen under acidic conditions in the endosome of the cell. After dissociation from the antigen, FcRn is used to circulate the antibody in the plasma again to bind and neutralize another antigen molecule again. Therefore, one pH-dependent antibody can repeatedly bind and neutralize multiple antigen molecules.

Recently, it has been reported that antibody recovery properties can be achieved by focusing on the difference in calcium (Ca) ion concentration between plasma and endosomes, and the use of antibodies (hereinafter also referred to as "calcium ions") that exhibit calcium-dependent antigen-antibody interactions Concentration-dependent antibody ") (WO2012 / 073992). (Hereinafter, a pH-dependent antibody and a "calcium ion concentration-dependent antibody" are collectively referred to as a "pH / Ca concentration-dependent antibody.")

By binding to FcRn, the IgG antibody has a long residence time in plasma. The binding between the IgG antibody and FcRn is strong under acidic pH conditions (eg, pH 5.8), but hardly binds under neutral pH conditions (eg, pH 7.4). IgG antibodies are brought into cells non-specifically, and bound to FcRn under the acidic pH conditions of the endosome, thereby returning to the cell surface. This IgG is then dissociated from FcRn under plasma neutral pH conditions.

It has been reported that pH-dependent antibodies modified to increase binding to FcRn under neutral pH conditions have the ability to repeatedly bind and eliminate antigen molecules from plasma; therefore administration of this antibody can eliminate antigens from plasma (WO2011 / 122011). According to this report, pH-dependent antibodies modified to increase binding to FcRn under neutral pH conditions (e.g., pH 7.4) compared to those that include PH-dependent antibodies in the Fc region of natural IgG antibodies can further accelerate antigen elimination (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 will no longer be recovered from the endosome to plasma, which will significantly endanger the retention of the antibody in the plasma. Related to this, there are reports that a method of increasing FcRn binding under acidic pH conditions can be used as a method to improve the plasma retention of IgG antibodies. Introduction of amino acid modifications to the Fc region of IgG antibodies to enhance their FcRn binding in acidic pH conditions can enhance the efficiency of endosome recovery to plasma, thereby improving plasma retention. For example, the modifications M252Y / S254T / T256E (YTE; Dall'Acqua et al., J. Biol. Chem. 281: 23514-235249 (2006)), M428L / N434S (LS; Zalevsky et al., Nat. Biotechnol. 28: 157-159 (2010)) and N434H (Zheng et al., Clin. Pharm. & Ther. 89 (2): 283-290 (2011)) have been reported to have longer antibody half-lives than natural IgG1.

However, in addition to concerns that the incidence of immunogenicity or agglutination of such antibodies, including Fc region variants with increased FcRn binding at neutral or acidic pH conditions, may worsen, there have been reports of therapeutic antibody administration In previous patients, the binding to anti-drug antibodies (hereinafter also referred to as "pre-existing ADA") (such as rheumatoid factors) increased (WO2013 / 046722, WO2013 / 046704). WO2013 / 046704 reports that Fc region variants containing specific mutations (expressed as two residue modifications of Q438R / S440E according to EU numbering) increased binding to FcRn under acidic pH conditions, and compared to unmodified natural Fc, Significantly reduced binding was shown for rheumatoid factors. However, WO2013 / 046704 does not specifically prove that this Fc region variant has better plasma retention than antibodies with natural Fc region.

Therefore, a safe and more favorable Fc region variant that has further improved plasma retention and does not show binding for pre-existing ADA is needed.

Antibody-dependent cytotoxicity (hereinafter referred to as "ADCC"), complement-dependent cytotoxicity (hereinafter referred to as "CDC"), antibody-dependent cell phagocytosis (ADCP) by phagocytosis of target cells mediated by IgG antibodies, according to Reporters function as effectors of IgG antibodies. In order for IgG antibodies to mediate ADCC 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 Within the disclosed range, it is also called "Fcγ receptor", "FcgR", "Fc gamma receptor" or "FcγR"). 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 a balance of the respective affinity of antibodies for activated receptors including FcyRIa, FcyRIIa, FcyRIIIa, or FcyRIIIb and inhibitory receptors including FcyRIIb is important in optimizing antibody effector functions.

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

Recently, it has been reported that introduction of amino acid modifications to the Fc region of IgG antibodies to increase the activity of antibodies targeted to soluble antigens and binding 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). And also identified Fc region variants with little change in FcγRIIb binding activity to the natural IgG antibody but reduced activity to other activated FcγRs (WO2014 / 163101).

Compared to antibodies with FcRn-mediated recovery mechanisms, the plasma retention of soluble antigens is very short, and thus soluble antigens can be demonstrated by binding to antibodies with such recovery mechanisms (for example, antibodies that do not have a pH / Ca concentration dependency) Increased plasma retention and plasma concentration. Therefore, for example, if a soluble antigen in plasma has multiple types of physiological functions, even if one type of physiological function is blocked due to antibody binding, the plasma concentration of the antigen may still worsen the pathogenic symptoms caused by other physiological functions. Retention and / or increased plasma concentration. In this case, in addition to using the method of antibody modification exemplified above to accelerate antigen elimination, such as a method using multiple pH / Ca concentration-dependent antibodies to form a multivalent immune complex with multiple antigens, there have been reports of increased For FcRn, FcγR (s), complement receptor binding method (WO2013 / 081143).

Even if the Fc region is not modified, it has been reported that by modifying amino acid residues to change the charge of amino acid residues that may be exposed to the surface of the variable region of the antibody to increase or decrease the isoelectric point (pI) of the antibody, it will be possible to Regardless of the type of target antigen or antibody, control the half-life of the antibody in the blood without substantially reducing the antigen-binding activity of the antibody (WO2007 / 114319: technology for replacing amino acids mainly in FR; WO2009 / 041643: mainly for CDRs Acid-based technology). These documents show that the plasma half-life of an antibody can be extended by reducing 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 modification of the charge of specific amino acid residues can promote the entry of antigens into cells, especially the charge of specific amino acid residues in the CH3 domain. In order to increase the isoelectric point value of the antibody, there are also records showing that this modification should not interfere with the binding to FcRn (WO2014 / 145159). It has also been reported that modification of the amino acid residue charge in the constant region of the antibody (mainly the CH1 domain) to reduce the isoelectric point value can extend the half-life of the antibody in the plasma, and the combination of amino acid residue mutations can increase FcRn Binding can enhance binding to FcRn and prolong the plasma half-life of the antibody (WO2012 / 016227).

However, it is not clear if combining modification techniques designed to increase or decrease the isoelectric point value of antibodies with other modification techniques to increase or decrease the binding of FcRn or FcγR (s), will there be an increase in antibody plasma retention or From blood The effect of plasma to eliminate antigen.

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

ECM is reported to be involved in in vivo kinetics of proteins administered in vivo. The blood concentration of VEGF-Trap molecules administered subcutaneously as a fusion protein of VEGF receptor and Fc has been tested (Holash et al., Proc. Natl. Acad. Sci., 99 (17): 11393-11398 (2002) ). The subcutaneously administered VEGF-Trap molecule with a high electrical point has a low plasma concentration, and therefore has low bioavailability. Modified VEGF-Trap molecules with amino acid substitutions that lower the isoelectric point have higher plasma concentrations and their bioavailability can be improved. In addition, the change in bioavailability is related to the binding strength to ECM. It can be seen that the bioavailability of VEGF-Trap molecules administered subcutaneously depends on 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 antibody molecules that do not bind to ECM have better plasma retention than antibodies that bind to ECM.

As mentioned above, techniques have been reported to reduce extracellular matrix binding in order to improve the bioavailability of proteins in vivo and plasma retention. In contrast, the advantages of increasing the binding of antibodies to ECM have not yet been elucidated.

Human IL-8 (Interleukin 8) is a member of the chemokine family and is 72 or 77 amino acid residues in length. The term "chemotactin" is a general term for molecular weight 8-12kDa and a family of 4 cysteine-containing proteins that form intermolecular disulfide bonds. According to the cysteine arrangement, chemokines are divided into CC chemokines, CXC chemokines, C chemokines, and CA3C chemokines. IL-8 is classified in CXC chemokines, also known as CXCL8.

IL-8 exists in solution as monomeric and homodimeric. The IL-8 unit body includes an anti-parallel Beta plate with a structure in which the C-terminal Alpha spiral 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 Between 2 disulfides. The IL-8 homodimer is stabilized by non-covalent interactions between Beta units of the two units, because there is no covalent bonding between the molecules of the homodimer.

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

In normal tissues, chemokines are generally undetectable or weakly detectable, but are strongly detected at the site of inflammation, and involve the use of white blood cells to promote the involvement of the inflammation site to induce inflammation. IL-8 is a proinflammatory chemokine. It is known to activate neutrophils, promote the expression of cell adhesion molecules, and increase the adhesion of neutrophils to vascular endothelial cells. IL-8 also has neutrophil chemotactic ability. IL-8 produced in injured tissue will promote the chemotaxis of neutrophils that adhere to vascular endothelial cells into the tissue and induce inflammation with the infiltration of neutrophils. . IL-8 is also known to be a potent angiogenic factor for endothelial cells and is involved in promoting tumor angiogenesis.

Inflammatory diseases associated with increased (e.g., excessive) IL-8 levels, including inflammatory diseases of the skin, such as inflammatory keratinization (e.g., psoriasis), atopic dermatitis, contact dermatitis; 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 liver B , C, alcoholic hepatitis, drug-induced allergic hepatitis; inflammatory nephropathy such as glomerulonephritis; inflammatory respiratory diseases such as bronchitis and asthma; inflammatory chronic vascular diseases such as atherosclerosis; multiple sclerosis Disease, oral ulcers, vocal cord inflammation, and related inflammation caused by artificial organs and / or artificial blood vessels. Increased (eg excessive) levels of IL-8 are also associated with malignancies, such as ovarian, lung, prostate, gastric, breast, melanoma, head and neck cancer, and kidney cancer; sepsis due to infection; cystic fibrosis; And pulmonary fibrosis. (See, for example, Russo et al., Exp. Rev. Clin. Immunol. 10 (5): 593-619 (2014) (No, hereby incorporated by reference in its entirety).

Against 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, there is only one pharmaceutical composition including an IL-8 antibody, which is a mouse anti-IL-8 antibody for external use as a psoriasis. New anti-IL-8 antibodies for the treatment of diseases are expected.

In an non-exclusive aspect, the undefined purpose of the disclosed embodiment of A is to provide molecules with improved pharmacokinetic properties for antibodies, such as ion concentration-dependent antigen-binding properties, to improve antibody half-life and / or from plasma Clear the antigen.

In an unexclusive state, the undefined purpose of the disclosed embodiment of B is to provide a safer and more favorable variant of the Fc region, which has a longer half-life and reduced binding to pre-existing anti-drug antibodies (ADAs) .

In an non-exclusive aspect, an undefined object of the embodiment of C is to provide an anti-IL-8 antibody that has a pH-dependent binding affinity for IL-8. An additional embodiment relates to an anti-IL-8 antibody, which has the effect of eliminating IL-8 more quickly when administered to an individual than a reference antibody. In another embodiment, it is disclosed that the C line is an anti-IL-8 antibody that can stably maintain its IL-8 neutralizing activity when administered to an individual. In some embodiments, the anti-IL-8 antibody displays lower immunogenicity. In other embodiments, it is disclosed that C is a method for manufacturing and using the above-mentioned anti-IL-8 antibody. Another non-limiting purpose of revealing C is to provide new anti-IL-8 that can be included in pharmaceutical compositions.

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

In a non-exclusive aspect, within the scope of disclosure B provided here, the inventors performed Fc region variants that do not bind to anti-drug antibodies (pre-existing ADA) and can improve plasma retention with safety and benefits Precision research. Knot The inventors have unexpectedly found that the substitution of amino acid residues to Ala (A) according to EU numbering at 434 and two specific residue mutations (represented by Q438R / S440E according to EU numbering) are used as amino acid residue mutation combinations. The Fc region variant is ideal for maintaining significantly reduced binding to rheumatoid factors and simultaneously achieving plasma retention of antibodies.

In a non-exclusive state, within the scope of disclosure C provided herein, the inventors have produced 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 have identified that when administered to an individual, a pH-dependent anti-IL-8 antibody has a faster elimination effect of IL-8 than a reference antibody. In some embodiments, the present disclosure is about a pH-dependent anti-IL-8 antibody that can stably maintain its IL-8 neutralizing activity. In an additional non-limiting embodiment, the pH-dependent anti-IL-8 antibody has lower immunogenicity and excellent performance levels.

Also in the category of revealing C, the inventors successfully obtained an anti-IL-8 antibody including an Fc region, whose Fc region has an increased FcRn binding affinity at an acidic pH compared to the natural Fc region's FcRn binding affinity. In an alternative aspect, the inventors successfully obtained an anti-IL-8 antibody including an Fc region, the binding affinity of the Fc region to the pre-existing ADA is reduced compared to the binding affinity of the natural Fc region to the pre-existing ADA. . In an alternative aspect, the inventors successfully obtained an anti-IL-8 antibody in the Fc region, whose plasma half-life is increased compared to the natural plasma Fc region. In an alternative aspect, the inventors successfully obtained a pH-dependent anti-IL-8 antibody including the Fc region, which has a binding affinity to the effector receptor compared to the binding of the natural Fc region to the effector receptor Reduced affinity. In a different aspect, the inventors identified a nucleic acid encoding the anti-IL-8 antibody described above. In another aspect, The inventors obtained a host containing the above nucleic acid. In another aspect, the inventor has developed a method for manufacturing the above-mentioned anti-IL-8 antibody, which includes the step of culturing the aforementioned host. In another aspect, the inventors have developed a method for promoting the elimination of IL-8 relative to a reference antibody in an individual, including the step of administering to the individual an anti-IL-8 antibody as described above.

In one embodiment, it is disclosed that A is related but not limited to:

[1] An antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ionic concentration conditions, wherein the isoelectric point (pI) of the antibody is modified 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 according to [1] or [2], wherein the antigen-binding domain is a domain in which the antigen-binding activity is higher in a high ion concentration condition than in a low ion concentration condition.

[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, for the antigen, the ratio of the acidic pH range to the KD of the neutral pH range, that is, KD (acidic pH range) / KD (neutral pH range), is 2 or more high.

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

[7] The antibody according to any one of [1] to [6], which can promote elimination of antigen from plasma compared to before modification of the antibody.

[8] The antibody according to any one of [1] to [7], wherein the extracellular matrix binding activity of the antibody is improved compared to that before the modification of the antibody.

[9] The antibody according to any one of [1] to [8], wherein the amino acid residue is modified by amino acid residue substitution.

[10] The antibody of any one of [1] to [9], wherein the amino acid residue modification is selected from the group consisting of: (a) replacing a negatively charged amino acid residue to become uncharged Amino acid residues, (b) replacing negatively charged amino acid residues into positively charged amino acid residues, and (c) replacing uncharged amino acid residues into positively charged amino acid residues 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 to the variable region and / or the constant region Modification of amino acid residues in the region.

[12] The antibody according to [11], wherein the variable region includes a complementarity determining region (CDR) and / or a framework region (FR).

[13] The antibody of [12], wherein the variable region includes a heavy chain variable region and / or a light chain variable region, and the CDR or FR is selected according to Kabat numbering from the group consisting of One position has at least one amino acid residue modified: (a) 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, FR in the heavy chain variable region 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, Positions 108, 110, and 112; (b) positions 31, 61, 62, 63, 64, 65, and 97 of the CDR of the heavy chain variable region; (c) position 1 of FR of the light chain variable region 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 of the CDRs of the light chain variable region , 26, 27, 52, 53, 54th, 55th, and 56th.

[14] The antibody of [13], wherein at least one amino acid residue in the CDR and / or FR selected from one of the positions of the group is modified: (a) the heavy chain is variable Districts FR 8, 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105; (b) the Positions 31, 61, 62, 63, 64, 65, and 97 of the CDR of the heavy chain variable region; (c) 16, 18, 37, 41, 42, 45, 65, FR of the variable region of the light chain Positions 69, 74, 76, 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.

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

[16] The antibody of [15], wherein at least one amino acid residue in the constant region selected from one of the positions of the group consisting of is modified: according to EU No. 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434, and 443.

[17] The antibody of [16], wherein at least one amino acid residue in the constant region selected from one of the positions of the group consisting of is modified: according to EU No. 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 an Fcganma receptor (FcγR) binding activity, and the FcγR binding activity at neutral pH conditions is greater than that of a natural 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 activated 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 increased and the binding activity to the activated FcγR is reduced compared to IgG, which is a natural reference antibody in the constant region, which is a different reference antibody.

[21] The antibody of any one of [1] to [20], wherein the constant region has FcRn binding activity, and is a different reference antibody compared to IgG, which is only natural in the constant region, at neutral pH Conditions (e.g., pH 7.4) the FcRn binding activity of the constant region is increased.

[22] The antibody of any one of [1] to [21], wherein the antibody is a multispecific 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 according to any one of [1] to [23].

[25] The pharmaceutical composition according to [24], which is used for promoting the antigen from plasma eliminate.

[26] The pharmaceutical composition according to [24] or [25], which is used to improve the binding of an antibody to an extracellular matrix.

[27] A nucleic acid encoded as an antibody according to any one of [1] to [23].

[28] A vector comprising a nucleic acid as in [27].

[29] A host cell comprising a vector such as [28].

[30] A method for manufacturing an antibody, the antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, comprising the steps of: culturing a host cell as in [29], and collecting the antibody from the cell culture.

[30A] A method for manufacturing an antibody, the antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ionic concentration conditions, including the steps of modifying at least one amino acid residue that may be exposed on the surface of the antibody to increase such Electric point (pI).

[30B] The method for producing an antibody according to [30A], wherein at least one amino acid residue is modified in CDR or FR and selected from one of the group consisting of: (a) according to Kabat numbering: 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 46 in the FR of the heavy chain variable region, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112; (b) the variable region of the heavy chain Positions 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) the CDRs of the light chain variable region 24th, 25th, 26th, 27th, 52nd, 53nd, 54th, 55th, and 56th; or (II) one of the positions in the constant region selected from the group consisting of: 196, 253 according to the EU numbering , 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) replacing a negatively charged amino acid residue into an uncharged one Amino acid residues; (b) replacing negatively charged amino acid residues into positively charged amino acid residues; (c) replacing uncharged amino acid residues into positively charged amino acid residues Residues; (d) substitution or insertion of histidine at CDR or FR.

[32] The method for producing an antibody according to any one of [30], [30A], or [30B], further optionally including any one or more of the following steps: (a) the selection can promote Antibodies that eliminate antigen from plasma; (b) select antibodies that have enhanced binding activity to the extracellular matrix; (c) select antibodies that have enhanced FcγR binding activity under neutral pH conditions (e.g., pH 7.4); (d) Select antibodies that have enhanced FcγRIIb binding activity under neutral pH conditions (e.g. pH 7.4); (e) Select antibodies that have maintained or enhanced FcγRIIb binding activity and have reduced binding to one or more activated FcγRIIb Activity The suitable FcyR is preferably selected from the group consisting of: FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb, and FcyRIIa; (f) selecting antibodies that have enhanced FcRn binding activity under neutral pH conditions (e.g. pH 7.4); (g) selecting Antibodies with increased isoelectric point (pI); (h) Confirm the isoelectric point (pI) of the collected antibodies, and then select antibodies with increased isoelectric point (pI); and (i) select the antigen-binding activity to change according to ion concentration conditions Or increased antibodies.

In an alternative embodiment, it is disclosed that A is about but 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 modification site of the group as defined in [15] or [16] is modified to be constant Area.

[A2] An antibody such as [A1], further comprising a heavy chain variable region and / or a light chain variable region, the variable region having a CDR and / or FR, and selected from and selected from the group consisting of [13] or [14] At least one amino acid residue in the same modification site group of the defined modification site group is modified to CDR and / or FR.

[A3] An antibody having a constant region selected from the group consisting of at least one amino acid residue in the same group of modification sites as the group defined in [15] or [16], which is modified in the constant region to Increase its isoelectric point value.

[A4] The antibody of [A3], further comprising a heavy chain variable region and / or a light chain variable region, the variable region having a CDR and / or FR, selected from and selected from the group consisting of [13] or [14] At least one amino acid residue in the same modification site group of the defined modification site group is modified to CDR and / or FR.

[A5] An antibody comprising an antigen-binding domain whose antigen-binding activity changes in accordance with ionic concentration conditions, the antibody has a constant region and is selected from the same modification site as the modification site in the group as 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 comprising a heavy chain variable region and / or a light chain variable region, the variable region having a CDR and / or FR, selected from and defined in [13] or [14] At least one amino acid residue in the same modification site group of the same modification site group is modified in CDR and / or FR.

[A7] The use of an antibody according to any one of [1] to [23] and [A1] to [A6] is for the manufacture of a medicine that promotes elimination of an antigen from plasma.

[A8] The use of an antibody according to any one of [1] to [23] and [A1] to [A6] is for the manufacture of a medicine that increases the binding of extracellular matrix.

[A9] The use of an antibody according to any one of [1] to [23] and [A1] to [A6] for eliminating an antigen from plasma.

[A10] Use of an antibody according to any one of [1] to [23] and [A1] to [A6] for increasing extracellular matrix binding.

[A11] An antibody obtained by a method for producing an antibody according to any one of [30], [30A], [30B], [31], and [32].

According to various embodiments, the disclosure A includes part or all of one or more of which are described in [1] to [30], [30A], [30B], [31], [32], and [A1] to [A11] The combination of the elements, provided that such a combination does not technically conflict with ordinary technical knowledge in the technical field. For example, in some embodiments, it is disclosed that A includes a method of making a modified antibody, the antibody comprising The antigen-binding domain that eliminates the antigen includes: (a) modifying at least one amino acid residue that may be exposed on the surface of the antibody, which is: (I) selected from the group consisting of: CDR or FR according to Kabat numbering Position of group: (a) 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42 in the FR of the heavy chain variable region , 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112; (b) 31, 61, 62, 63, 64, 65, and 97 positions in the CDR of the heavy chain variable region; (c) 1, 3, 7, 8, 9, 11 in the FR of the light chain variable region , 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 in the CDR of the light chain variable region , 54, 55, and 56; or (II) a position in the constant region 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; (b) modify the antigen-binding domain so that the obtained antigen-binding activity changes according to the ion concentration conditions, wherein (a) and (b ) Can be performed simultaneously or sequentially; (c) culturing the host cell to express a nucleic acid encoding the modified antibody; and (d) collecting the modified antibody from the host cell culture.

In another embodiment, the method may optionally further include one or more of the following steps: (e) selecting an antibody that promotes elimination of the antigen from the plasma compared to the modification of the antibody; (f) selecting an extracellular matrix Antibodies with enhanced binding activity; (g) antibodies with enhanced FcγR binding activity selected at neutral pH conditions (e.g. pH 7.4); (h) antibodies with increased FcγRIIb binding selected at neutral pH conditions (e.g. pH 7.4) Active antibodies; (i) Select antibodies that have maintained or enhanced FcγRIIb binding activity and have reduced binding activity to one or more activated FcγR. The activated FcγR is preferably selected from the group consisting of: FcγRIa , FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa; (j) select antibodies with enhanced FcRn binding activity under neutral pH conditions (such as pH 7.4); (k) select antibodies with increased isoelectric point (pI); (l) ) Confirm the isoelectric point (pI) of the collected antibodies, and then select antibodies with increased isoelectric point (pI); and (m) select antibodies whose antigen-binding activity changes or increases according to ionic concentration conditions.

Another embodiment of Disclosure A is, for example, not limited to: [D1] a method of making a modified antibody that has a prolonged or shortened half-life in plasma compared to the modified antibody, including the following steps: (a) before the modification A nucleic acid encoded by the antibody to change The charge of at least one amino acid residue in one position of 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; (b) culture the host cell to express the nucleic acid; and (c) collect the antibody from the host cell culture.

[D2] A method for extending or shortening the half-life of an antibody in plasma, comprising the steps of modifying at least one amino acid residue at a position selected from the group consisting of: 196, 253, 254 according to EU numbering , 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 is about but not limited to: [33] An Fc region variant, including an FcRn binding domain, the FcRn binding domain includes: (a) Ala at position 434 according to EU numbering; 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 at position 434; Arg or Lys 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: Ile or Leu at position 428 according to EU numbering; and / or Ile, Leu, Val, Thr, or Phe is at 436.

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

[37] The Fc region variant of any one of [33] to [36], wherein the FcRn binding domain comprises a combination of amino acid substitutions selected from the group consisting of: 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; / Y436V / Q438R / S440E; N434A / Y436V / Q438R / S440D; N434A / Y436V / Q438K / S440E; N434A / Y436V / Q438K / S440D; N434A / R435H / F436T / Q438R / S440E; N434A / R435H / F436T / Q438R / ; N434A / R435H / F436T / Q438K / S440E; N434A / R435H / F436T / Q438K / S440D; N434A / R435H / F436V / Q438R / S440E; N434A / R435H / F436V / Q438R / S440D; N434A / R435H / F436V / Q438R / S440D; N434A / R435H / F436V / Q438K ; N434A / R435H / F436V / Q438K / S440D; M428L / N434A / Q438R / S440E; M428L / N434A / Q438R / S440D; M428L / N434A / Q438K / S440E; M428L / N434A / Q438K / S440D; M428L / N434A / Y436T / 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 / N428A Y436V / Q438K / S440D; L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, according to the EU numbering method.

[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 / Y440T / Q438R ; And L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E, in accordance with EU numbering.

[39] The Fc region variant according to any one of [33] to [38], wherein the FcRn-binding activity is increased under acidic pH conditions (for example, pH 5.8) compared to the Fc region of natural IgG.

[40] The Fc region variant according to any one of [33] to [39], wherein the binding activity of the anti-drug antibody (ADA) is not significantly improved at neutral pH conditions compared to the Fc region of natural IgG. .

[41] The Fc region variant of [40], wherein the anti-drug antibody (ADA) is a rheumatoid factor (RF).

[42] The Fc region variant according to any one of [33] to [41], wherein the plasma clearance (CL) is reduced, the plasma retention time is increased, Or the plasma half-life (t1 / 2) is increased.

[43] The Fc region variant of any one of [33] to [42], wherein the plasma retention is increased compared to a reference Fc region variant including the following amino acid substitution combination: N434Y / Y436V / Q438R / S440E, according to EU numbering.

[44] An antibody comprising an Fc region variant according to any one of [33] to [43].

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

[46] A pharmaceutical composition comprising an antibody such as [44] or [45];

[47] The pharmaceutical composition 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 a nucleic acid as in [48].

[50] A host cell comprising a vector such as [49].

[51] A method for producing an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant, comprising the steps of: culturing a host cell as in [50], and then collecting the Fc region variant or the Antibodies to this variant.

[52] The method for producing an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant as described in [51], which more optionally includes any one or more of the following steps: Fc region of IgG, an Fc region variant that has improved FcRn binding activity under acidic pH conditions; (b) Choose a Fc region that has no binding activity against drug-resistant antibodies (ADA) at neutral pH conditions compared to the Fc region of natural IgG Significantly enhanced Fc region variants; (c) Selection of Fc regions with increased plasma retention compared to Fc regions of native IgG Variants; and (d) selecting antibodies that include Fc region variants that facilitate elimination of the antigen from plasma compared to reference antibodies that include the Fc region of native IgG.

[53] A method for producing an Fc region variant comprising an FcRn binding domain or an antibody comprising the variant, comprising the steps of substituting an amino acid such that the obtained Fc region variant or an antibody containing the variant is numbered according to EU Methods include Ala at 434; Glu, Arg, Ser, or Lys at 438; and Glu, Asp, or Gln at 440.

In one embodiment, the disclosure B is not limited to:

[B1] The use of an Fc region variant according to any one of [33] to [43] or an antibody such as [44] or [45], which is a medicine for increasing plasma retention.

[B2] The use of an Fc region variant according to any one of [33] to [43] or an antibody such as [44] or [45], which is to produce an Fc region at a neutral pH compared to a natural IgG Condition medicines that do not significantly increase the binding activity of anti-drug antibodies (ADA).

[B3] Use of an Fc region variant according to any one of [33] to [43] or an antibody such as [44] or [45] for the purpose of increasing retention in plasma.

[B4] The use of an Fc region variant such as any one of [33] to [43] or an antibody such as [44] or [45], for neutral pH compared to the Fc region of natural IgG, Conditions did not significantly increase the binding activity of anti-drug antibodies (ADA).

[B5] An Fc region variant or an antibody containing the variant is obtained by a method for producing an antibody according to any one of [51], [52], and [53].

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

In one embodiment, it is disclosed that C is, for example, not limited to: [54] an isolated anti-IL-8 antibody that binds to human IL-8, including at least one of the following positions (a) to (f) 1 amino acid substituted and bound to IL-8 in a pH-dependent manner: (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2, including SEQ ID NO: 68 Amino acid sequence; (c) HVR-H3, including the amino acid sequence of SEQ ID NO: 69; (d) HVR-L1, including the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2, including the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3, including SEQ ID NO : Amino acid sequence of 72.

[55] The anti-IL-8 antibody according to [54], comprising: tyrosine at position 9 in the amino acid sequence of SEQ ID NO: 68, and 11 at 11 in the amino acid sequence of SEQ ID NO: 68 Arginine at position 3, and amino acid substitution of tyrosine at position 3 in the amino acid sequence of SEQ ID NO: 69.

[56] The anti-IL-8 antibody according to [54] or [55], further comprising the alanine at position 6 of the amino acid sequence of SEQ ID NO: 68 and the amino acid sequence of SEQ ID NO: 68 Amino acid substitution of glycine at position 8.

[57] The anti-IL-8 antibody according to any one of [54] to [56], comprising aspartic acid at position 1 in the amino acid sequence of SEQ ID NO: 71, and at SEQ ID NO : Leucine at position 5 of the amino acid sequence of 71, amino acid substitution of glutamic acid at position 1 of the amino acid sequence of SEQ ID NO: 72.

[58] The anti-IL-8 antibody according to any one of [54] to [57], including (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 67, (b) HVR-H2, Including the amino acid sequence of 5EQ ID NO: 73, and (c) HVR-H3, including the amino acid sequence of SEQ ID NO: 74.

[59] The anti-IL-8 antibody according to any one of [54] to [58], including (a) HVR-L1, including the amino acid sequence of SEQ ID NO: 70, (b) HVR-L2, Including the amino acid sequence of SEQ ID NO: 75, and (c) HVR-L3, including the amino acid sequence of SEQ ID NO: 76.

[60] The anti-IL-8 antibody according to any one of [54] to [59], comprising a heavy chain variable region of SEQ ID NO: 78 and a light chain variable region of SEQ ID NO: 79.

[61] The anti-IL-8 antibody according to any one of [54] to [60], comprising an Fc region having at least one property selected from the following (a) to (f): (a) relative to the native Fc The binding affinity of the region to FcRn is increased under acidic pH conditions; (b) the binding affinity for pre-existing ADA is reduced relative to the native Fc region, and the binding affinity for pre-existing ADA is reduced; c) Plasma half-life is increased relative to the plasma half-life of the native Fc region; (d) Plasma clearance is reduced in the Fc region relative to the plasma clearance rate of the natural Fc region; (e) Targeting effector receptors relative to the native Fc region The binding affinity of the Fc region to the effector receptor decreases; (f) the binding to the extracellular matrix increases.

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

[63] The anti-IL-8 antibody according to [62], comprising an Fc region comprising an amino acid substitution selected from the group consisting of one or more of the following: L235R, G236R, S239K, A327G, A330S , P331S, M428L, N434A, Y436T, Q438R and S440E.

[64] The anti-IL-8 antibody according to [63], wherein the Fc region includes the following amino acid substitutions: L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E.

[65] The anti-IL-8 antibody according to [63], wherein the Fc region includes the following amino acid substitutions: L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E.

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

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

[68] An isolated nucleic acid encoding an anti-IL-8 antibody according to any one of [54] to [67].

[69] A vector comprising a nucleic acid as described in [68].

[70] A host cell comprising a vector such as [69].

[71] A method for producing an anti-IL-8 antibody, comprising the step of culturing a host such as [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], for use in a pharmaceutical composition.

[75] The use of the anti-IL-8 antibody according to any one of [54] to [67], for the treatment of a condition in which an excessive amount of IL-8 is present.

[76] The use of the anti-IL-8 antibody according to any one of [54] to [67], for the manufacture of a pharmaceutical composition for a condition in which an excessive amount of IL-8 is present.

[77] A method of treating a patient having a condition with excess IL-8, comprising the step of administering an anti-IL-8 antibody to any one of the subjects of [54] to [67].

[78] A method for promoting the elimination of IL-8 from an individual, including the following steps: An individual is administered an anti-IL-8 antibody according to any one of [54] to [67].

[79] A pharmaceutical composition comprising the anti-IL-8 antibody according to any one of [54] to [67], which antibody binds to IL-8 and to an extracellular matrix.

[80] A method for manufacturing an anti-IL-8 antibody, the antibody comprising: a variable region having pH-dependent IL-8 binding activity, including the following steps: (a) evaluating the anti-IL-8 antibody and extracellular matrix (B) selecting an anti-IL-8 antibody that strongly binds to the extracellular matrix, (c) culturing a host including a vector encoding a nucleic acid for the antibody, and (d) isolating the antibody from the culture solution.

In an alternative embodiment, C is disclosed about:

[C1] Use of an 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] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is for inhibiting the accumulation of biologically active IL-8.

[C3] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for producing a pharmaceutical composition for inhibiting angiogenesis.

[C4] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is for inhibiting angiogenesis.

[C5] Use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for producing a pharmaceutical composition for promoting the inhibition of neutrophil migration .

[C6] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is for inhibiting the promotion of neutrophil migration.

[C7] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for inhibiting the accumulation of biologically active IL-8.

[C8] A method for inhibiting the accumulation of biologically active IL-8 by administering an anti-IL-8 antibody such as [54] to [67] and [C26] to [C31] to an individual.

[C9] A pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, including an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] .

[C10] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for suppressing angiogenesis.

[C11] A method of inhibiting angiogenesis in an individual, comprising the following steps:

For an individual, an anti-IL-8 antibody such as [54] to [67] and [C26] to [C31] is administered.

[C12] A pharmaceutical composition for inhibiting angiogenesis, including an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31].

[C13] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is used for inhibiting the promotion of neutrophil migration.

[C14] A method for inhibiting neutrophil migration promotion in an individual, comprising administering anti-IL such as [54] to [67] and [C26] to [C31] to the individual -8 antibody steps.

[C15] A pharmaceutical composition for inhibiting neutrophil migration promotion in an individual, including an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] .

[C16] the anti-IL-8 antibody as in any one of [54] to [67] and [C26] to [C31] Body, is used to treat conditions where there is an excess of IL-8.

[C17] Use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], which is used for manufacturing a medicine for treating a condition in which there is an excess of IL-8 combination.

[C18] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] for the treatment of a condition in which there is an excess of IL-8.

[C19] A method for treating a condition in which there is an excess of IL-8 in an individual, comprising the step of administering an anti-IL such as [54] to [67] and [C26] to [C31] to the individual -8 antibody.

[C20] A pharmaceutical composition for treating a condition in which there is an excess of IL-8 in an individual, including anti-IL-8 as described in any one of [54] to [67] and [C26] to [C31] antibody.

[C21] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is used to promote elimination of IL-8.

[C22] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is for treating a pharmaceutical composition for promoting the elimination of IL-8.

[C23] The use of an anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] is to promote elimination of IL-8.

[C24] A method for promoting elimination of IL-8 in an individual, including the following steps: An anti-IL-8 antibody such as any of [54] to [67] and [C26] to [C31] is administered to the individual.

[C25] A pharmaceutical composition for promoting the elimination of IL-8, comprising the following steps: An anti-IL-8 antibody such as any of [54] to [67] and [C26] to [C31] is administered to the individual.

[C26] An anti-IL-8 antibody comprising an Fc region comprising an amino acid substitution 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.

[C27] The anti-IL-8 antibody of [C26], which includes an Fc region having at least one property selected from the following (a) to (f): (a) for FcRn relative to the native Fc region The binding affinity for FcRn is increased under acidic pH conditions; (b) the binding affinity for pre-existing ADA is reduced relative to the native Fc region, and the binding affinity for pre-existing ADA is reduced; (c) relative The plasma half-life in the natural Fc region is increased; (d) the plasma clearance of the Fc region is reduced relative to the plasma clearance of the natural Fc region; (e) the binding affinity for the effector receptor relative to the natural Fc region The binding affinity of the Fc region to the effector receptor is reduced; and (f) the binding to the extracellular matrix is increased.

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

[C29] An anti-IL-8 antibody as in [C28], comprising an Fc region comprising an amino acid substitution comprising one or more amino acids selected from the group consisting of: (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E in accordance with EU numbering; or (b) L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

[C30] The anti-IL-8 antibody according to [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 according to [C26], comprising: a heavy chain containing the amino acid sequence of SEQ ID NO: 80 and a light chain containing the amino acid sequence of SEQ ID NO: 82.

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

[C33] A vector comprising a nucleic acid such as [C32].

[C34] A host cell comprising a vector such as [C33].

[C35] A method for producing an anti-IL-8 antibody, comprising culturing a host cell such as [C34].

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

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

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

[C39] A method for promoting elimination of IL-8 from an individual, comprising the step of administering to the individual an anti-IL-8 antibody such as [C26] to [C31].

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

[C41] The method for inhibiting IL-8 as in [C40], wherein the method inhibits the biological activity of IL-8.

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

Figure 1 shows changes in human IL-6 receptor concentration in the plasma of human FcRn transgenic mice. The mouse was administered a human IL-6 receptor binding antibody, which will bind to humans in a pH-dependent manner. IL-6 receptor, and its constant region is natural IgG1 (Low_pI-IgG1), or an antibody is administered, and the isoelectric point value of the antibody's variable region increases (High_pI-IgG1).

Figure 2 shows changes in the human IL-6 receptor concentration in the plasma of human FcRn transgenic mice. The human FcRn transgenic mice were individually administered with human IL-6 receptor binding antibodies. The antibodies were pH-dependent Binding to the human IL-6 receptor in a sexual manner and conferring binding to FcRn at a neutral pH (Low_pI-F939); and when the antibody is administered, the isoelectric point value of the variable region of the antibody increases (Middle_pI-F939) , High_pI-F939).

Figure 3 shows changes in the human IL-6 receptor concentration in the plasma of human FcRn transgenic mice. The human FcRn transgenic mice were individually administered with human IL-6 receptor binding antibodies. The antibodies were pH-dependent It binds to human IL-6 receptor in a sexual manner, and its FcγR binding increases at neutral pH (Low_pI-F1180), and when the antibody is administered, the isoelectric point value of the variable region of the antibody increases (Middle_pI-F1180 , High_pI-F1180).

Figure 4 shows the change of human IL-6 receptor concentration in the plasma of human FcRn gene transgenic mice, and the soluble human IL-6 receptor concentration in plasma remained stable. Individual human FcRn gene transgenic mice A human IL-6 receptor binding antibody was administered, and the antibody bound to the human IL-6 receptor in a pH-dependent manner, and its constant region was natural IgG1 (Low_pI-IgG1); and an antibody was administered, which Including an Fc region variant, wherein the Fc region in the antibody has increased FcRn binding at neutral pH conditions (Low_pI-F 11); and the antibody is administered, and the isoelectric point of the variable region of the antibody Values increased (High_pI-IgG1, High_pI-F11).

Figure 5 shows the extent of extracellular matrix binding (Low_pI-IgG1, Middle_pI-IgG1, and High_pI-IgG1) of the three types of antibodies with different isoelectric point values that bind to the human IL-6 receptor in a pH-dependent manner, And the degree of extracellular matrix binding (Low_pI (NPH) -IgG1 and High_pI (NPH) -IgG1) of the 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 category of disclosure A described herein, "NPH" means pH-independent.

Figure 6 shows the relative range of soluble human FcγRIIb binding (measured by BIACORE (registered trademark)) of the antibody. The antibody includes a variant of the Fc region whose isoelectric point has been modified by modifying 1 of the constant region of the Ab1H-P600 antibody. With an increase in amino acid residues, the antibody binds to IgE in a pH-dependent manner. The Ab1H-P600 is set to 1.00.

Figure 7 shows the relative value of the rate of antibody uptake into the hFcγRIIb-expressing cell line. The antibody includes a variant of the Fc region whose isoelectric point has been modified by modifying an amino acid in the constant region of the Ab1H-P600 antibody Residues. The Ab1H-P600 is set to 1.00.

Figure 8 shows the degree of binding of Fv4-IgG1 with the Fc region of natural human IgG1 to rheumatoid factors in the serum of each RA patient.

Figure 9 shows the extent of binding of Fv4-YTE of Fc region variants with increased FcRn binding to rheumatoid factors in the serum of each RA patient.

Figure 10 shows the degree of binding of Fv4-LS with increased FcRn-binding Fc region variants to rheumatoid factors in the serum of each RA patient.

Figure 11 shows the extent 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 of Fc region variants with increased FcRn binding to rheumatoid factors in the serum of each RA patient.

Figure 13 shows the extent of binding of Fv4-F1848m of Fc region variants with increased FcRn binding to rheumatoid factors in the serum of each RA patient.

Figure 14 shows the extent of binding of Fv4-F1886m of Fc region variants with increased FcRn binding to rheumatoid factors in the serum of each RA patient.

Figure 15 shows the extent of binding of Fv4-F1889m of Fc region variants with increased FcRn binding to rheumatoid factors in the serum of each RA patient.

Figure 16 shows the extent of binding of Fv4-F1927m of Fc region variants with increased FcRn binding to rheumatoid factors in the serum of each RA patient.

Figure 17 shows the extent of binding of Fv4-F1168m of the Fc region variant with increased FcRn binding to rheumatoid factors in the serum of each RA patient.

Figure 18 shows the average value of the binding of Fv4-IgG1 to rheumatoid factors in the serum of each RA patient. Fv4-IgG1 has the natural Fc region of human IgG1, and this antibody each includes a novel Fc region variant. This Fc Regions have Fc region variants that increase binding to each FcRn.

Figure 19 shows the change in the concentration of each anti-human IgE antibody in the plasma of Malay monkeys. It was administered with OHB-IgG1, which is an anti-human IgE antibody and has a natural human IgG1 Fc region. Each of these antibodies has a novel Fc region. Variants, each Fc region has an Fc region variant with increased binding to FcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m).

Figure 20 shows the changes in the concentration of anti-human IL-6 receptor antibodies in the plasma of human FcRn transgenic mice. Fv4-IgG1 was administered as an anti-human IL-6 receptor antibody and has natural human IgG1. The Fc region, or Fv4-F1718 was administered, which increased FcRn binding under acidic pH conditions.

Figure 21 shows the sensogram of IL-8 for the combination of H998 / L63 and Hr9 at pH 7.4 and pH 5.8 measured by Biacore.

Figure 22 shows changes in human IL-8 concentration in mouse plasma when H998 / L63 or H89 / L118 was administered as a mixture of 2 mg / kg and human IL-8 in mice.

Figure 23 shows changes in human IL-8 concentration in mouse plasma when H89 / L118 was administered as a mixture of 2 mg / kg or 8 mg / kg and human IL-8 in mice.

Figure 24 shows changes in human IL-8 concentration in mouse plasma when H89 / L118 or H553 / L118 was administered as a mixture of 2 mg / kg or 8 mg / kg and human IL-8 in mice.

Figure 25A shows the relative change in the concentration-dependent chemiluminescence of the antibody Hr9, H89 / L118, or H553 / L118 before storage in plasma.

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

Figure 25C shows that after storage in plasma for 2 weeks, antibodies Hr9, H89 / L118 Or H553 / L118 antibody concentration-dependent relative change in chemiluminescence.

Figure 26 shows predicted frequencies 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 for each anti-IL-8 antibody (H496 / L118, H496v1 / L118, H496v2 / L118, H496v3 / L118, H1004 / L118, or H1004 / L395) predicted by EpiMatrix, and The predicted frequency of ADA for other pre-existing therapeutic antibodies.

Figure 28A shows the relative change in antibody concentration-dependent chemiluminescence with antibody Hr9, H89 / L118, or H1009 / L395-F1886 before storage in plasma.

Figure 28B shows the change in the relative value of antibody concentration-dependent chemiluminescence with antibody Hr9, H89 / L118, or H1009 / L395-F1886 after stored in plasma for one week.

Figure 28C shows the change in the relative value of antibody concentration-dependent chemiluminescence with antibody Hr9, H89 / L118, or H1009 / L395-F1886 after stored in plasma for 2 weeks.

Fig. 29 shows changes in human IL-8 concentration in mouse plasma when H1009 / L395, H553 / L118, and H998 / L63 and human IL-8 were administered as a mixture to mice.

Figure 30 shows the extent to which the extracellular matrix binds when Hr9, H89 / L118 or H1009 / L395 is added to the extracellular matrix alone and as a mixture 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 separately administered or human IL-8 is administered to human FcRn transgenic mice in a mixture, small Changes in antibody concentration in rat plasma.

Figure 32 shows the predicted frequencies of ADA for H1009 / L395 and H1004 / L395 predicted by EpiMatrix and the predicted frequencies 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. It was administered to H89 / L118-IgG1, which has the variable region of H89 / L118 and the Fc region of natural 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 the antibody, which has a variable region of H1009 / L395, and its Fc region is a variant (F1886m, F1886s or F1974m) against each FcyR.

Figure 35 shows changes in human IL-8 concentration in mouse plasma. A mixture of anti-IL-8 antibody and human IL-8 was administered to human FcRn gene transgenic mice. In this case, the anti-IL-8 antibody is H1009 / L395-IgG1 (2mg / kg), including the variable region of H1009 / L395 and the Fc region of natural human IgG1, or H1009 / L395-F1886s (2, 5 Or 10 mg / kg), including the variable region of H1009 / L395 and the modified Fc region.

Figure 36 shows the change in antibody concentration in the plasma of Malay monkeys, which were administered to Hr9-IgG1 or H89 / L118-IgG1, both of which include the Fc region of natural human IgG1, or to H1009 / L395-F1886s or H1009 / L395-F1974m, both of which include a modified Fc region.

Figure 37 shows that in the case of antibody variable region modification in C57BL6J mice IgE plasma concentration time curve for some of the anti-IgE antibodies.

Figure 38 (Figures 38A-38D) shows the Octet sensing pattern 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 the C5 plasma concentration time curve of some anti-C5 bispecific antibodies in C57BL6J mice for antibody variable region modification.

Figure 40 shows the time course of IgE plasma concentration of some anti-IgE antibodies in C57BL6J mice for antibody constant region modification.

Non-limiting embodiments that disclose A, B, or C are described below. All the implementations described in the following examples are intended to be correctly understood, and are not limited to any patent practice, regulations, rules, or narrow interpretations of the content described in the examples in countries that want to obtain patent rights for this application.

Reveal A or Reveal B

In some embodiments, it is disclosed that A is about an antibody, including an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, and whose isoelectric point (pI) is at least 1 amino acid residue that may be exposed on the surface of the antibody. And increase (also known as "ion concentration-dependent antibody with increasing isoelectric point value" in the category of revealing A; and the antigen-binding domain of this antibody is also called "ion concentration-dependent antigen-binding domain with increasing isoelectric point value" "). This invention is based in part on the inventor's unexpected discovery that elimination of antigen 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 surface of the antibody Disability (For example, when the antibody is administered in vivo); and the binding of the antibody to the extracellular matrix can be increased using an ion concentration-dependent antibody whose isoelectric point value is increased (raised). This invention is also based in part on the inventor's 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 an isoelectric point (pI ) An antibody that is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody (also known as "antibody with increased isoelectric point value" in the context of revealing A); and the isoelectric point (pI) An antibody that is reduced (reduced) by modifying at least one amino acid residue that may be exposed on the surface of the antibody (also referred to as "antibody with reduced isoelectric point value" in the context of disclosure A. This invention belongs to disclosure A and therefore Categorized in pioneering inventions, can lead to significant technological innovations in this technical field (such as the medical field).

Of course, for example, an antibody includes an antigen-binding domain and its isoelectric point is increased by modifying at least one amino acid residue that may be exposed on the surface of the antibody, and the antigen-binding activity of the antigen-binding domain is further modified according to ionic concentration conditions The change is also included in the category of disclosure A described here (herein, within the scope of this disclosure A, such an antibody is also referred to as an "ion concentration-dependent antibody having an increased isoelectric point value").

Of course, for example, an antibody includes an ion concentration-dependent antigen-binding domain, and the charge of at least one amino acid residue that may be exposed on the surface of the antibody is different from that before the modification of the antibody (natural antibody (such as natural Ig antibody, preferably Is a natural IgG antibody), or at least one amino acid residue at the corresponding position of a control or parent antibody (such as before antibody modification or before antibody library construction or construction), and the net antibody pI increases, Included in the scope of the disclosure A disclosed herein (the disclosure A described here, such antibodies are also referred to as "isoelectric Spot-value-increasing ion concentration-dependent antibodies ").

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

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

When used in revealing the context of A and B, "modified amino acid", "modified amino acid residue" or equivalent words can be understood as, but not limited to, one molecule of the amino acid sequence of a chemical modification or Multiple (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) specific amino acids (residues), or additions, deletions, substitutions to the antibody amino acid sequence Or insert one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids. Amino acid additions, deletions, substitutions, or insertions can be performed on a nucleic acid encoded as an amino acid sequence, such as by using a site Targeting mutation (Kunkel et al., Proc. Natl. Acad. Sci. USA 82: 488-492 (1985)) or overlapping extension PCR; maturation via affinity of the antibody, or dragging using the heavy or light chain of the antibody; or Antigen panning-based selection using phage libraries is used (Smith et al., Method Enzymol. 217: 228-257 (1993)); and these are performed separately or in appropriate combinations. Such amino acid modification is not limited but is preferably performed by replacing one or more amino acid residues of the antibody amino acid sequence with different amino acids (individually). Addition, deletion, substitution or insertion and modification of amino acid sequences using humanization or chimerization can be performed using methods known in the art. Modification or modification of amino acids (residues), such as amino acid additions, deletions, substitutions, or insertions, can also be performed on the antibody variable region or antibody constant region of an antibody for the production of an antibody that discloses a recombinant antibody for A or B.

In one embodiment, within the scope of disclosures A and B described herein, substituted amino acids (residues) refer to substitutions with different amino acids (residues) and can be designed to modify, for example, (a) to (c ) Matters: (a) the polypeptide backbone structure in the region of the patch or helical configuration; (b) the charge or hydrophobicity of the target site; or (c) the size of the side chain.

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

Substitution of amino acid residues in the same group is called conservative substitution, and substitution of amino acid residues between different groups is called no non-conservative substitution. The amino acid residue substitution may be a conservative substitution, a non-conservative substitution, or a combination thereof. There are some suitable methods that can be used to replace amino acids with amino acids other than natural amino acids. (Wang et al., Annu. Rev. Biophys. Biomol. Struct. 35: 225-249 (2006); Forster et al., Proc. Natl. Acad. Sci. USA 100 (11): 6353-6357 (2003)). For example, a cell-free translation system can be used, including tRNA, in which an unnatural amino acid is linked to an amber suppressor tRNA (Clover Direct (Protein Express)) complementary to the UAG codon that is a stop codon.

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

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

Within the scope of disclosure A and B described herein, the term "prolonged half-life in plasma" or "shortened half-life in plasma" or equivalent terms of the target molecule (which may be an antigen or an antibody) may be used in addition to In addition to the half-life (t1 / 2) parameter expression, it is expressed in any other parameters, such as the average residence time in plasma, the clearance rate (CL) in plasma, and the area under the concentration curve (AUC) (Pharmacokinetics: Enshuniyoru Rikai (Understanding through practice) Nanzando). These parameters can be specifically evaluated using non-separated analysis using the experimental procedure attached to WinNonlin (Pharsight). Those skilled in the art know that these parameters are normally related to each other.

Within the scope of disclosures A and B described herein, "antigenic determinant" refers to the antigenic determinant in the antigen and refers to the site on the antigen to which the antigen-binding domain of the antibody binds. Therefore, an epitope can be defined by, for example, a 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 specified according to its specific sugar chain structure. It was revealed that the antigen-binding domains of A and B can bind to a single epitope or different epitopes on the antigen.

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

Due to the epitope configuration, the amino acids that make up this epitope generally do not exist continuously in the primary sequence. Antibodies recognize the three-dimensional epitope configuration of a peptide or protein. Methods for determining the 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 natural immunoglobulins ("Ig") for short), and molecules and variants derived therefrom, such as Fab, Fab ', F (ab') ₂ , Diabody antibody, ScFv (Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; WO93 / 11161; Peer et al., Nature Nanotechnology 2: 751-760 (2007)), low molecular weight antibodies (minibodies) (Orita et al., Blood 105: 562-566 (2005)), scaffold proteins, single-arm antibodies (including embodiments of all single-arm antibodies described in WO2005 / 063816), Multispecific antibodies (such as bispecific antibodies: antibodies specific for two different epitopes, including antibodies that recognize different antigens, and antibodies that recognize different epitopes on the same antigen). Within the scope of disclosures A and B described herein, "bispecific antibodies" are not limited, but can be prepared, for example, as antibody molecules with a common L chain described in WO2005 / 035756, or by the method described in WO2008 / 119353 Mixing a common type of antibody with a constant region similar to IgG4 results in an exchange between the two types of antibodies (known in the art as a "Fab-arm exchange" method). In an alternative embodiment, it may be an antibody having a structure in which a heavy chain variable region and a light chain variable region are linked together into a single chain (eg, sc (Fv) ₂ ). Or it can be an antibody-like molecule (such as scFv-Fc), which is obtained by linking the Fc region (constant region lacking the CH1 domain) to scFv (or sc (Fv) ₂ ), and the heavy chain variable region (VH) is linked To the light chain variable region (VL). A multispecific antibody composed of scFv-Fc having a (scFv) ₂ -Fc structure, and the first and second polypeptides are each a VH1-linker-VL1-Fc and a VH2-linker-VL2-Fc. Alternatively, it can be an antibody-like molecule in which a single domain antibody is linked to the Fc region (Marvin et al., Curr. Opin. Drug Discov. Devel. 9 (2): 184-193 (2006)), an Fc fusion protein (such as immunoadhesin ) (US2013 / 0171138), its functional fragments, functionally equivalent substances, and its sugar chain modified variants. Here, a natural IgG (for example, natural IgG1) refers to a polypeptide having the same amino acid sequence as a naturally occurring IgG (for example, natural IgG1), and belongs to the class of antibodies substantially encoded by the immunoglobulin gamma gene. Natural IgG can be its spontaneous mutant and the like.

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

For example, an IgG antibody can be digested with papain to cut into 2 units of Fab (region) and 1 unit of Fc (region). Papaya enzymes will cut the hinge region (also referred to as "A" and "B") "Hinge"), the heavy chain Fab region is linked to the Fc region. Generally, the Fab region contains an antigen-binding domain. Phagocytic cells such as white blood cells and macrophages have receptors that bind to the Fc region (Fc receptor), and can recognize and phagocytose this antigen through Fc receptor antibodies that bind to an antigen (opsonization) . At the same time, the Fc region is involved in the intermediate immune response, such as ADCC or CDC, and has effector function, that is, the antibody induces a response when it binds to the antigen. This antibody effector function is known to vary depending on the type of immunoglobulin (structural isotype). The Fc region of the IgG class may be indicated as, for example, a cysteine at position 226 or proline at position 230 (EU numbering) to the C-terminus; however, the Fc region is not limited thereto. The Fc region can be appropriately obtained by partially digesting the monoclonal antibody IgG1, IgG2, IgG3, or IgG4 antibody with a protease such as pepsin, and then extracting the adsorbed fraction from the protein A or protein G column.

Within the scope of disclosure A and B described herein, the amino acid residue positions of the variable regions (CDR and / or FR) of the antibody are shown by Kabat, and the amino acid residue positions of the constant region or Fc region are Kabat's amino acid positions are expressed in accordance with EU numbering (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991).

In the context of the disclosures A and B described herein, "library" refers to molecules (populations) such as polyantibodies that have sequence variability, wherein each sequence may be the same or different from each other; polyfused polypeptides containing the antibody; or encode Nucleic acids or polynucleotides for these amino acid sequences are detailed in WO2013 / 125667 (e.g. paragraph 0121-0125). The library may contain, for example, an antibody molecule of at least 10 ^4, preferably at least ¹⁰⁵ antibody molecule, more preferably at least ¹⁰⁶ antibody molecules, particularly preferably at least ¹⁰⁷ or more antibody molecules. The library may be a phage library. The term "mainly comprising" means that the antibody can have different antigen-binding activities, corresponding to a certain portion of most independent colonies of different sequences in the library. In one embodiment, an animal that has been immunized based on a specific antigen, an infected patient, a person who has been vaccinated to have an increased antibody titer in the blood, or a lymphocyte from cancer or an autoimmune disease The immune library constructed by the antibody gene can be suitably used as a random variable region library. In an alternative embodiment, an unaltered library containing unaltered sequences (unbiased antibody sequences in stock) constructed from antibody genes derived from lymphocytes of healthy people 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)). An amino acid sequence containing an unaltered sequence may refer to an acquirer from an unaltered library. In an alternative embodiment, the CDR sequence from the genomic DNA V gene or the reconstructed functional V gene CDR sequence can be replaced with a synthetic library containing a set of synthetic oligonucleotides encoding a sequence of an appropriate length codon set as appropriate Random variable region library. In this case, sequence variation is also observed in the CDR3 gene, and it is also possible to replace only the heavy chain CDR3 sequence. The standard method for generating amino acid diversity in the variable region of an antibody can be to increase the variation of the position of amino acid residues that may be exposed on the surface of the antibody.

In one embodiment, if the antibody of A or B is disclosed, for example, having a structure that is substantially equivalent or similar to a natural Ig antibody, it generally has a variable region ("V region") [heavy chain variable region ("VH region") and Light chain variable region ("VL region")] and constant region ("C region") ["heavy chain constant region (" CH region ") and light chain constant region (" CL region ")]. The CH region is subdivided 3: CH1 to CH3. Generally, the Fab region of the heavy chain contains the VH region and CH1, and the Fc region of the heavy chain generally includes CH2 and CH3. Generally, the hinge region is located between CH1 and CH2. The variable region is generally complementary Determining regions ("CDR") and framework regions ("FR"). Generally, the VH region and the VL region each have three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR4). Generally, the 6 CDRs of the variable regions of the heavy and light chains interact to 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 has the ability to recognize and bind to antigens.

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

In one embodiment, if it is revealed that the antibody of A or B has a heavy chain, for example, the heavy chain may be any of the γ chain, the μ chain, the α chain, the δ chain, and the ε chain, or may be derived from any one. The antibody of A or B has a light chain. For example, the light chain may be a κ chain or a λ chain, or derived from one of them. Within the scope of disclosure A and B described herein, this antibody can be of any structural isotype (such as IgG, IgM, IgA, IgD, or IgE) and any subclass (such as human IgG1, IgG2, IgG3, IgG4, IgA1) And IgA2; mouse IgG1, IgG2a, IgG2b, and IgG3) or any of them, but it is not limited thereto.

Within the scope of the disclosures A and B described herein, the "antigen-binding domain" may have any structure as long as it binds to the antigen of interest. Such domains may include, for example, the variable regions of the antibody heavy and light chains (e.g., 1 to 6 CDRs); a module of about 35 amino acids called the A domain, which is included in Avimer, a cell membrane present in the body Proteins (WO2004 / 044011 and WO2005 / 040229); Adnectin, which contains a 10Fn3 domain, binds to the protein in glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925); Affibody, which has 58 amine groups as constituting protein A Scaffold of the IgG binding domain of the acid 3 helix bundle (WO1995 / 001937); Designed Ankyrin Repeat Protein (DARPin), which is the region exposed on the surface of the Ankyrin repeat (AR) molecule, has the following Structure: Primary unit with a turn consisting of 33 amino acid residues, 2 anti-parallel spirals, and repeating stacked loops (WO2002 / 020565); Anticalin, etc., which are 4 loop regions and support 8 The center of the antiparallel strand twists one side of the tube structure, and apolipoproteins such as neutrophil gelatinase-associated apolipoprotein (NGAL) are highly conserved (WO2003 / 029462); and by Ma Recessed area formed by the parallel plate structure in the shoe-like structure, The structure is formed by stacking repeats of the leucine-rich repeat (LRR) module of the variable lymphoglobulin receptor (VLR), which has no immunoglobulin structure and is used as a jawless vertebrate such as the lamprey and Blind eel acquired immune system (WO2008 / 016854). It is revealed that the A or B antigen binding domain should preferably include 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 the disclosure A, the “ion concentration” is not particularly limited, and refers to a hydrogen ion concentration (pH) or a metal ion concentration. Here, the "metal ion" may 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, and IV other than carbon and silicon. Elements, group VIII elements, such as iron and platinum group elements, elements belonging to subgroup A of groups V, VI, and VII, and metal elements such as antimony, bismuth, and thallium ions. Metal atoms have the property of releasing electrons to become cations. Called free tendency. Metals with strong free hydrogenation are speculated to be chemically active.

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

In one embodiment of the disclosure A, the "ion concentration condition" may focus on the difference in the biological behavior of the ion concentration-dependent antibody between low ion concentration and high ion concentration. Also, "its antigen-binding activity varies depending on the ion concentration conditions" may mean that the antigen-binding activity of the ion-concentration-dependent antigen-binding domain of A or B or the ion-concentration-dependent antibody is changed between low ion concentration and high ion concentration. Such situations include, for example, but not limited to, higher (stronger) or lower (weaker) antigen-binding activity at higher ion concentrations than at lower ion concentrations.

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

In one embodiment that discloses the context of A, an ion concentration-dependent antigen binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen binding domain with an increased isoelectric point value, and an ion concentration-dependent antibody with an increased isoelectric point value. Can be obtained from a library mainly including antibodies with different sequences (variability), the antigen-binding domain of the antibody contains at least one amino acid residue that will cause the antigen-binding domain or the antibody's antigen-binding activity to change according to the ion concentration conditions base. This antigen-binding domain may suitably be located within a light chain variable region (which may be modified) and / or a heavy chain variable region (which may be modified). In order to construct a library, such a light or heavy chain variable region can be combined with a heavy or light chain variable region that has been constructed to form a random variable region sequence library. If the ion concentration is hydrogen or calcium ion concentration, non-limiting examples of the library include, for example, a library of heavy chain variable regions that has been constructed to constitute a random variable region sequence library, and a library of light chain variable region sequences in which the light chain is variable The germ cell sequence of the region sequence is, for example, the amino acid residue of SEQ ID NO: 1 (Vk1), SEQ ID NO: 2 (Vk2), SEQ ID NO: 3 (Vk3), or SEQ ID NO: 4 (Vk4). At least one amino acid residue that changes antigen-binding activity depending on the ion concentration. If the ion concentration is a calcium ion concentration, the library includes, for example, the sequence of the heavy chain variable region of SEQ ID NO: 5 (6RL # 9-IgG1) or SEQ ID NO: 6 (6KC4-1 # 85-IgG1) and the constructed composition. A random variable region sequence library of light chain variable regions or a combination of light chain variable regions with germ cell sequences.

In one embodiment, if the ion concentration is a calcium ion concentration, the high calcium ion concentration is not particularly limited to a specific value; but the concentration may be selected from between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, and between 200 μM With 2 mM, or between 400 μM and 1 mM. Preferably, it is 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 may 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, it is selected from a concentration between 1 μM and 5 μM, which is close to 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 according to the conditions of metal ion concentration (such as calcium ion concentration) can be easily determined according to known methods, for example, using the method disclosed in A or the method described in WO2012 / 073992 method. For example, the antigen-binding activity of this antigen-binding domain or an antibody containing this domain can be measured and compared at low and high calcium ion concentrations. In this case, the conditions other than the calcium ion concentration are preferably the same. The 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, in conditions of HEPES buffer at 37 ° C, or using BIACORE (GE Healthcare) or other equipment.

In an embodiment that discloses the context of A, the ion concentration dependent antigen binding domain, ion concentration dependent antibody, ion concentration dependent antigen binding domain with increased isoelectric point value, or ion concentration dependent antibody with increased isoelectric point value. Antigen-binding activity is 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 condition of low calcium ion concentration to the antigen-binding activity under the condition of high calcium ion concentration is not limited; however, the KD (dissociation constant) of the antigen under the condition of low calcium ion concentration relative to the high calcium ion concentration The KD ratio of the conditions, that is, KD (3 μM Ca) / KD (2 mM Ca) is preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. KD (3μM Ca) / KD (2 The upper limit value of mM Ca) is not limited, and may be any value, such as 400, 1000, or 10,000.

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, an 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 cytometer.

Alternatively, for example, the dissociation rate constant (kd) can be used as another indicator to represent the ratio of binding activity. If the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator of the ratio of antigen-binding activity, the ratio of the dissociation rate constant (kd) in the condition of low calcium ion concentration to the dissociation rate constant (kd) in 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, still 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, such as 50, 100, or 200.

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

In one embodiment, the method for producing or screening an antigen-binding activity of a calcium-ion-dependent antigen-binding domain or a calcium-ion-dependent antibody or a library thereof under conditions of high calcium ion concentration conditions is higher than under conditions of low calcium ion concentration is not limited . Methods include, for example, methods described in WO2012 / 073992 (eg, paragraph 0200-0213).

This method may include, for example: (a) determine the antigen-binding activity of the 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) select at low calcium ion concentration The antigen-binding activity of the condition is lower than the antigen-binding domain or antibody of the antigen-binding activity under the condition of high calcium ion concentration.

Alternatively, the method may include, for example: (a) contacting the antigen with an antigen-binding domain or antibody or a library thereof under conditions of high calcium ion concentration; (b) contacting the antigen-binding domain or antibody that binds to the antigen in step (a) with low calcium Ionic concentration conditions; and (c) an antigen-binding domain or antibody that is isolated in step (b).

Alternatively, the method may include, for example: (a) contacting an antigen with an antigen-binding domain or antibody or its library under conditions of low calcium ion concentration; (b) selected in step (a) not binding to the antigen or having low antigen-binding capacity Antigen-binding domain or antibody; (c) allowing the antigen-binding domain or antibody selected in step (b) to bind to this antigen under conditions of high calcium ion concentration; and (d) antigen-binding isolated from step (c) that binds to the antigen Domain or antibody.

Alternatively, the method may include, for example: (a) contacting an antigen-binding domain or an antibody or a library thereof with an immobilized antigen column under high calcium ion concentration conditions; (b) washing out from the column under low calcium ion concentration conditions in a step (a) Binding to the tube The antigen-binding domain or antibody of the column; and (c) the antigen-binding domain or antibody isolated from step (b).

Alternatively, the method may include, for example: (a) passing the antigen-binding domain or antibody or its library under low calcium ion concentration conditions through a column of immobilized antigen to collect the antigen-binding domain or antibody washed out without binding to the column; ( b) allowing the antigen-binding domain or antibody collected in step (a) to bind to the antigen under conditions of high calcium ion concentration; and (c) isolating the antigen-binding domain or antibody that binds to the antigen in step (b).

Alternatively, the method may include, for example: (a) contacting an antigen with an antigen-binding domain or antibody or a library thereof under conditions of high calcium ion concentration; (b) obtaining an antigen-binding domain or antibody that binds to the antigen in step (a); c) incubating the antigen-binding domain or antibody obtained in step (b) at a low calcium ion concentration; and (d) isolating the antigen-binding domain or antibody, which has a weaker antigen-binding activity in step (c) than that in step (b) Selection criteria.

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

In the context of revealing A, in one embodiment, the antigen-binding domain or antibody as a starting material may be a modified antigen-binding domain or antibody that is modified by modifying at least one amino acid residue that may be exposed on its surface Increase in charge Isoelectric point value. In an alternative embodiment, if an amino acid that changes the binding activity of an ion concentration-dependent antigen-binding domain is introduced into the sequence, at least one amino acid residue may be exposed to the antigen-binding domain or the surface of the antibody. The charge modification is introduced 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 by fusion tumors obtained by immunizing animals, or B cells from immunized animals or Its library; or an antigen-binding domain, antibody, or library obtained by introducing natural or unnatural amino acid mutations that can chelate calcium (described below) (e.g., a library with increased calcium-chelable amino acids, Or a library of calcium chelate amino acids introduced at specific sites).

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

In an embodiment that reveals the context of A, if the ion concentration is a calcium ion concentration, an amino acid having metal chelating activity can be used as the ion point dependent antigen-binding domain or ion concentration dependent that has an increased isoelectric point value. Amino acid of the binding activity of the sex antigen binding domain. For example, any amino acid can be suitably 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 preferably include, but are not limited to, Ser (S), Thr (T), Asn (N), Gln (Q), Asp (D), and Glu (E).

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

Troponin C, calmodulin, parvalbumin, and myosin light chains are known to have multiple calcium-binding sites. It is speculated that the molecular evolution came from a common origin. In one embodiment, one or more of the light chain CDR1, CDR2, and CDR3 can be designed to include a combination thereof Phantom. For the above purpose, for example, cadherin domains; EF hands contained in calmodulin; C2 domains included in protein kinase C; Gla domains included in coagulation factor IX; asialoglycoprotein receptors or manna Type C lectin of the sugar binding receptor; A domain contained in the LDL receptor; Annexin; thrombospondin type-3 domain; and EGF-like domain.

In one embodiment, if the ion concentration is a hydrogen ion concentration (pH), the concentration conditions of protons, ie, hydrogen nuclei, are used synonymously with the hydrogen index (pH). If the hydrogen ion activity of the aqueous solution is represented by aH ⁺ , the pH is defined as -1og10aH ⁺ . If the ionic strength of the aqueous solution is low (for example, 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 conditions may focus on the difference between the biological behavior of pH-dependent antibodies in high hydrogen ion concentration (acidic pH range) and low hydrogen ion concentration (neutral pH range) against hydrogen ion concentration conditions or pH conditions. For example, in the context of revealing A, "the antigen-binding activity under conditions of high hydrogen ion concentration (acidic pH range) is lower than that under conditions of low hydrogen ion concentration (neutral pH range)" means an ion concentration-dependent antigen The antigen-binding activity of a binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen with an increased isoelectric point value, or an ion concentration-dependent antibody with an increased isoelectric point value should be selected from the group consisting of pH 4.0 to pH 6.5, and preferably pH 4.5 to pH 6.5, more preferably pH 5.0 to pH 6.5, and more preferably pH 5.5 to pH 6.5, more preferably pH 6.7 to pH 9.5, more preferably pH 7.0 than a pH selected from pH 6.7 to pH 10.0 The pH is weak to pH 9.0, and more preferably pH 7.0 to pH 8.0. Ideally, the above expression means: the pH in the living body in the early endosome, the antigen-binding activity is weaker than the activity of the plasma pH in the living body; and in particular it may refer to: the antigen-binding activity of the antibody, for example at pH 5.8 is weaker than for example pH 7.4.

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

Within the scope of disclosure A, "neutral pH range" (also known as "low hydrogen ion concentration", "high pH", "neutral pH condition", or "neutral pH") is not particularly limited unless the context specifically indicates 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, for example, pH 7.0 can be used.

Within the scope of the disclosure A, "acid pH range" (also referred to as "high hydrogen ion concentration", "low pH", "acid pH condition" or "acid pH") is not particularly limited to a specific value unless the context specifically indicates otherwise ; 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 is connected to the living hydrogen ion concentration in the early endosome, but for convenience of measurement, for example, pH 6.0 can be used.

In an embodiment that reveals the context of A, if the ion concentration is Antigen-binding activity of hydrogen ion concentration, ion concentration-dependent antigen binding domain, ion concentration-dependent antibody, ion concentration-dependent antigen binding domain with increased isoelectric point value, or ion concentration-dependent antibody with increased isoelectric point value, in Sexual pH conditions are higher than in acidic pH conditions. 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 dissociation constant (KD) of the antigen at acidic pH is relative to the KD at neutral pH. The ratio, that is, KD (acid pH range) / KD (neutral pH range), (for example, KD (pH 5.8) / KD (pH 7.4)) may be 2 or more; 10 or more; or 40 or more. The upper limit of KD (acid pH range) / KD (neutral pH range) is not limited, and may be any value, such as 400, 1000, or 10,000.

In an alternative embodiment, the dissociation rate constant (kd) can also be used as an indicator to represent the aforementioned binding activity ratio. If the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator of the binding activity ratio, the ratio of the dissociation rate constant (kd) under high hydrogen ion concentration conditions to the dissociation rate constant (kd) under low hydrogen ion concentration conditions, That is, kd (acidic pH range) / kd (neutral pH range) may be 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit value of kd (acid pH range) / kd (neutral pH range) is not limited, and may be any value, such as 50, 100, or 200.

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

In one embodiment, the pH-dependent antigen-binding domain or pH-dependent The method of the lysing antibody or its library is not limited. Such methods include, for example, the methods described in WO2009 / 125825 (eg, paragraph 0158-0190).

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

Alternatively, the method may include, for example: (a) contacting an antigen with an antigen-binding domain or an antibody or a library thereof at neutral pH conditions; (b) in an acidic pH condition, which will bind step (a) to the antigen-binding domain of the antigen or Antibody incubation; and (c) isolating the antigen-binding domain or antibody dissociated in step (b).

Alternatively, the method may include, for example: (a) contacting an antigen with an antigen-binding domain or antibody or a library thereof under acidic pH conditions; (b) selecting an antigen that does not bind to the antigen or has low antigen-binding capacity in step (a) Domain or antibody; (c) allows the antigen to bind to the antigen-binding domain or antibody selected in step (b) at neutral pH; and (d) isolates the antigen-binding domain or antibody that binds to this antigen in step (c) .

Or this method may include, for example: (a) The antigen-binding domain or antibody or its library is contacted with a column that has been immobilized with the antigen at a neutral pH; (b) The antigen-binding domain that is bound to the column at step (a) is washed out from the column at an acidic pH Or antibody; and (c) the antigen-binding domain or antibody eluted from step (b) separately.

Alternatively, the method may include, for example: (a) allowing the antigen-binding domain or antibody or its library to pass through a column of immobilized antigen under acidic pH conditions to collect the washed-out unbound antigen-binding domain or antibody; (b) The neutral pH conditions allow the antigen-binding domain or antibody collected in step (a) to bind to this antigen; and (c) the antigen-binding domain or antibody that is isolated from step (b) to bind to this antigen.

Alternatively, the method may include, for example: (a) contacting the antigen with an antigen-binding domain or antibody or a library thereof at a neutral pH condition; (b) obtained in step (a) the antigen-binding domain or antibody that binds to the antigen; (c) ) Cultivating the antigen-binding domain or antibody obtained in step (b) under acidic pH conditions; and (d) isolating the antigen-binding domain or antibody whose antigen-binding activity in step (c) is weaker than that selected in step (b) Guidelines.

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

In the context of revealing A, in one embodiment, the antigen-binding domain or antibody as a starting material may be a modified antigen-binding domain or antibody that is modified by modifying at least one 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 changes the binding activity of an ion concentration-dependent antigen-binding domain is introduced into the sequence, at least one amino acid residue may be exposed to the antigen-binding domain or the surface of the antibody. The charge modification is introduced 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 by fusion tumors obtained by immunizing animals, or B cells from immunized animals or Its library; or an antigen-binding domain, antibody, or library obtained by introducing a natural or unnatural amino acid mutation with a side chain having pKa 4.0-8.0 (described below) (e.g., one with a side chain having pKa 4.0-8.0 Library of natural or unnatural amino acid mutations, or a library of natural or non-natural amino acid mutations with pKa 4.0-8.0 side chains introduced at specific sites). Such a preferred antigen-binding domain may have, for example, an amino acid sequence in which at least one amino acid residue is substituted with an amino acid having a side chain pKa of 4.0-8.0 and / or inserted with a side chain pKa of 4.0-8.0 amino acids as described in WO2009 / 125825.

In an embodiment that reveals the context of A, the site for introducing a mutation with an amino acid having a side chain pKa of 4.0-8.0 is not limited. This mutation can be introduced at any site, as long as the antigen-binding activity is compared to that before substitution or insertion. It becomes weaker in the acidic pH range than in the neutral pH range (KD (acid pH range) / KD (neutral pH range) value increases or kd (acid pH range) / kd (neutral pH range) value increases)) . If the antibody has a variable region or CDR, this site can be in the variable region or CDR Inside. The number of substituted or inserted amino acids may be appropriately determined by those having ordinary knowledge in the technical field; and the number may be one or more. In addition to the substitution or insertion described above, other amino acids may be deleted, added, inserted, and / or substituted or modified. Substitution or insertion of amino acids with side chains pKa 4.0-8.0 can be performed in a random manner using scanning methods, such as histamine scanning, in which propylamine in alanine scanning known to those skilled in the art is known Replace acid with histidine, and / or select from the antigen-binding domain or antibody because these amino acids are randomly substituted or inserted into the mutation. Compared to the mutation, the KD (acid pH range) / KD (neutral pH range) value or Antibodies or libraries with increased kd (acidic pH range) / kd (neutral pH range) values.

Furthermore, the antigen-binding domain or antibody should preferably have no significant decrease, no substantial decrease, substantially equal or increase in antigen-binding activity in the neutral pH range before and after these mutations; and in other words, the activity can be maintained at least 10% or higher, preferably It is 50% or higher, more preferably 80% or higher, and even more preferably 90% or higher or higher. If the binding activity of the antigen-binding domain or antibody is reduced due to substitution or insertion of the amino acid of pKa 4.0-8.0, the binding activity can be substituted, deleted, added, or inserted into one or more amino acids outside the substitution or insertion site. Of the site while recovering or increasing.

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

In one embodiment, within the scope of disclosure A, arbitrary amino acid residues can be suitably used as amino acid residues that change the antigen-binding activity of the antigen-binding domain or antibody as a condition of hydrogen ion concentration. Specifically, such amino acid residues may include those having a side chain pKa of 4.0-8.0. Electron-donating amino acids include, for example, natural amino acids, such as His (H) and Glu (E), and non-natural 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 6.0-7.0, especially His (H).

Within the scope of disclosure A, unless otherwise specified and unless inconsistent with the context, it should be understood that the isoelectric point (pI) can be a theoretically or experimentally determined isoelectric point, also known as "pI".

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

In one embodiment, is it compared to the antibody before modification (natural antibody (such as natural Ig antibody, preferably natural IgG antibody) or reference antibody (such as antibody modification or library construction or antibody under construction)) The isoelectric point value of an antibody with an increased isoelectric point value or an antibody that reveals A can be determined by using the above method. In addition, or in place of the above method, it can be used from, for example, mice, rats, rabbits, and dogs. Antibody pharmacokinetic test of human, monkey or human plasma, group The combination is determined by, for example, BIACORE, cell proliferation analysis, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), or fluorescence immunoassay.

Within the scope of record 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 a solvent molecule (typically mostly a water molecule). However, the side chain does not necessarily have to be completely in contact with the solvent molecule. If even a part of the side chain is in contact with the solvent molecule, the amino acid residue is still defined as the "amino acid on the surface". The amino acid residues located on the surface of the polypeptide can include amino acid residues located close to the surface of the antibody, so they can have a mutual charge effect with even amino acid residues with only a part of the side chain in contact with the solvent molecule. Those skilled in the art can use commercial software to make homology models of polypeptides or antibodies using, for example, homology simulation methods. Alternatively, methods such as X-ray crystallization can be used. The amino acid residues that may 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 Insight II program (Accelrys). The exposed surface area 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 parts that can be exposed to the surface can be determined using software suitable for protein simulation and the three-dimensional structure information obtained from this antibody. Available software for this purpose includes, for example, SYBYL Biopolymer Module software (Tripos Associates). The algorithm requires user input 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 the surface exposed area and area for personal computers (Pacios, Comput.Chem18 (4): 377- 386 (1994); J. Mol. Modelling. 1: 46-53 (1995)). Based on the above information, as described above, an appropriate amine for the surface of the polypeptide constituting the antibody can be selected. Acid residues.

Methods to increase the isoelectric point of a protein, such as reducing the number of amino groups (such as aspartic acid and glutamic acid) with negatively charged side chains at neutral pH and / or increasing the positively charged side chains Acid number (such as arginine, lysine, and histidine). Amino acid residues with negatively charged side chains have a negative charge expression of -1 at pH conditions, which is sufficiently larger than their side chain pKa, a theory well known to those skilled in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the negative charge of the side chain under neutral pH conditions (such as a solution at pH 7.0) is expressed as -1. In contrast, amino acid residues with a positively charged side chain are positively expressed at +1 at pH conditions, which is sufficiently lower than their side chain pKa. For example, the theoretical pKa of the side chain of arginine is 12.5, and the positive charge of the side chain under neutral pH conditions (such as a solution at pH 7.0) is expressed as +1. The uncharged amino acid residues of the side chains at neutral pH conditions (e.g., solutions at pH 7.0) are known to have 15 types of natural amino acids, namely alanine, cysteine, phenylalanine, glycine, Isoleucine, leucine, methionine, aspartic acid, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. It should of course be understood that the amino acid used to change 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 a neutral pH condition (such as a solution at pH 7.0), the charge of the protein of interest can be modified by +1. For example, due to the amino acid sequence of the protein, aspartic acid (Residue) or glutamic acid (residue) (the side chain of which is negatively charged-1) is replaced with an amino acid (residue) having an uncharged side chain. You can also add +1 to charge modification of the protein of interest, for example, by replacing an amino acid (residue) with an uncharged side chain with arginine or lysine (its side chain has a positive charge +1) for amine group Acid (residue). In addition, a charge modification of +2 can be imparted to the protein of interest, for example, by substituting aspartic acid or glutamic acid (the side chain has a negative charge of -1) to arginine or lysine (the side chain of which has a positive charge) +1). Or in order to increase the isoelectric point of the protein, the amino acid sequence of the protein can be added or inserted into the amino acid with an uncharged side chain and / or the amino acid with a positively charged side chain, or it can be deleted from The amino acid sequence of a protein is an amino acid with an uncharged side chain and / or an amino acid with a negatively charged side chain. It should be understood that in addition to the charge from the side chain, for example, the charge from the main chain of the N-terminal and C-terminal amino acid residues of the protein (NH ^{3+ of the} N-terminal amino group and COO ^- of the carbonyl group of the C-terminal) . Therefore, the isoelectric point of a protein can also be increased by some additions, deletions, substitutions or insertions of functional groups from the main chain.

Those skilled in the art can understand the net charge obtained by modifying one or more amino acid sequences (residues), focusing on the presence or strength of the charge of the amino acid (residues). The effect of the isoelectric point of a protein or protein is not only (or essentially) dependent on the amino acid sequence itself or the type of target antigen that constitutes the antibody, but rather on the amino acid residues that are added, deleted, substituted or inserted Types and numbers.

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

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

The pH concentration of biological fluids (such as plasma) is in the neutral pH range. Without being bound by a specific theory, it is believed that in biological fluids, as the isoelectric point value increases, the net positive charge of an antibody with an increased isoelectric point increases, so the antibody will be more strongly subjected to a net charge than an antibody that has not increased the isoelectric point as Physiological and chemical Coulomb interactions on the surface of negative endothelial cells; binding this antibody through non-specific binding and entering the cell, resulting in shortening the antibody's plasma half-life or enhancing antigen elimination from plasma. Increasing the isoelectric point of the antibody to increase the uptake of the antibody (or antigen / antibody complex) into the cell and / or intracellular permeability is thought to reduce the concentration of the antibody in the plasma, reduce the bioavailability of the antibody, and / or Shorten the half-life of this antibody in plasma; and these phenomena are expected to occur generally in vivo, regardless of cell type, tissue type, organ type, etc. 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 in to cell.

In one embodiment, an antibody of increased isoelectric point is manufactured or screened. Methods are described, for example, in WO2007 / 114319 (e.g. paragraph 0060-0087), WO2009 / 041643 (e.g. paragraph 0115-), WO2014 / 145159 and WO2012 / 016227. Such a method may include, for example: (a) modifying a nucleic acid encoding an antibody that includes at least one amino acid residue that may be exposed on the surface of the antibody, modifying the charge of the amino acid residue and increasing the antibody, etc. Electrical point value; (b) culturing the host cell to express the nucleic acid; and (c) collecting antibodies from the host cell culture. Or this method may include, for example: (a ') a modification encoded to include at least the surface of the antibody that may be exposed A nucleic acid of an amino acid residue antibody to modify the charge of this amino acid residue; (b ') culture the host cell to express the nucleic acid; (c') collect the antibody from the host cell culture; and (d ') (Confirm or measure as needed) and select an antibody that has an increased isoelectric point compared to the antibody before modification. Here, the antibody or the reference antibody before modification as a starting material may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying the amino acid residue, an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence.

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

In an alternative embodiment, this method can be, for example, a method of making a multispecific antibody, the antibody comprising a first polypeptide and a second polypeptide and optionally a third polypeptide and a fourth polypeptide, including: (A) Modification of the first polypeptide and / or the second polypeptide and optionally the third polypeptide A nucleic acid of a polypeptide and / or a fourth polypeptide, any one or more of which includes at least one amino acid residue that may be exposed on the surface of the polypeptide to modify the charge of the amino acid residue to increase the isoelectricity of the antibody Point value; (B) culturing host cells to express the nucleic acid; and (C) collecting multispecific antibodies from the host cell culture.

Alternatively, the method may include, for example: (A ') modifying a nucleic acid encoding a first polypeptide and / or a second polypeptide and optionally a third polypeptide and / or a fourth polypeptide, any one or more of which include possible exposure to At least one amino acid residue on the surface of the polypeptide to modify the charge of the amino acid residue so as to increase the charge of the amino acid residue; (B ') culture the host cell to express the nucleic acid; (C' ) Collecting multispecific antibodies from the host cell culture; and (D ') (confirm and if necessary) select antibodies that have an increased isoelectric point compared to the one before the antibody modification.

The antibody or the pre-modified antibody or reference antibody used as the starting material herein may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying the amino acid residue, an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence.

Alternatively, the method may simply include culturing the host cells obtained in step (B) or (B ') and collecting antibodies 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 same multimer, the same multimer of the fourth polypeptide, or the different multimers of the third and fourth polypeptides).

In an alternative embodiment, this method can be, for example, a method of making a humanized or human antibody with reduced half-life in plasma, including: a group consisting of a CDR selected from human-derived CDRs, animal-derived CDRs other than human, and synthetic CDR Group of CDRs; human-derived FRs; and human constant region antibodies, (I) modifying at least one amino acid residue on at least one region selected from the group consisting of CDR, FR, and constant regions The amino acid residues corresponding to the corresponding amino acid residues before the modification have different charges, so that the isoelectric point of this antibody is increased.

Alternatively, the method may include, for example, a CDR including a group selected from human-derived CDRs, animal-derived CDRs, and synthetic CDRs; human-derived FR; and human constant region antibodies. (I ') modification is selected from CDRs. At least one amino acid residue in at least one region of the group consisting of FR, constant region and constant region may be exposed on the surface to become an amino acid residue having a different charge in the corresponding amino acid residue before the modification; and

(II ') (confirm and, if necessary) select an antibody with an increased isoelectric point compared to that before modification of the antibody.

The antibody or the pre-modified antibody or reference antibody used as the starting material herein may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying the amino acid residue, an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence.

Alternatively, for example, a pre-existing antigen-binding domain or antibody, a pre-existing library (phage library, etc.); an antibody prepared by a fusion tumor obtained by immunizing an animal, or a B cell or a library thereof from an immunized animal; or according to the above In any of the embodiments, the at least one amino acid residue that may be exposed on the surface modifies the antigen-binding domain antibody or an antigen-binding domain or antibody library with an increased isoelectric point of the above-mentioned antigen-binding domain. Body or its library.

In one embodiment of the antibody disclosed by A, the isoelectric point value may suitably be increased by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more or increased by at least 0.6, 0.7, 0.8, 0.9 or more And significantly shorten the half-life of this antibody in plasma, the isoelectric point value can be increased 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 (natural antibody (such as natural Ig antibody, preferably natural IgG antibody) or control or parent antibody ( For example, antibodies before modification or library construction or at the time of construction)). Those with ordinary knowledge in this technical field may consider the balance between pharmacological effects and toxicity, such as the number of antibodies or antigen-binding domains or the isoelectric point of the antigen, and routinely determine the optimal isoelectricity of the antibody to be disclosed in accordance with the application. Point value. Without being bound by a particular theory, it is believed that revealing that the antibody of A is beneficial in one embodiment, not only shuttles between plasma and cellular endosomes and repeatedly binds as a single antibody molecule due to the presence of an ion concentration-dependent antigen binding domain For multiple antigens, and because the isoelectric point increases, the net positive charge of this antibody increases, allowing this antibody to be quickly taken up into cells. These characteristics can shorten the half-life of the antibody in the plasma, increase the extracellular matrix binding activity of the antibody, or enhance elimination of the antigen from the plasma. The optimum isoelectric point value can be determined to benefit from these characteristics.

In an embodiment that reveals the context of A, compare this antibody before modification or before modifying at least 1 amino acid residue to increase the isoelectric point (natural antibody (such as natural Ig antibody, preferably natural IgG Antibodies) or control or parental antibodies (such as antibodies before antibody modification or library construction or during construction), which can be ion concentration-dependent antibodies). It was revealed that the antibody of A should improve the elimination of antigen from plasma by at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times. , 4.25 times, 4.5 times, 4.75 times, 5 times, 5.5 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 this antibody is administered To the living body) or its extracellular matrix binding activity may be increased, for example, by at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times, 4.75 times or 5 times or more.

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

In one embodiment, it is evaluated whether the extracellular matrix binding activity of the antibody of A is revealed compared to that before modification or modification of the antibody (native antibody (eg, a natural Ig antibody, which may be a natural IgG antibody) or a control or parent antibody (E.g. The antibody before antibody modification or library construction or at the time of construction) may be an ion concentration-dependent antibody or antibody with an increased isoelectric point). The analysis method is not limited. For example, the assay can be performed using an ELISA system, which detects the binding between the antibody and the extracellular matrix, adds the antibody to the extracellular matrix immobilization plate, and adds a labeled antibody against the antibody. Alternatively, as described in Examples 1 to 4 and WO2012 / 093704, electrochemiluminescence (ECL) can also be used, which can detect the extracellular matrix binding ability with high sensitivity. This method can be performed, for example, using an ECL system, in which a mixture of an antibody and a ruthenium antibody is added to an extracellular matrix-immobilized plate, and the binding between this antibody and the extracellular matrix is measured based on the electrochemiluminescence of ruthenium. The concentration of the antibody to be added can be appropriately set; the concentration to be added can be high to increase the sensitivity of detecting extracellular matrix binding. Such an extracellular matrix may be derived from an animal or a plant as long as it contains glycoproteins such as collagen, proteoglycans, fibronectin, laminin, entactin, fibrin, and perlecan ; It should be extracellular matrix from animals. For example, extracellular matrices from animals such as human, mouse, rat, monkey, rabbit or dog can be used. For example, a human-derived extracellular matrix can be used as an indicator of antibody pharmacodynamics in human plasma. The conditions for assessing the binding of the extracellular matrix of the antibody should be a neutral pH range, about pH 7.4, which is a physiological condition; however, the conditions need not necessarily be a neutral range, and the binding can also be in an acidic pH range (e.g., about pH 6.0) Evaluation. Alternatively, when assessing the extracellular matrix binding of an antibody, the assay can be performed in the co-presence of the antibody-bound antigen molecule and assess the binding activity of this antigen-antibody complex to the extracellular matrix.

In one embodiment, it is disclosed that the antibody of A is (substantially) compared to this antibody before modification or modification of at least one amino acid residue to increase pI (natural antibody (such as natural Ig antibody, preferably natural IgG antibody) or reference antibody (e.g. Such as antibodies before modification or before library construction or during construction)) can retain its antigen-binding activity. In this case, "(substantially) retain this antigen-binding activity" may mean at least 50% or more, preferably 60% or more, and more preferably 70 compared to the binding activity of the antibody before modification or modification. % Or 75% or more, and preferably 80%, 85%, 90% or 95% or more. Or reveal that the antibody of A need only maintain the binding activity to the extent that it can tolerate its function when binding to the antigen; therefore, the affinity determined at 37 ° C under physiological conditions may be, for example, 100 nM or less, preferably 50 nM or less, more It is preferably 10 nM or less, and still more preferably 1 nM or less.

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

Such substituted amino acid residues may include, for example, substitution of an amino acid residue having a negatively charged side chain in the amino acid sequence of the antibody of interest to an amino acid residue having an uncharged side chain, an uncharged Substitution of amino acid residues in side chains to amino acid residues with positively charged side chains, and substitution of amino acid residues in negatively charged side chains to amino acid residues with positively charged side chains , Can be implemented individually or in appropriate combinations. Insertion or addition of amino acid residues may include, for example, insertion or addition of an amino acid having an uncharged charge and / or insertion or addition of an amino acid having a positively charged side chain to the amino acid sequence of the antibody of interest , Which can be implemented individually or in appropriate combinations. Deleting an amino acid residue may include, for example, deleting an amino acid having an uncharged charge and / or deleting an amino acid having a negatively charged side chain in the amino acid sequence of the antibody of interest, which may be implemented individually or in appropriate combinations.

Those of ordinary skill in the art can appropriately combine one or more of these amino acid sequences of the antibody of interest with such additions, deletions, substitutions, and insertions. Modifications that cause a reduction in the local charge of amino acid residues are also acceptable, as long as the net isoelectric point of the antibody of A is revealed to increase. For example, if necessary, an antibody whose isoelectric point value has increased (too much) can be modified to (slightly) decrease the isoelectric point. It is also acceptable to perform modification of at least one amino acid residue at the same time or at different times for other purposes (for example, to increase antibody stability or reduce immunogenicity) to reduce the local charge of the amino acid residue. Such antibodies include antibodies constructed for specific purposes.

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

As detailed in Examples 1 to 4, at neutral pH (eg, pH 7.0), lysine and arginine are present as residues in the antibody, almost 100% are positively charged, while histidine is Existing as a residue in antibodies, only about 9% are 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, it is disclosed that the antibody of A preferably has a variable region and / or a constant region. The variable region should preferably have a heavy chain variable region and / or a light chain variable region, and / or should have a CDR (such as one or more of CDR1, CDR2, and CDR3) and / or FR (such as FR1, FR2, One or more of FR3 and FR4). The constant region should have a heavy chain constant region and / Or light chain constant region, in terms of sequence and type, such as an IgG type constant region (preferably human IgG1, human IgG2, human IgG3 or human IgG4 type constant region, human kappa chain constant region, and 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 may modify a single amino acid or a combination of multiple amino acids. Ideally, a combination of multiple amino acid substitutions can be introduced into the amino acid site of the antibody. There are no restrictions, but it is preferable that such a plurality of amino acid substitutions be introduced to the positions which are close to each other in three dimensions. When an amino acid that may be exposed on the surface of the antibody (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 (such as Lys (K) or Arg (R)) is used, for example, one Or multiple amino acids (depending on the state, which can include amino acids embedded inside the antibody molecule) are replaced with positively charged amino acids to create a dense state of local positive charge in the three-dimensional proximity. Here, "stereo-proximity "Site" is not particularly limited; it may mean that one or more amino acid substitutions are introduced within, for example, 20 angstroms, preferably within 15 angstroms, and more preferably within 10 angstroms. Whether the amino acid substitution site of interest is exposed to the antibody molecule Whether or not the amino acid-substituted site is close to other amino acid-substituted sites or the pre-existing amino acid described above can be evaluated by known methods such as X-ray crystallography.

In addition to the method described above, a method of providing a plurality of positively charged sites that are three-dimensionally close to each other may include using an amino acid that is originally positively charged in a natural IgG constant region. Such amino acids include, for example, spermines at positions 255, 292, 301, 344, 355, and 416 according to the EU numbering; and 121, 133, 147, 205, 210, 213, 214, and 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360, 370, 392, 409, 414, and lysines at positions 439 and 439. Multiple positive charges can be obtained by substituting these positively charged amino acids in positions that are sterically close to the positively charged amino acids.

If it is revealed that the antibody of A has a variable region (which can be modified), amino acid residues (that may still be exposed on the surface) that are not masked by antigen binding can be modified, and / or no amino acid modification is introduced At sites that are blocked by antigen binding, amino acid modifications that do not (substantially) inhibit antigen binding can be performed. If the amino acid residues of the ion concentration-dependent binding domain that may be exposed on the surface of the antibody molecule have been modified, the amino acid residues of the antigen binding domain can be modified so that the modification does not (substantially) decrease and can be changed according to the ion concentration conditions The binding activity of the amino acid residue of the antibody's antigen-binding activity (for example, at the calcium-binding motif or the histidine insertion site and / or the histidine substitution site) or the amino acid residue can be modified in accordance with the ion concentration conditions Sites other than amino acid residues that alter the antigen-binding activity of the antibody. On the other hand, if the amino acid residues present in the ion concentration-dependent binding domain that can be exposed on the surface of the antibody molecule have been modified, amino acid residues that can change the antigen-binding activity of the antibody according to the ion concentration conditions can be selected The type or position is such that the isoelectric point of the antibody does not fall below acceptable levels. If the isoelectric point of the antibody is below an acceptable level, the overall isoelectric point of the antibody can be increased by modifying at least one amino acid residue of the antibody molecule that may be exposed on the surface.

Without limitation, the FR sequence having a higher isoelectric point is preferably selected from a human germline FR sequence or a sequence of an equivalent region. These amino acids may sometimes be modified.

If it is revealed that the antibody of A has an FcγR binding domain (which can be a binding domain for any of the following FcγR isoforms and paratypes) and / or a constant region (which can be modified) of the FcRn binding domain, at least 1 of this constant region Amino acids that may be exposed on the surface The modification site of the residue may be an amino acid residue of an amino acid residue other than the FcγR binding domain and / or the FcRn binding domain, if necessary. Or if the modification site is selected from amino acid residues in the FcγR-binding domain and / or FcRn-binding domain, a site that does not (substantially) affect the binding activity or binding affinity for FcγR and / or FcRn, or The effect is a biologically or pharmacologically acceptable site.

In one embodiment, at least 1 amino acid is modified by modifying at least one amino acid residue that may be exposed on the surface of the variable region (which may be modified) to produce an isoelectric point-increasing antibody that reveals A The positions of the residues are not limited; however, such positions may be selected from the group consisting of: (a) FRs 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) Positions of the light chain variable region FR of 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108; and (d) 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, and in terms of side chain charge, for example, Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N), but is not limited thereto. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the amino acid positions described above are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of the aforementioned amino acid positions are modified.

In one embodiment, among the positions to be modified, The combination of the lower position and other positions that have the effect of increasing the isoelectric point value of the antibody to increase the isoelectric point value of the antibody that reveals A increases. Such an auxiliary position for increasing the isoelectric point value may be, for example, for a light chain variable region, selected from the group consisting of the following according to Kabat numbering: 27, 52, 56, 65, and 69.

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

When at least one amino acid residue modification site is selected, for example, from the group consisting of the above, the amino acid type of the modified heavy chain variable region may be, for example: (a) for the 8-position: 8K, 8R , 8Q, 8G, 8S or 8N; (b) for 13 bits: 13K, 13R, 13Q, 13G, 13S or 13N; (c) for 15 bits: 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 bits: 41K, 41R, 41Q, 41G, 41S or 41N; (h) for 43 bits: 43K, 43R, 43Q, 43G, 43S or 43N; (i) for 44 bits: 44K , 44R, 44Q, 44G, 44S, or 44N; (j) for 63 bits: 63K, 63R, 63Q, 63G, 63S, or 63N; (k) for 64 bits: 64K, 64R, 64Q, 64G, 64S, or 64N; (l) for 77 bits : 77K, 77R, 77Q, 77G, 77S, or 77N; (m) for 82 bits: 82K, 82R, 82Q, 82G, 82S, or 82N; (n) for 82a bits: 82aK, 82aR, 82aQ, 82aG, 82aS, or 82aN ; (O) for 82b: 82bK, 82bR, 82bQ, 82bG, 82bS, or 82bN; (p) for 83: 83K, 83R, 83Q, 83G, 83S, or 83N; (q) for 84: 84K, 84R, 84Q, 84G, 84S or 84N; (r) for 85 bits: 85K, 85R, 85Q, 85G, 85S or 85N; or (s) for 105 bits: 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 any of the aforementioned amino acid positions is modified. In some embodiments, a combination of 1-20, 1-15, 1-10, or 1-5 of any of the aforementioned amino acid positions is modified.

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

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

Similarly, the amino acid type of the light chain variable region modification is, for example: (a) 16-position: 16K, 16R, 16Q, 16G, 16S or 16N; (b) 18-position: 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 bits: 27K, 27R, 27Q, 27G, 27S or 27N; (g) for 37 bits: 37K, 37R, 37Q, 37G, 37S or 37N; ( h) For 41 bits: 41K, 41R, 41Q, 41G, 41S or 41N; (i) For 42 bits: 42K, 42R, 42Q, 42G, 42S or 42N; (j) For 45 bits: 45K, 45R, 45Q, 45G, 45S or 45N; (k) for 52 bits: 52K, 52R, 52Q, 52G, 52S or 52N; (l) for 53 bits: 53K, 53R, 53Q, 53G, 53S or 53N; (m) for 54 bits : 54K, 54R, 54Q, 54G, 54S, or 54N; (n) for 55 bits: 55K, 55R, 55Q, 55G, 55S, or 55N; (o) for 56 bits: 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 bits: 76K, 76R, 76Q, 76G, 76S or 76N; (t) for 77 bits: 77K, 77R, 77Q, 77G, 77S or 77N; (u) for 79 bits: 79K , 79R, 79Q, 79G, 79S or 79N; and (v) for the 107th position: 107K, 107R, 107Q, 107G, 107S or 107N. In some embodiments, a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than any of the aforementioned amino acid positions is modified. In some embodiments, a combination of 1-20, 1-15, 1-10, or 1-5 of the aforementioned amino acid positions is modified.

Non-limiting examples of combinations of amino acid position modification in the light chain variable region are, for example, positions 24 and 27 according to Kabat numbering; positions 25 and 26; 41 and 42 42; 76; 52 and 56; 65 and 79; 74 and 77; 76 and 79; any 2 or more positions selected from the group consisting of: 16, 24, and 27 Position; any 2 or more positions selected from the group consisting of: 24, 27, and 37 positions; any 2 or more positions selected from the group consisting of: 25, 26, and 37 Position; any 2 or more positions selected from the group consisting of positions: 27, 76, and 79 positions; any 2 or more positions selected from the group consisting of positions: 41, 74, and 77 Position; any 2 or more positions selected from the group consisting of positions: 41, 76, and 79 positions; any 2 or more positions selected from the group consisting of positions: 24, 27, 41, And 42 positions; any 2 or more positions selected from the group consisting of: 24, 27, 52, and 56 positions; any 2 or more positions selected from the group consisting of: 24, Positions 27, 65, and 69; any 2 or more positions selected from the group consisting of: positions 24, 27, 74, and 77; positions selected from Any 2 or more positions in the group: 24, 27, 76, and 79; selected from any 2 or more positions in the group consisting of: 25, 26, 52, and 56; selected from Any 2 or more positions in the following group of positions: 25, 26, 65, and 69; any 2 or more positions in the group of positions: 25, 26, 76, and 79 Position; any 2 or more positions selected from the group consisting of: 27, 41, 74, and 77 positions; any 2 or more positions selected from the group consisting of: 27, 41, 76, and 79; any 2 or more positions selected from the group consisting of positions: 52, 56, 74, and 77; any 2 or more positions selected from the group consisting of positions: Positions 52, 56, 76, and 79; any 2 or more positions selected from the group consisting of: positions 65, 69, 76, and 79; any positions selected from the group consisting of positions 2 or more positions: 65, 69, 74, and 77 positions; any 2 or more positions selected from the group consisting of: 18, 24, 45, 79, and 107 positions; selected from positions consisting of Any 2 or more positions in the group: 27, 52, 56, 74, and 77; any 2 or more positions selected from the group consisting of: 27, 52, 56, 76, and 79 positions; any 2 or more positions selected from the group consisting of: 27, 65, 69, 74, and 77 positions; any 2 or more positions selected from the group consisting of: 27 , 65, 69, 76, and 79; any two or more positions selected from the group consisting of: 41, 52, 56, 74, and 77; selected from the group formed of Any 2 or more positions: 41, 52, 56, 76, and 79; any 2 or more positions selected from the group consisting of positions: 41, 65, 69, 74, and 77; selected from Any 2 or more positions in the following group of positions: 41, 65, 69, 76, and 79; any 2 or more positions in the group of positions: 24, Positions 27, 41, 42, 65, and 69; any 2 or more positions selected from the group consisting of positions: 24, 27, 52, 56, 65, and 69 positions; selected from the group consisting of positions Any 2 or more positions in the group: 24, 27, 65, 69, 74, and 77; any 2 or more positions selected from the group consisting of: 24, 27, 65, 69, 76 , And 79; selected from any 2 or more of the group consisting of: 24, 27, 41, 42, 74, and 77; selected from any 2 or more of the group consisting of Multi-position: 24, 27, 52, 56, 74, and 77; selected from any 2 or more positions in the group consisting of: 24, 27, 41, 42, 76, and 79; selected from Any 2 or more positions in the group of positions: 24, 27, 52, 56, 76, and 79; any 2 or more positions in the group of positions: 24, 27, 74, 76, 77, and 79; selected from Any 2 or more positions in the following group of positions: 52, 56, 65, 69, 74, and 77; or any 2 or more positions in the group consisting of: 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, and in terms of side chain charge, for example, Lys (K), Arg (R), Gln (Q ), Gly (G), Ser (S), or Asn (N), but not limited to this.

The specific combination may be, for example, 24R / 27Q; 24R / 27R; 24K / 27K; 25R / 26R; 25K / 26K; 41R / 42K; 42K / 76R; 52R / 56R; 65R / 79K; 74K / 77R; 76R / 79K; 16K / 24R / 27R; 24R / 27R / 37R; 25R / 26R / 37R; 27R / 76R / 79K; 41R / 74K / 77R; 41R / 76R / 79K; 24R / 27R / 41R / 42K; 24R / 27R / 52R / 56R ; 24R / 27R / 52K / 56K; 24R / 27R / 65R / 69R; 24R / 27R / 74K / 77R; 24R / 27R / 76R / 79K; 25R / 26R / 52R / 56R; 25R / 26R / 52K / 56K; 25R / 26R / 65R / 69R; 25R / 26R / 76R / 79K; 27R / 41R / 74K / 77R; 27R / 41R / 76R / 79K; 52R / 56R / 74K / 77R; 52R / 56R / 76R / 79K; 65R / 69R / 76R / 79K; 65R / 69R / 74K / 77R; 18R / 24R / 45K / 79Q / 107K; 27R / 52R / 56R / 74K / 77R; 27R / 52R / 56R / 76R / 79K; 27R / 65R / 69R / 74K / 77R; 27R / 65R / 69R / 76R / 79K; 41R / 52R / 56R / 74K / 77R; 41R / 52R / 56R / 76R / 79K; 41R / 65R / 69R / 74K / 77R; 41R / 65R / 69R / 76R / 79K; 24R / 27R / 41R / 42K / 65R / 69R; 24R / 27R / 52R / 56R / 65R / 69R; 24R / 27R / 65R / 69R / 74K / 77R; 24R / 27R / 65R / 69R / 76R / 79K ; 24R / 27R / 41R / 42K / 74K / 77R; 24R / 27R / 52R / 56R / 74K / 77R; 24R / 27R / 41R / 42K / 76R / 79K; 24R / 27R / 52R / 56R / 76R / 79K; 24R / 27R / 74K / 76R / 77R / 79K; 52R / 56R / 65R / 69R / 74K / 77R; or 52R / 56R / 65R / 69R / 76R / 79K.

In WO2007 / 114319 or WO2009 / 041643, according to the theory Evidence, homology models, or experimental techniques explaining or demonstrating that the effect of increasing the isoelectric point of some of the amino acid residues of the variable region is not exclusively (or substantially) dependent on the amino acid sequences that make up the antibody The type of antigen or target antigen depends on the type and number of amino acid residues substituted. It has been demonstrated that even after some amino acid modifications, the antigen-binding activity against several types of antigens is (substantially) maintained or at least maintained by those with ordinary knowledge in the technical field to expect a high probability.

For example, WO2009 / 041643 specifically points out that in the heavy chain FR of the humanized glycican 3 antibody shown in SEQ ID NO: 8, the modified site of the amino acid residue that may be exposed on the surface is in accordance with Kabat numbering method 1, 3 , 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87 , 89, 90, 107, 110, 112, and 114. It has also been reported that amino acid residues at position 97 according to Kabat numbering are desirable because they are 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 the antibody are ideal. Also disclosed is the humanized glycican 3 antibody shown in SEQ ID NO: 9 of the amino acid residues 1, 3, 7, 8, 9, 11, 12, 16, 17, 18 of the light chain FR of Kabat numbering , 20, 22, 43, 44, 45, 46, 48, 49, 50, 54, 62, 65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90 , 105, 108, 110, 111, and 112 are ideal. It has also been revealed that amino acid residues at positions 24, 27, 33, 55, and 59 of the light chain CDR of this antibody are ideal. Also WO2009 / 041643 specifically specifies that 31, 64, and 65 of the amino acid residues of the heavy chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 10 according to Kabat numbering are ideal sites because they can be Allows modification of amino acid residues that may be exposed on the surface to maintain antigen binding activity Sex. It is also disclosed that the anti-human IL-6 receptor antibody shown in SEQ ID NO: 11 is preferably at positions 24, 27, 53, and 55 of the amino acid residues of the light chain CDR according to Kabat numbering. It is also specifically pointed out that the 31 position of the amino acid residue of the heavy chain CDR of the anti-human IL-6 receptor antibody shown in SEQ ID NO: 12 according to Kabat numbering is an ideal site because it may allow the modification to be exposed on the surface The amino acid residues on the amino acid residue to maintain the antigen-binding activity. It is also revealed that the anti-human IL-6 receptor antibody shown in SEQ ID NO: 13 is preferably at positions 24, 53, 54 and 55 of the amino acid residues of the light chain CDR according to Kabat numbering. WO2009 / 041643 also discloses that the 61, 62, 64 and 65 positions of the amino acid residues of the heavy chain CDRs of the heavy chain CDR according to Kabat numbering of the anti-human glycican 3 antibody shown in SEQ ID NO: 14 are ideal sites because they can tolerate Modifications may expose amino acid residues on the surface to maintain antigen-binding activity. It is also revealed that positions 24 and 27 of the amino acid residues of the light chain CDR of the anti-human glycican3 antibody shown in SEQ ID NO: 15 according to Kabat numbering are ideal sites. It is also revealed that positions 61, 62, 64, and 65 of the amino acid residues of the heavy chain CDR of the anti-human IL-31 receptor antibody shown in SEQ ID NO: 16 according to Kabat numbering are ideal sites because they allow Modifications may expose amino acid residues on the surface to maintain antigen-binding activity. WO2009 / 041643 also discloses that the anti-human IL-31 receptor antibody shown in SEQ ID NO: 17 is an ideal site for positions 24 and 54 of the amino acid residue of the light chain CDR according to Kabat numbering. Similarly, WO2007 / 114319 reports antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and hB26-p15, manufactured by modifying the charge of one or more amino acid residues that may be exposed on the surface. The isoelectric point of hB26-PF showed a change in isoelectric point when compared with that before modification or modification of this antibody. It had equivalent binding activity to its antigen's factor IXa or factor X. It has also been reported that if these antibodies are administered to mice, each The isoelectric point of an antibody is highly correlated with its clearance rate (CL) in plasma, retention time in plasma, and half-life (T1 / 2) in plasma. WO2007 / 114319 also proves that the amino acid residues at positions 10, 12, 23, 39, 43, 97, and 105 of the variable region are ideal as 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 antibody constant regions (preferably human constant regions, more preferably human Ig type constant regions, and more preferably human IgG type constant regions) can be used. Region, but not limited to this) The homology simulation method of the constructed homology model is used to identify the amino acid residues of the constant region of the antibody that may be exposed on the surface to determine the antibody that reveals the increased isoelectric point to reveal A Modification site of at least 1 amino acid residue. The modification site of at least one amino acid residue of the constant region that may be exposed on the surface is not limited; however, this site should be 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 group consisting of 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 modified at each position may be selected from the above amino acids. As far as the side chain charge is concerned, for example, Lys (K), Arg (R), Gln (Q) or Asn (N) is not limited. herein. When selecting at least 1 amino acid residue modification The site is selected from the above group, for example, the modified amino acid type of each site may be as follows: at position 254: 254K, 254R, 254Q, or 254N; at position 258, 258K, 258R, 258Q, or 258N; at 281 Position: 281K, 281R, 281Q, or 281N; at position 282: 282K, 282R, 282Q, or 282N; at position 285: 285K, 285R, 285Q, or 285N; at position 309: 309K, 309R, 309Q, or 309N; at position 311: 311K, 311R, 311Q, or 311N; at 315: 315K, 315R, 315Q, or 315N; at 327: 327K, 327R, 327Q, or 327N; at 330: 330K, 330R, 330Q, or 330N; at 342: 342K, 342R, 342Q, or 342N; at position 311: 343K, 343R, 343Q, or 343N; at position 345: 345K, 345R, 345Q, or 345N; at position 356: 356K, 356R, 356Q, or 356N; at position 358: 358K, 358R, 358Q or 358N; at position 359: 359K, 359R, 359Q or 359N; at position 361: 361K, 361R, 361Q or 361N; at position 362: 362K, 362R, 362Q or 362N; at position 384: 384K, 384R, 384Q or 384N; at 385: 385K, 385R, 385Q or 385N; at 386: 386K, 386R, 386Q or 386N; at 387: 387K, 387R, 387Q or 387N; At 389: 389K, 389R, 389Q, or 389N; At 399: 399K, 399R, 399Q, or 399N; At 400: 400K, 400R, 400Q, or 400N; At 401: 401K, 401R, 401Q, or 401N; At 402 Position: 402K, 402R, 402Q, or 402N; at position 413: 413K, 413R, 413Q, or 413N; at position 418: 418K, 418R, 418Q, or 418N; at position 419: 419K, 419R, 419Q, or 419N; at position 421: 421K, 421R, 421Q, or 421N; at 433: 433K, 433R, 433Q, or 433N; at 434: 434K, 434R, 434Q, or 434N; and at 443: 443K, 443R, 443Q, or 443N.

In an alternative embodiment, the modification site of the 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 the EU numbering method.

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

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

Among them, G1m1, 17 and G1m3 are allotypes known to human IgG1, for example. The amino acid sequence of this subtype is different: G1m1, 17 are aspartic acid at position 356 according to EU numbering, leucine at position 358, and G1m3 is glutamic acid at position 356 according to EU numbering and Methionine. However, there are no reports suggesting significant differences in the function and properties of these reported paratypes in the main antibodies. Therefore, those skilled in the art can easily predict various evaluations using specific subtypes, and the results are not limited to the subtypes used but examples can be obtained and the same effect can be predicted in any subtype. In the scope of disclosure A and B described here, when expressed as "human IgG1", "human IgG2", "human IgG3", or "human IgG4", this subtype is not limited to a specific subtype and may include all reported subtypes .

In revealing an alternative or further embodiment of A or B, the light chain constant region of an antibody may include a kappa chain (IgK) type or a lambda chain (IgL1, IgL2, IgL3, IgL6, or IgL7) type constant region. The light chain constant region may 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, some paratypic sequences are due to the polymorphism of the human kappa chain constant region and 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, there are no reports suggesting significant differences in the function and properties of these reported paratypes in the main antibodies. Therefore, those with ordinary knowledge in the technical field can easily understand the specificity in the scope of disclosure A and B when referring to the records here. Subtypes, arbitrary subtypes are expected to have the same effect (hereinafter also collectively referred to as the natural (human) IgG (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. Therefore, if it is revealed that the antibody of A or B is, for example, an IgG antibody or a molecule derived therefrom, this antibody may include a natural IgG (IgG1, IgG2 , IgG3 or IgG4 type) (hereinafter also collectively referred to as the natural (human) IgG (type) Fc region) Fc region contained in the constant region. The Fc region of a natural IgG may refer to an Fc region having the same amino acid sequence as a naturally occurring IgG Fc region. Specific examples of the Fc region of natural human IgG may include the aforementioned human IgG1 constant region (SEQ ID NO: 18), human IgG2 constant region (SEQ ID NO: 19), human IgG3 constant region (SEQ ID NO: 20), or human The Fc region contained in the IgG4 constant region (SEQ ID NO: 21) (Fc region of the IgG class may refer to, for example, from 226-cysteine to the C-terminus in accordance with EU numbering or 230-position proline in accordance with the EU numbering to C side).

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

Within the scope of disclosure A, WO2013 / 081143 reports, for example, that it can form a multivalent immune complex with a multimeric antigen, an ion concentration-dependent antibody (multivalent antigen-antibody complex), and recognizes two or more antigens in the haploid. Multi-epitope can form a multispecific immune concentration-dependent antibody or multi-antibody binding site ion concentration-dependent antibody (multivalent antigen-antibody complex) capable of forming a multivalent immune complex, which can bind more strongly to FcγR, FcRn, Complement receptor, due to at least 2 or more multivalent constant regions (may be modified) included in this antibody molecule Or the Fc region (which can be modified) has avidity (the sum of the binding strength between the multiple epitope and the multiple antibody binding site), so the antibody can be taken up into the cell faster. Therefore, by modifying at least one amino acid residue that can be exposed on the surface of this antibody to increase the isoelectric point, the above ion concentration-dependent antibody can form a multivalent immune complex with a polymorphic antigen or a haploid antigen. As an antibody that reveals A (an ion concentration-dependent antibody with an increased isoelectric point value). Those with ordinary knowledge in this technical field should understand that the ion concentration-dependent antibody that can increase the isoelectric point value of a polyvalent immune complex with a polymorphic antigen or a haploid antigen compared to an isoelectric point that cannot form a polyvalent immune complex. Increased ion concentration-dependent antibodies can be quickly taken up into cells. Those of ordinary skill in the art can also understand that in one embodiment, the activity of the antibody that reveals A to bind to FcRn and / or FcγR can be increased under neutral pH conditions. In this case, it can interact with polymorphic antigens or units. Ion concentration-dependent antibodies with increased isoelectric point values for the formation of multivalent immune complexes by antigens are taken up into cells faster and faster.

In one embodiment, the antibody disclosed by A may be a one-armed antibody (including all embodiments of the one-armed antibody described in WO2005 / 063816). Generally, a one-armed antibody is an antibody lacking two Fab regions normally found in IgG antibodies, and can be produced without limitation, for example, by the method described in WO2005 / 063816. There is no limitation. The structure of an IgG antibody with a heavy chain is, for example, VH-CH1-hinge-CH2-CH3. If the Fab region is cut closer to the N-terminus than the hinge (such as VH or CH1), the antibody will Expressed by including additional sequences, when the Fab region is cut closer to the C-terminus than the hinge (such as CH2), this Fc region will have an incomplete form. Therefore, there is no limitation. From the viewpoint of stability of the antibody molecule, the one-arm antibody is preferably manufactured by cutting the hinge region (hinge) of one of the two Fab regions of the IgG antibody. More suitable In order to cleave the heavy chain, an intramolecular disulfide bond is used to link the uncut heavy chain. 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. If the antibody with an increased isoelectric point value introduced by the ion concentration-dependent antigen-binding domain is a one-armed antibody, the antibody has a half-life in plasma compared to an antibody that does not increase the isoelectric point value of the ion-concentration-dependent antigen-binding domain. It can be further shortened, the antibody uptake by the cell can be further enhanced, the elimination of the antigen from the plasma can be further enhanced, or the affinity of the antibody for the extracellular matrix can be further increased.

Without being bound by a specific theory, if the cell uptake acceleration effect of a one-arm antibody is expected in one embodiment, it can be conceived, but not limited to, the isoelectric point value of the soluble antigen is lower than the isoelectric point value of the antibody. The net isoelectric point of a complex composed of an antibody and an antigen can be calculated using a known method 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 larger the isoelectric point value of the soluble antigen, the larger the net isoelectric point of the complex. When a common IgG antibody molecule (with 2 Fabs) binds to a single low isoelectric point soluble antigen compared to two low isoelectric point soluble antigens, the latter complex has a lower net isoelectric point. When such a general-type antibody is converted into a one-armed antibody, only one antigen can bind to a single molecule of this antibody; the reduction of the isoelectric point of the complex caused by binding to 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, it is converted into a one-arm antibody compared to a normal antibody, allowing the isoelectric point of the complex to increase, thereby accelerating the uptake into the cell.

There is no limitation. When the isoelectric point value of the Fab of an IgG antibody molecule (with 2 Fabs) is generally lower than the isoelectric point value of Fc, conversion to a one-arm antibody will increase the number of complexes formed by the one-arm antibody and the antigen. Net isoelectric point. Also, when the implementation is so turned To become a single-arm antibody, from the viewpoint of the stability of the single-arm antibody, one of the Fabs should be cut in the hinge region located at the boundary between the Fab and the Fc. In this case, by selecting a site that increases the isoelectric point value of the one-arm antibody to a desired level, it is expected that the isoelectric point value will increase effectively.

Therefore, those with ordinary knowledge in the technical field can understand (or essentially) depend on the antibody amino acid sequence 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 theory isoelectric point value) and the theoretical isoelectric point value of soluble antigen, and predict the relationship between the theoretical isoelectric point value of the antibody, in order to transform this antibody into a one-arm antibody, the isoelectric point value of the antibody can be Increased, and consequently, cellular uptake of this antigen can be accelerated.

In one embodiment, it is disclosed that the antibody of A or B may be a multispecific antibody, and the multispecific antibody may be, but is not limited to, a bispecific antibody. The multispecific antibody may be a multispecific antibody comprising a first polypeptide and a second polypeptide. The "multispecific antibody containing the first polypeptide and the second polypeptide" herein 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, and more preferably a variable region containing a CDR and / or a FR. 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 include a third polypeptide and a fourth polypeptide, each containing a light chain variable region and preferably a light chain constant region. In this case, the first to fourth polypeptides can be assembled together to form a multispecific antibody.

In one embodiment, if it is revealed that the antibody of A is a multispecific antibody and the multispecific antibody contains a constant region of a heavy chain to reduce its isoelectric point value, for example, the following sequence can be used: IgG2 or IgG4 sequence at position 137; 196th IgG1, IgG2 or IgG4 sequence; 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 at position 268 Sequence; 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; or IgG1, IgG2 or IgG4 sequence. To increase the isoelectric point value simultaneously, 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 it is revealed that the antibody of A has 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 can be IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions that originally have different isoelectric points. Alternatively, an isoelectric point difference can be introduced between the two heavy chain constant regions. In order to introduce such a difference in isoelectric point in the constant region, the modification site of at least one amino acid residue may be the position as described above or according to EU numbering. The heavy chain constant region described in WO2009 / 041643 is selected from the following: The position of the group: 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435. Or the amino acid residue at position 297 which is the glycation site can be modified to remove sugar Chain, because the removal of sugar chains from the constant region of the heavy chain causes differences in isoelectric points.

In one embodiment, it is disclosed that the antibody of A or B may be a multiple antibody or a monoclonal antibody, and preferably a monoclonal antibody of mammalian origin. Monoclonal antibodies include producers of host cells produced by fusion tumors or transformed by genetic engineering techniques with expression vectors bearing antibody genes. The antibody of this disclosure A or B may be, for example, an antibody such as a chimeric antibody, a humanized antibody, or an antibody produced by affinity maturation or a molecule derived therefrom.

In one embodiment, it is disclosed that the antibodies of A or B may be derived from any animal species (such as humans; or non-human animals such as mice, rats, hamsters, rabbits, monkeys, malay monkeys, rhesus monkeys, and Arabian baboons) without limitation. , Chimpanzee, goat, sheep, dog, cow or camel) or any bird; and the antibody should preferably be from a human, monkey or mouse.

In one embodiment, it is disclosed that the antibody of A or B may be an Ig antibody, preferably an IgG antibody.

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

Within the scope of disclosure A, "FcγR" may refer to a receptor protein that can bind to the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a molecule or Fc region variant derived therefrom, and may include a substance substantially composed of One or more or all members of the protein family encoded by the FcyR gene. In humans, this family includes but is not limited to FcγRI (CD64) including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) Including isotype FcγRIIa (including subtypes H131 (H type) and R131 (type R)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) includes isotype FcγRIIIa (including paratype Types V158 and F158) and FcγRIIIb (including paratypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and all unidentified human FcγRs and FcγR isoforms and paratypes. In addition, FcγRIIb1 and FcγRIIb2 have been reported to be cleavable variants of human FcγRIIb (hFcγRIIb). There is also a report on a cleavage variant called FcγRIIb3 (Brooks et al., J. Exp. Med, 170: 1369-1385 (1989)). In addition to the methods described above, hFcγRIIb includes, for example, all cleavage variants registered in 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 genetic 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 gene polymorphisms to be reported in the future.

The FcγR may be from any organism, and may be from human, mouse, rat, rabbit or monkey, but is not limited. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), and all unidentified mouse FcγRs, isotypes and paratypes with FcγR. Such an ideal FcyR includes, for example, human FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16), or FcyRIIIB (CD16). FcγR is a membrane type in vivo, so it can be used in experimental lines after being artificially converted to an appropriate soluble type.

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

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

FcγRI (CD64) including FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16) including FcγRIIIa (including paraforms V158 and F158), the α chain system that binds to the Fc region of IgG and the common γ chain with ITAM The ITAM transmits an activation signal in the cell. FcγRIIIb (including paratype FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPI anchor protein. The cytoplasmic domain of FcγRIIa (CD32) including FcγRIIa (including paratypes H131 and R131) and FcγRIIc isoforms contains ITAM. These receptors appear on many immune cells such as macrophages, obese cells, and antigen-presenting cells. The activation signals transmitted by these receptors binding to the Fc region of IgG will promote the phagocytosis of macrophages, produce inflammatory interleukins, degranulate obese cells, and increase antigen-presenting cellular functions. Within the scope of A and B, FcγR capable of transmitting the activation signal as described above is also referred to as activated FcγR.

The cytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM, which transmits inhibitory signals. In B cells, the cross-linking between FcγRIIb and B cell receptor (BCR) will inhibit the activation signal from BCR, resulting in Inhibits BCR production of antibodies. In macrophages, the cross-linking of FcyRIII and FcyRIIb inhibits phagocytosis and the ability to produce inflammatory interleukins. In the category of A and B, FcγR capable of transmitting such an inhibitory signal as described above is also disclosed, and is also referred to as an inhibitory Fcγ receptor.

Reveal whether the binding activity of an antibody or Fc region (variant) to various FcγRs in the category described in A is increased, (substantially) maintained or decreased compared to the antibody or Fc region (variant) before modification. There are methods known in the art that are generally known to those skilled in the art. Such a method is not particularly limited, and the method described in the examples can be used, for example, BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, which is mainly based on the surface plasmon resonance (SPR) phenomenon. ). Alternatively, for example, ELISA and fluorescence activated cell selection (FACS) and ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) can be used. In these assays, the extracellular domain of human FcyR can be used (e.g., WO2013 / 047752).

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

Within the scope of revealing A, in one embodiment, if revealing A or The antibody of B has a constant region (which can be modified). The constant region may have an Fc region or an Fc region variant (preferably a human Fc region or a human Fc region variant), and the scope of A and B disclosed in this document is disclosed here. Within the FcγR binding domain and FcRn binding domain.

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

In one embodiment, when it is revealed that the antibody of A has FcγR-binding activity, it is desirable that the antibody's FcγR-binding activity at neutral pH conditions is increased compared to a reference antibody containing a natural IgG constant region. Considering the viewpoint of comparing the FcγR binding activity between the two, it is not limited but it should be revealed that the antibody of A and the reference antibody containing the natural IgG constant region have the same amino acid sequence in this region (such as the variable region), rather than this It is revealed that the constant region of the antibody of A is modified at one or more amino acid residues.

In one embodiment, when it is revealed that the antibody of A has FcγR binding activity or increased FcγR binding activity under neutral pH conditions (such as pH 7.4), without being limited by theory, it is believed that this antibody combination possesses the following properties: The nature of the shuttle between the intracellular body and the cell is based on the ionic concentration-dependent antigen-binding domain, which repeatedly binds to multiple antigens with a single antibody molecule; by increasing the isoelectric point and positive charge of the entire antibody, it is quickly The nature of uptake into cells; and the nature of rapid uptake into cells by increasing FcγR binding activity at neutral pH conditions. Results 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 the plasma can be further promoted; therefore, it is beneficial to disclose the antibody of A. It is commonly known in the art Experts can routinely determine the optimal isoelectric point value of this antibody to benefit from these properties.

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

In one embodiment of Disclosure A, the starting FcyR binding domain preferably includes, for example, a (human) IgG Fc region or a (human) IgG constant region. As long as a variant of the starting Fc region or the starting constant region can bind to human FcγR at a neutral pH range, any Fc region or constant region can be used as the starting Fc region or the starting constant region. The Fc region or constant region obtained by further modifying the starting Fc region or the starting constant region of the Fc region or the constant region of the amino acid residue that has been modified from the Fc region or the constant region can also be obtained as the Fc revealing A Zone or constant zone. The starting Fc region or the starting constant region may refer to the polypeptide A composition comprising itself, a starting Fc region or a starting constant region, or an amino acid sequence encoding a starting Fc region or a starting constant region. The starting Fc region or starting constant region may include a known Fc region or a known constant region made using recombinant technology. The origin of the starting Fc region or the starting constant region is not limited, and can be obtained from a non-human animal or a human organism. Again the initiating FcrR binding domain can be obtained from Malay monkeys, marmosets, rhesus monkeys, chimpanzees or humans. The starting Fc region or the starting constant region should preferably be obtained from human IgG1; however, it is not limited to a specific IgG class. This means that the Fc region of human IgG1, IgG2, IgG3 or IgG4 can be used as an appropriate starting FcγR binding domain, and also means the IgG class or subclass from any organism within the scope of disclosure A described herein An Fc region or constant region of a subclass can be used as the starting Fc region or the starting constant region. Examples of natural IgG variants or modifications disclosed in publicly known literature are, for example, Strohl, Curr. Opin. Biotechnol. 20 (6): 685-691 (2009); Presta, Curr. Opin. Immunol. 20 (4): 460-470 (2008); Davis et al., Protein Eng. Des. Sel. 23 (4): 195-202 (2010); WO2009 / 086320, WO2008 / 092117; WO2007 / 041635; and WO2006 / 105338, but not Limited to this.

In one embodiment, the amino acid residues of the starting FcγR binding domain, the starting Fc region, or the starting constant region may include, for example, one or more mutations: Amino acid residues; insertion of one or more amino acid residues into the starting Fc region or the starting constant region; or deletion of one or more amines from the starting Fc region or the starting constant region Acid residues. The amino acid sequence of the modified Fc region or constant region is preferably a portion of the amino acid sequence containing an Fc region or constant region that does not occur naturally. Such a variant must have less than 100% sequence identity or similarity to the starting Fc region or the starting constant region. For example, relative to the amino acid sequence of the starting Fc region or the starting constant region, about 75% of this variant is less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, still more preferably about 90% to less than 100%, still more preferably about 95% to less than 100% of the amino group Acid sequence identity or similarity. In a non-limiting example, it is revealed that the modified Fc region or constant region of A is different from the starting Fc region or the starting constant region by at least one amino acid.

In one embodiment, an Fc region or a constant region having FcγR binding activity in an acidic pH range and / or a neutral pH range may be included in the antibody disclosed in A, and may be obtained by any method. Specifically, a variant of an Fc region or a constant region having an FcγR-binding activity in a neutral pH range can be obtained by an amino acid of a human IgG antibody that can be used as a starting Fc region or a starting constant region. Suitable modified IgG antibody Fc regions or IgG antibody constant regions may include, for example, the Fc region or constant region of human IgG (IgG1, IgG2, IgG3, or IgG4, or a variant thereof), and spontaneous mutants produced therefrom. For the Fc region or constant region of human IgG1, human IgG2, human IgG3 or human IgG4 antibodies, there are some paratypic sequences due to gene polymorphisms described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, but Use either of them in Reveal A. In particular, for human IgG1 sequences, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM.

Within the scope of revealing A, in another embodiment, the modification to other amino acids is not limited as long as this variant has FcγR binding activity in the neutral pH range. Such modified amino acid positions are reported in, for example: WO2007 / 024249, WO2007 / 021841, WO2006 / 031370, WO2000 / 042072, WO2004 / 029207, WO2004 / 099249, WO2006 / 105338, WO2007 / 041635, WO2008 / 092117, WO2005 / 070963, WO2006 / 020114, WO2006 / 116260, WO2006 / 023403, WO2013 / 047752, WO2006 / 019447, WO2012 / 115241, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101, WO2013 / 118858, and WO2014 / 030750.

The amino acid modified site that increases the FcγR binding activity of the constant or Fc region in the neutral pH range may include, for example, one or more positions selected from the group consisting of EU numbering positions, 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 are described in WO2013 / 047752L. Such amino acid residue modification may increase FcγR binding in the Fc region or constant region of an IgG antibody at neutral pH conditions. WO2013 / 047752 states that an ideal modification of an IgG-type constant region or an Fc region is, for example, a modification of one or more amino acid residues selected from the group consisting of: according to EU numbering, the amino acid at position 221 becomes Lys or Tyr; amino acid at position 222 becomes any of Phe, Trp, Glu, and Tyr; amino acid at position 223 becomes any of Phe, Trp, Glu, and Lys; 224 The amino acid at position IX becomes any of Phe, Trp, Glu, and Tyr; the amino acid at position 225 becomes any of toGlu, Lys, and Trp; the amino acid at position 227 becomes toGlu, Gly, Any of Lys, and Tyr; the amino acid at position 228 becomes any of Glu, Gly, Lys, and Tyr; the amino acid at position 230 becomes any of Ala, Glu, Gly, and Tyr ; The amino acid at position 231 becomes any of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 232 becomes any of toGlu, Gly, Lys, and Tyr; the amino group at position 233 The acid becomes any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 becomes Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 becomes Ala , Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 becomes Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, G Any of ln, 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, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Any of Thr, Val, Trp, and Tyr; the amino acid at position 239 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val Any one of Trp, Trp, and Tyr; the amino acid at position 240 becomes any of toAla, Ile, Met, and Thr; the amino acid at position 241 becomes Asp, Glu, Leu, Arg, Trp, and Any of Tyr; the amino acid at position 243 becomes toGlu, Leu, Gln, Any of Arg, Trp, and Tyr; the amino acid at position 244 becomes His; the amino acid at position 245 becomes Ala; the amino acid at position 246 becomes any of Asp, Glu, His, and Tyr The amino acid at position 247 becomes any of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; the amino acid at position 249 becomes Glu, His, Gln, and Any of Tyr; amino acid at 250 becomes Glu or Gln; amino acid at 251 becomes Phe; amino acid at 254 becomes any of Phe, Met, and Tyr; 255 The amino acid becomes any of Glu, Leu, and Tyr; the amino acid at position 256 becomes any of Ala, Met, and Pro; the amino acid at position 258 becomes Asp, Glu, His, Ser Either of Tyr and Tyr; the amino acid at position 260 becomes any of Asp, Glu, His, and Tyr; the amino acid at position 262 becomes any of Ala, Glu, Phe, Ile, and Thr The amino acid at position 263 becomes any of Ala, Ile, Met, and Thr; the amino acid at position 264 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn , Pro, Gln, Arg, Ser, Thr, Trp, and Tyr ; The amino acid at position 265 becomes any of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 266 becomes any of Ala, Ile, Met, and Thr; the amino acid at position 267 becomes Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln , Arg, Thr, Val, Trp, and Tyr; the amino acid at position 268 becomes Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, And Trp; the amino acid at position 269 becomes any of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 270 becomes Glu, Phe, Gly, His, Ile, Leu, Any of 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 Tyr; the amino acid at position 272 becomes Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Any of 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, 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, Gln, Arg, Ser, Thr, Val, and Trp; the amino acid at position 279 becomes Ala; the amino acid at position 280 becomes Ala, Gly, His, Lys, Any of Leu, Pro, Gln, Trp, and Tyr; amino acid change at position 281 Any of Asp, Lys, Pro, and Tyr; the amino acid at position 282 becomes any of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 283 becomes Ala, Gly, His , Ile, Lys, Leu, Met, Pro, Arg, and Tyr; the amino acid at position 284 becomes any of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino group at position 285 The acid becomes any of Asp, Glu, Lys, Gln, Trp, and Tyr; the amino acid at position 286 becomes any of Glu, Gly, Pro, and Tyr; the amino acid at position 288 becomes Asn Any of Asp, Glu, and Tyr; the amino acid at position 290 becomes any of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid at position 291 becomes Any of Asp, Glu, Gly, His, Ile, Gln, and Thr; 292 The amino acid at position IX becomes 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, Any of Ser, Thr, Val, Trp, and Tyr; the amino acid at position 294 becomes Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp Any one of Tyr 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 One; the amino acid at position 296 becomes Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val; the amino acid at position 297 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 298 becomes Ala, Asp , Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 299 becomes Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, Any of Tyr; the amino acid at position 300 becomes Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; 301 The amino acid at position A becomes any of Asp, Glu, His, and Tyr; the amino acid at position 302 becomes Ile; the amino acid at position 303 becomes any of Asp, Gly, and Tyr; 304 The amino acid at position IX becomes any of Asp, His, Leu, Asn, and Thr; the amino acid at position 305 becomes any of Glu, Ile, Thr, and Tyr; the amino acid at position 311 becomes Is any of Ala, Asp, Asn, Thr, Val, and Tyr; the amino acid at position 313 becomes Phe; the amino acid at position 315 becomes Leu; the amino acid at position 317 becomes Glu or Gln; the amino acid at position 318 becomes His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position 320 becomes Asp, Phe, Gly, His, Ie, Leu, Asn, Pro, Ser, Thr, Val Any one of Trp, Trp, and Tyr; the amino acid at position 322 becomes any of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; The amino acid becomes I1e; the amino acid at position 324 becomes any of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; the amine at position 325 The base acid becomes any of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amine group at position 326 The acid becomes any of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and 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 Tyr; the amino acid at position 328 becomes Ala, Asp, Glu, Phe , Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, T Any of rp and Tyr; the amino acid at position 329 becomes Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Any of Tyr; the amino acid at position 330 becomes Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr One: the amino acid at position 331 becomes any 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 Tyr; the amino acid at position 333 becomes Ala, Asp , Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Any of Ser, Thr, Val, and Tyr; the amino acid at position 334 becomes any of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; the amino acid at position 335 becomes Asp, Any of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 becomes any of Glu, Lys, and Tyr; The amino acid at position 337 becomes any of Glu, His, and Asn; the amino acid at position 339 becomes any of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr. One: the amino acid at position 376 becomes Ala or Val; the amino acid at position 377 becomes Gly or Lys; the amino acid at position 378 becomes Asp; the amino acid at position 379 becomes Asn ; Amino acid at 380 becomes Ala, Asn, and Ser; Amino acid at 382 becomes Ala or Ile; Amino acid at 385 becomes Glu; Amino acid at 392 becomes Thr; The amino acid at position 396 becomes Leu; the amino acid at position 421 becomes Lys; the amino acid at position 427 becomes Asn; the amino acid at position 428 becomes Phe or Leu; the amino acid at position 429 Becomes Met; amino acid change at position 434 It is Trp; 436 becomes a bit of the amino acid lle; and 440 becomes the amino acid Gly, His, Ile, Leu, and Tyr any one. The number of amino acids to be modified is not particularly limited. Amino acid modification can be performed at only one position or at two or more positions. Combinations of amino acid modifications at 2 or more positions are shown in Table 5 of WO2013 / 047752. These amino acid residue modifications can also be appropriately introduced into an antibody that reveals A.

In one embodiment, this reveals that the (FcγR binding domain) of the antibody of A is directed toward (human) FcγR, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and any one or more of FcγRIIIb, which can have higher binding activity than natural Binding activity of IgG (the Fc region or constant region) or a reference antibody containing the starting Fc region or the starting constant region. For example, the FcγR-binding activity of the antibody (FcγR-binding domain) of A is compared The FcγR binding activity on the reference antibody may be 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more 90% or more, 95% or more, 100% or more, 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more 170% or more, 175% or more, 180% or more, 185% or more, 190% or more or 195% or more, or 2 times or more than the FcγR binding activity of the reference antibody 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 FcγR (FcγRIIb-1 and / or FcγRIIb-2) may be greater than for active FcγR (FcγRIa: FcγRIb; FcγRIc; FcγRIc; FcγRIIIa, including Subtype V158; FcγRIIIa including subtype F158; FcγRIIIb including subtype FcγRIIIb-NA1; FcγRIIIb including subtype FcγRIIIb-NA2; FcγRIIa including subtype H131; or FcγRIIa including subtype R131) is an increase in binding activity Level.

In one embodiment, it is disclosed that the antibody of A may have binding activity to FcyRIIb (including FcyRIIb-1 and FcyRIIb-2).

In one embodiment, the ideal FcγR-binding domain of A is also disclosed to include an FcγR-binding domain that has a greater binding activity to a particular FcγR than other FcγRs (FcγR-binding domains, selective FcγR-binding activity). If using antibodies (or their Fc Region as an FcyR binding domain), a single antibody molecule can only bind to a single FcyR molecule. Therefore, a single antibody molecule bound to a state of inhibiting FcγR cannot bind to other activated FcγRs, and a single antibody molecule bound to a state of activated FcγR cannot bind to other activated FcγR or inhibitory FcγR.

As mentioned above, the activating FcγR preferably includes, for example, FcγRI (CD64), such as FcγRIa, FcγRIb, or FcγRIc; and FcγRIII (CD16) such as FcγRIIIa (such as paraform V158 or F158) or FcγRIIIb (such as paraform FcγRIIIb-NA1 or FcγRIIIb-NA2) . Inhibitory FcyR preferably includes, for example, FcyRIIb (such as FcyRIIb-1 or FcyRIIb-2).

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

Whether or not the FcγR binding domain has a selective binding activity can be evaluated by comparing the respective binding activities for FcγR determined by the above method, for example, by comparing the KD value against activated FcγR by the KD value against inhibitory FcγR The value (ratio) is more specifically a comparison of the FcγR selectivity index shown by the following formula:

[Formula 1] FcγR selectivity index = KD value against active FcγR / against KD value of inhibitory FcγR

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

The FcyR selectivity index may 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 greater, 260 or greater, 270 or greater, 280 or greater, 290 or greater, 300 or greater, 310 or greater, 320 or greater, 330 or greater, 340 or greater, 350 Or greater, 360 or greater, 370 or greater, 380 or greater, 390 or greater, 400 or greater, 410 or greater, 420 or greater, 430 or greater, 440 or greater, 450 or greater, 460 or greater, 470 or greater, 480 or greater, 490 or greater, 500 or greater, 520 or greater, 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 greater, 760 or greater, 780 or greater, 800 or greater, 820 or greater, 840 or greater, 860 or greater, 880 or greater, 900 or greater, 920 or greater, 940 or greater, 960 or greater, 980 or greater, 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, 6500 or greater, 7000 or greater, 7500 or greater, 8000 or greater, 8500 or greater, 9000 or greater, 9500 or greater, 10,000 or greater, or 100,000 or greater; but not limited thereto.

In one embodiment, it is preferred to use human IgG (IgG1, IgG2, IgG3 or IgG4) in accordance with EU numbering at positions 238 or 328, each of which is an Fc region-containing variant or a constant region variant of Asp or Glu ( Antibodies) are disclosed as antibodies to Fc region-containing variants or constant region variants, as disclosed in WO2013 / 125667, WO2012 / 115241, and WO2013 / 047752, specifically for FcγRIIb-1 and / or FcγRIIb-2 Compared to FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, including V158, FcyRIIIa, including F158; FcyRIIIb, including FcyRIIIb-NA1; FcyRIIIb, including FcyRIIIb-NA2; FcyRIIa, including H131; FcyRIIa, Including paratype R131, and / or FcγRIIc has higher binding activity. In such an embodiment, the antibody of A is disclosed for all activated FcyRs (herein, selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb, FcyRIIa) and FcyRIIb. Binding activity, and its FcγRIIb binding activity is maintained or increased compared to a reference antibody containing a natural IgG constant region or a natural IgG Fc region, and / or its binding activity is reduced for all activated FcγRs.

One embodiment of the antibody of this disclosure A containing an Fc region variant or a constant region variant is that the binding activity for FcγRIIb can be maintained or increased compared to a reference antibody with a constant region or Fc region of a natural IgG , Its binding activity to FcγRIIa (type H) and FcγRIIa (type R) may be reduced. Such antibodies have increased binding selectivity for FcyRIIb compared to FcyRIIa.

Within the scope of disclosure A, the degree of the "reduction in binding activity to all activated FcγRs" may be, but is not limited to, 99% or less, 98% or less, 97% or less, 96% or Less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84 % Or less, 82% or less, 80% or less, 78% or less, 76% or less, 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.

In the scope disclosed in A, the "maintenance or increase of FcγRIIb binding activity", the "maintenance or increase of FcγRIIb binding activity", or "maintenance or increase of FcγRIIb binding activity" may be, but is not limited to, 55% or greater, 60 % Or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 87% or greater, 88% or greater, 89% or greater Large, 90% or larger, 91% or larger, 92% or larger, 93% or larger, 94% or larger, 95% or larger, 96% or larger, 97% or larger, 98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101% or greater, 102% or greater, 103% or greater, 104% or greater, 105% Or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 112% or greater, 114% or greater, 116% or greater Large, 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 Large, 195% or larger, 2 or larger, 3 or larger, 4 or larger, 5 or larger , 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 Times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or Larger, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, 10,000 times or more or 100,000 times or more .

In the category disclosed in A, the extent to which "the binding activity is reduced for FcγRIIa (H type) and FcγRIIa (R type)" or "the binding activity is decreased for FcγRIIa (H type) and FcγRIIa (R type)" may be, but is not limited to 99% or more Less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% Or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less 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 less.

In the category disclosed in A, it is preferable to modify the binding selectivity of more FcγRIIb than FcγRIIa (type R), and more suitable to increase the binding selectivity of FcγRIIb more than FcγRIIa (type H), as described in WO2013 / 047752. It is reported that such modified ideal amino acid substitutions may include, for example, in accordance with EU numbering: (a) the substitution of Gly at position 237 as Trp; (b) the substitution of Gly at position 237 by Phe; (c) the substitution of position 238 by Pro is Phe; (d) Asn substituted for 325 is Met; (e) Ser substituted for 267 is Ile; (f) Leu substituted for 328 is Asp; (g) Ser substituted for 267 is Val; (h) ) Replace Leu at position 328 with Trp; (i) Replace Ser at position 267 with Gln; (j) Replace Ser at position 267 with Met; (k) Replace Gly with position 236 as Asp; (l) Replace Ala with position 327 Is Asn; (m) Asn substituted for 325 is Ser; (n) Leu substituted for 235 is Tyr; (o) Val substituted for 266 is Met; (p) Leu substituted for 328 is Tyr; (q) Lep at 235 is Trp; (r) Leu at 235 is Phe; (s) Ser at 239 is Gly; (t) Ala at position 327 is Glu; (u) Ala at position 327 is Gly; (v) Pro at position 238 is Leu; (w) Ser at position 239 is Leu; (x) is substituted Leu at 328 is Thr; (y) Leu at 328 is Ser; (z) Leu at 328 is Met; (aa) Pro at 331 is Trp; (ab) Pro at 331 is Tyr (Ac) Phe substituted for 331 is Phe; (ad) Ala substituted for 327 is Asp; (ae) Leu substituted for 328 is Phe; (af) Pro is substituted for Leu at 271; (ag) is substituted for 267 Ser at position Glu is; (ah) Leu at position 328 is Ala; (ai) Leu at position 328 is Ile; (aj) Leu at position 328 is Gln; (ak) Leu at position 328 is Val; (al) Lys at 326 is Trp; (am) Lys at 334 is Arg; (an) His at 268 is Gly; (ao) His at 268 is Asn; (ap) at Ser 324 is Val; (aq) Val for 266 is Leu; (ar) Pro for G is 271; Gly; (as) Ile for 332 is Phe; (at) Ser for 324 is Ile; (at) au) Replace Glu at position 333 as Pro; (av) Replace Tyr at position 300 as Asp; (aw) Replace Ser at position 337 as Asp; (ax) Replace Tyr at position 300 as Gln; (ay) Replace glut at position 335 Thr is Asp; (az) takes Ser at position 239 is Asn; (ba) Lys at position 326 is Leu; (bb) Lys at position 326 is Ile; (bc) Ser at position 239 is Glu; (bd) Lys at position 326 is Phe ; (Be) Lys in position 326 is Val; (bf) Lys in position 326 is Tyr; (bg) Ser in position 267 is Asp; (bh) Lys in position 326 is Pro; (bi) in place of 326 (Bj) replaces Lys at position 334 is Ala; (bk) replaces Lys at position 334 is Trp; (bl) replaces His at position 268 is Gln; (bm) replaces at position 326 is Gln; (bn) Lys at position 326 is Glu; (bo) Lys at position 326 is Met; (bp) Val at position 266 is Ile; (bq) is replacement Lys at position 334 is Glu; (br) Tyr at position 300 is Glu; (bs) Lys at position 334 is Met; (bt) Lys at position 334 is Val; (bu) Lys at position 334 is Thr (Bv) Lys replacing 334 is Ser; (bw) Lys replacing 334 is His; (bx) Lys replacing 334 is Phe; (by) Lys replacing 334 is Gln; (bz) replacing 334 Lys at position Pro is; (ca) Lys at position 334 is Tyr; (cb) Lys at position 334 is Ile; (cc) Gln at position 295 is Leu; (cd) Lys at position 334 is Leu; (ce) Lys at position 334 is Asn; (cf) His at position 268 is Ala; (cg) Ser at position 239 is Asp; (ch) Ser at position 267 is Ala; (ci) at position 234 Lec is Trp; (cj) Leu replacing T234 is Tyr; (ck) Gly replacing 237 is Ala; (cl) Gly replacing 237 is Asp; (cm) Gly replacing 237 is Glu; ( cn) Gly at 237 is Leu; (co) Gly at 237 is Met; (cp) Gly at 237 is Tyr; (cq) Ala at 330 is Lys; (cr) is at 330 Ala is Arg; (cs) replaces Glu at position 233 as Asp; (ct) replaces 268 at position His is Asp; (cu) replaces 268 at position His is Glu; (cv) replaces 326 at position Lys is Asp; (cw ) Replaces 326 (Cx) replaces Lys at position 326 with Thr; (cy) replaces Val at position 323 with Ile; (cz) replaces Val at position 323 with Leu; (da) replaces position 323 with Val is Met; 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 a combination of 2 or more positions. Or such ideal modifications may include, for example, variants of the human constant region or human Fc region of human IgG (IgG1, IgG2, IgG3 or IgG4) as shown in Tables 14 to 15, 17 to 24, and 26 to 28 of WO2013 / 047752 , Which according to the EU numbering The amino acid at position 238 is Asp, and the amino acid at position 271 according to EU numbering is Gly. In addition, it can replace 233, 234, 237, 264, 265, 266, 267, 268, 269, 269, One or more of 272, 296, 326, 327, 330, 331, 332, 333, and 396 positions. In this case, this variant may include, but is not limited to, a variant of the human constant region or human Fc region, which contains one or more amino acids according to the EU numbering scheme: Asp at position 233, Tyr at position 234, and 237 Bit is Asp, Bit 264 is Ile, Bit 265 is Glu, Bit 266 is any of Phe, Met, and Leu; Bit 267 is any of Ala, Glu, Gly, and Gln; Bit 268 is Asp or Glu; 269 is Asp; 272 is any of Asp, Phe, Ile, Met, Asn, and Glu; 296 is Asp; 326 is Ala or Asp; 327 is Gly; 330 is Lys or Arg; 331 Position is Ser; Position 332 is Thr; Position 333 is any of Thr, Lys, and Arg; Position 396 is any of Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, and Tyr .

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

It is more appropriate for this variant to be 238 in accordance with EU numbering as Asp and have At least one amino acid selected from the following amino acid groups: according to EU numbering, Asp at position 233, Tyr at position 234, Phe at position 235, Asp at position 237, Ile at position 264, and Ile at position 265 Glu; 266 is Phe, Leu or Met; 267 is Ala, Glu, Gly or Gln; 268 is Asp, Gln or Glu; 269 is Asp; 271 is Gly; 272 is Asp, Phe, Ile, Met, Asn, Pro or Gln; 274 is Gln; 296 is Asp or Phe; 326 is Ala or Asp; 327 is Gly; 330 is Lys, Arg or Ser; 331 is Ser; 332 is Lys , Arg, Ser or Thr; 333 is Lys, Arg, Ser or Thr; 334 is Arg, Ser or Thr; 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; 409 is Arg; 419 is Glu.

In an alternative embodiment, the Fc region-containing variant or constant region variant of the disclosed A antibody can maintain the binding activity to FcγRIIb and to all Activating FcγRs, especially the binding activity to FcγRIIa (type R) is reduced. The ideal amino acid substitution site of such a variant may be as reported in WO2014 / 163101. For example, in addition to the amino acid position 238 in accordance with the EU numbering method, there is still at least one amino acid position selected from: according to the EU number 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 one amino acid selected from the group of amino acids: according to EU numbering, Phe at position 235; 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 is His or Tyr.

Within the scope of disclosure A, the level of "maintaining binding activity to FcγRIIb" may be, but not limited to, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more 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 Large, 104% or greater, 105% or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 120% or greater, 130% or more, 140% or more, 150% or more, 175% or more or 2 times or more.

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

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

Within the scope of the disclosures A and B, unlike FcγR, which belongs to the immunoglobulin superfamily, "FcRn", especially human FcRn, is structurally similar to polypeptides of the major histocompatibility complex (MHC) class I And shows sequence identity between 22% and 29% with MHC class I molecules (Ghetie et al., Immunol. Today 18 (12), 592-598 (1997)). The FcRn line is expressed as a heterodimer, which is composed of a soluble Beta or light chain (Beta2 microglobulin) complexed with the transmembrane alpha or heavy chain. Like MHC, the FcRn alpha chain contains three extracellular domains (α1, α2, and α3), and its short cytoplasmic domains bind proteins to the cell surface. α1 interacts with the α2 domain and the FcRn-binding domain of the Fc region of this antibody (Raghavan et al., Immunity 1: 303-315 (1994)).

FcRn is expressed in the maternal placenta and yolk sac of mammals and is involved in maternal-to-fetal IgG transmission. In addition, FcRn is expressed in the small intestine of newborn rodents, and FcRn is involved in transmitting maternal IgG from digested colostrum or milk across the bristle edges of the epithelium. FcRn is also expressed in various tissues and endothelial cell lines of various species. FcRn is also manifested in vascular endothelial, muscular vascular and hepatic sinusoids in adult humans. FcRn is believed to play a role in maintaining plasma IgG concentration by binding to IgG and recycling IgG to serum. In general, the binding of FcRn to IgG molecules is strictly pH-dependent. Optimal binding was observed in an acidic pH range below pH 7.0.

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

This precursor forms a complex with human Beta2-microglobulin in vivo. So it can be manufactured for proper use by known recombination performance techniques Soluble human FcRn that forms complexes with human Beta2-microglobulin in various experimental lines. Such soluble human FcRn can be used to evaluate the FcRn-binding activity of antibodies or Fc region variants. In revealing A or B, FcRn is not particularly limited as long as it is in a form capable of binding to the FcRn-binding domain; however, ideally FcRn may be human FcRn.

Within the scope of A and B disclosed herein, when an antibody or Fc region variant has FcRn-binding activity, it may have an "FcRn-binding domain", which is preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited, as long as the antibody has binding activity or affinity for FcRn at acidic pH and / or neutral pH; or may be a domain that has activity to directly or indirectly bind to FcRn. Such domains include, but are not limited to, Fc-region 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, which have the activity of indirect binding to FcRn. In revealing A or B, it is also possible to use a domain having FcRn binding activity in an acidic pH range and / or a neutral pH range. If this domain originally has FcRn binding activity in the acidic pH range and / or the neutral pH range, it can be used without further modification. If this domain has only weak or no FcRn binding activity in the acidic pH range and / or neutral pH range, the amino acid residues of this 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, an amino acid originally in a domain having FcRn-binding activity in an acidic pH range and / or a neutral pH range can be modified to further increase its FcRn-binding activity. The FcRn binding activity before and after the amino acid modification in the acidic pH range and / or the neutral pH range can be compared to find the amino acid modification of interest for the FcRn binding domain.

The FcRn-binding domain is preferably a region that directly binds to FcRn. So Desirable FcRn binding domains include, for example, the constant and Fc regions of an antibody. However, it can bind to a region having FcRn-binding activity such as a polypeptide of 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 for albumin or IgG. There is no limitation. In order to promote the elimination of antigen from plasma, an FcRn binding domain with a higher FcRn binding activity at neutral pH should be used. To promote antibody retention in plasma, an FcRn binding with a higher FcRn binding activity at acidic pH is preferred area. For example, an FcRn-binding domain that has greater FcRn-binding activity at neutral or acidic pH may be selected. Alternatively, the amino acid of the antibody or Fc region variant can be modified to carry FcRn binding activity at neutral or acidic pH. Alternatively, pre-existing FcRn binding activity at neutral or acidic pH can be increased.

Whether the FcRn binding activity of an antibody or an Fc region (variant) within the scope of A and B disclosed herein is increased, (substantially) maintained or decreased compared to the antibody or Fc region (variant) before modification Evaluation can be performed using known methods, such as those described in the examples herein, and, for example, BIACORE, Scatchard plot, and flow cytometer (see WO2013 / 046722). The extracellular domain of human FcRn can be used as the soluble antigen in these assays. Conditions other than pH at which the FcRn-binding activity of an antibody or an Fc region (variant) is measured can be appropriately selected by a person having ordinary skill in the art. This analysis method can be performed under conditions such as MES buffer and 37 ° C, as described in WO2009 / 125825. The FcRn-binding activity of an antibody or an Fc region (variant) can be evaluated, 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 based on the dissociation constant (KD), apparent dissociation constant (apparent KD), dissociation rate (kd), and apparent dissociation rate (Apparent kd) Evaluation.

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

In one embodiment, when it is revealed that the antibody of A or B has FcRn-binding activity, it has an FcRn-binding domain, and is 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 that has an activity of directly or indirectly binding to FcRn. In a specific embodiment, it is disclosed that antibodies of A or B preferably have an increased FcRn binding activity at neutral pH conditions compared to a reference antibody containing the constant region of a native IgG (see WO2013 / 046722). From the viewpoint of comparing the FcRn binding activity between the two, it is suitable but not limited: this reveals that the antibody of A or B and the reference antibody containing the constant region of the natural IgG, except that one or more amino acid residues have been modified Regions other than the constant region (such as the variable region) of the group have the same amino acid sequence.

In one embodiment, within the scope of disclosure A described herein, when the antibody of disclosure A has increased FcRn binding activity under neutral pH conditions, and is not bound by a specific theory, the antibodies of disclosure A may have a combination of 2 or more Multiple to The following properties: shuttle between plasma and cellular endosomes, with a single antibody molecule repeatedly binding to multiple antigens by having an ion concentration-dependent antigen-binding domain; by increasing the isoelectric point value and increasing the positive charge of the entire antibody, quickly Is taken up into cells; and is rapidly taken up into cells by increasing FcRn binding activity under neutral pH conditions. As a result, the half-life of the antibody in the plasma can be further shortened or the binding activity of the antibody to the extracellular matrix can be further increased, or the elimination of the antigen from the plasma can be further promoted. Those skilled in the art can determine the optimum isoelectric point value of the antibody of this disclosure A to benefit from this property.

Within the scope of disclosure A and B described here. According to Yeung et al. (J. Immunol. 182: 7663-7671 (2009)), in the acidic pH range (pH 6.0), the activity of natural human IgG1 binding to human FcRn is KD 1.7 μM, and in the neutral pH range This activity was hardly detected. Therefore, in order to increase the FcRn binding activity in the neutral pH range, the following antibodies should be used to reveal 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 or stronger than natural human IgG; preferably an antibody or constant region variant or Fc region variant, whose human FcRn binding activity is acidic The pH range is KD 2.0 μM or stronger, and its human FcRn binding activity is at a neutral pH range KD 40 μM or stronger; and more preferably an antibody or constant region variant or Fc region variant, whose human FcRn binding activity KD is 0.5 μM or more in the acidic pH range, and its human FcRn binding activity is KD 15 μM or more in the neutral pH range. The KD value was determined according to the method described in Yeung et al. (J. Immunol. 182: 7663-7671 (2009) (by immobilizing an antibody on a wafer and loading human FcRn as an analyte)).

Within the scope of disclosures A and B described in this document, A domain of any structure of FcRn serves as an FcRn binding domain. In this case, it is not necessary to introduce an amino acid modification to make the FcRn binding domain, or it is possible to increase the affinity for FcRn by introducing additional modifications.

Within the scope of disclosures A and B described herein, the starting FcRn-binding domain may include, for example, the Fc region or the constant region of (human) IgG. As long as the variant of the starting Fc region or the starting constant region can bind to FcRn in the acidic pH range and / or the neutral pH range, any Fc region or constant region can be used as the starting Fc region or the starting constant region . Alternatively, an Fc region or constant region obtained by modifying the starting Fc region or the starting constant region (whose Fc region or constant region has been modified) can also be suitably used as the Fc region or constant region. The starting Fc region or the starting constant region may include a known Fc region that is produced using recombinant. Depending on the context, the starting Fc region or the starting constant region may refer to the polypeptide itself, a composition containing the starting Fc region or the starting constant region, or an amino acid sequence encoded as the starting Fc region or the starting constant region. The origin of the starting Fc region or the starting constant region is not limited and can be obtained from any non-human animal or human organism. The initiating FcRn binding domain can be obtained from Malay monkeys, marmosets, rhesus monkeys, chimpanzees and humans. The starting Fc region or starting constant region can be obtained from human IgG1, but is not limited to any particular IgG class. This means that the Fc region of human IgG1, IgG2, IgG3 or IgG4 can be used as an appropriate starting FcRn binding domain, and Fc regions or constant regions of any IgG class or subclass from any organism can be used as the starting Fc region or As the starting constant region. Examples of natural 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 initiating FcRn binding domain, initiating Fc region, or initiating constant region may include, for example, one or more mutations: such as substitutions and initiating Fc regions Or the amino acid residue in the initial constant region is a mutation of a different amino acid residue; insert one or more amino acid residues into the amino acid residue in the initial Fc region or the initial constant region; Alternatively, one or more amino acid residues may be deleted from the amino acid residues of the starting Fc region or the starting constant region. The amino acid sequence of the Fc region or constant region is preferably modified to contain at least a portion of the amino acid sequence of the Fc region or constant region that does not occur naturally. Such a variant must have less than 100% sequence identity or similarity to the starting Fc region or the starting constant region. For example, the amino acid sequence of this variant and the amino acid sequence of the starting Fc region or the starting constant region are about 75% to less than 100%, more preferably about 80% to less than 100%, and more preferably about 85%. At least 100%, more preferably about 90% to less than 100%, and still more preferably about 95% to less than 100% identity or similarity. In one non-limiting example, the modified Fc region or constant region between A and B is disclosed to be different from the initial Fc region or the initial constant region by at least one amino acid.

Within the scope of the disclosures A and B described herein, an Fc region or a constant region having FcRn-binding activity in an acidic pH range and / or a neutral pH range can be obtained by any method. Specifically, variants of an Fc region or a constant region having FcRn binding activity in an acidic pH range and / or a neutral pH range can be modified by modifying a human IgG-type antibody that can serve as an initial Fc region or an initial constant region. Amino acids are obtained. Suitable modified IgG-type antibody Fc regions or constant regions include, for example, human IgGs (IgG1, IgG2, IgG3, and IgG4 and variants thereof), and mutants generated spontaneously are also included in this IgG Fc region or constant region. For the Fc region or constant region of human IgG1, human IgG2, human IgG3 and human IgG4 antibodies, some paratypic sequences due to the polymorphism of genes are described in "Sequences of proteins of "Immunological interest", NIH Publication No. 91-3242, any of which can be used to reveal A or B. In particular, for human IgG1 sequences, the amino acid sequence at positions 356 to 358 according to EU numbering can be DEL or EEM.

Revealing one of the embodiments of A or B, the modification into other amino acids is not particularly limited, as long as the obtained variant has an acidic pH range and / or a neutral pH range, and preferably has an FcRn binding activity in the neutral pH range. Amino acid modified sites that increase FcRn binding activity at neutral pH conditions are described in, for example, WO2013 / 046722. Such modified sites include, for example, one or more positions selected from the group consisting of an Fc region or a constant region of a human IgG antibody, 221 to 225, 227, 228, 230, 232, 233 to 241 in accordance with EU numbering , 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, modification of 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 may be performed individually for a single position or at 2 or more positions. The modification of these amino acid residues can enhance the Fc region or constant region of the IgG antibody in binding to FcRn at neutral pH conditions. These amino acid residue modifications can also be appropriately introduced into antibodies revealing A or B.

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

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

In one embodiment, the amino acid sequence of the antibody to be modified to reveal A or B should preferably contain a human sequence (found in natural human-derived antibodies), so that when the antibody is administered into the living body (preferably into the human body), the Immunogenicity of antibodies. Alternatively, after the modification, mutations may be introduced at positions other than the amino acid modification site so that one or more of the FR. (FR1, FR2, FR3, and FR4) replace the adult sequence. Methods for replacing FR adult sequences are known in the art and include, but are not limited to, Ono et al., Mol. Immunol. 36 (6): 387-395 (1999) reporter. Humanization method in This technical field is known and includes, but is not limited to, the 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 antibodies of A or B may include human germline framework sequences. When the framework sequence is completely human germline sequence, it is expected that the antibody, when administered to a human (for example, to treat or prevent a disease), will induce a small or no immunogenic response.

The FR sequence may preferably include, for example, a fully human FR sequence such as shown in V-Base (vbase.mrc-cpe.cam.ac.uk/). These FR sequences can be suitably used to reveal A or B. 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) ); And Cox et al. (Nat. Genetics 7: 162-168 (1994)). The ideal germline sequence can be appropriately selected from: Vκ, which is classified into 7 subgroups; Vλ, which is classified into 10 Subgroups; and VH, which is classified into 7 subgroups.

Fully human VH sequences preferably include, for example, VH sequences: subgroups VH1 (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, and VH1- 69); subgroups VH2 (such as VH2-5, VH2-26, and VH2-70); subgroups 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); subgroups 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 skilled in the art have ordinary knowledge. The antibody can be appropriately designed based on such sequence information. Ideally, other fully human FR sequences or sequences of sequences equivalent thereto may be used.

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

The fully human Vλ sequence preferably includes, for example: V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1 -18, V1-19, V1-20, and V1-22 are classified into subgroups 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 subgroups VL2; V3-2, V3-3, and V3-4 are classified as subgroups VL3; V4-1, V4-2, V4-3, V4-4 and V4-6 are classified as subgroup VL4; or V5-1, V5-2, V5-4 and V5-6 are classified as subgroup VL5 (Kawasaki et al. Genome Res. 7: 250- 261 (1997)).

In general, these FR sequences may differ from one or more amino acid residues. These FR sequences can be used in modifying 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, for example, the aforementioned Kabat et al. (1991); Wu et al. (J. Exp. Med. 132: 211-250 (1970)).

Within the scope of the disclosures A and B described herein, "flexible residues" may refer to amino acid residue variations at locations where high amino acid diversity is present, where the light or heavy chain is variable Compare the known amino acid sequences and / or natural antibody or antigen binding domains with some different amino acids. Positions showing high diversity are generally located in the CDR. The information provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) is useful for deciding where high diversity of known and / or natural antibodies is. Still other online databases (vbase.mrc-cpe.cam.ac.uk/, bioinf.org.uk/abs/index.html) provide a collection of many light and heavy chain sequences and their locations. Such sequence and position information is useful for determining flexible residue positions. There is no limitation, for example, when the amino acid residue at a particular position has variability, it is preferably 2 to 20, 3 to 19, 4 to 18, 5 to 17, 6 to 16, 7 to 15, 8 to 14, 9 to 13 or 10 to 12 amino acid residues, this position can be judged to show (high) diversity.

In one embodiment, it is understood that when the antibody of A or B contains all or a portion of the light chain variable region and / or the heavy chain variable region, the antibody may include one or more suitable flexible residues as necessary. For example, heavy chain and / or light chain variable region sequences selected with FR sequences originally contained amino acid residues that would change the antigen-binding activity of the antibody according to the ion concentration (hydrogen ion concentration or calcium ion concentration) conditions. It is designed to contain other amino acid residues in addition to these amino acid residues. In this case, the number and position of the flexible residues, for example, may not be limited to a specific embodiment, as long as the antigen-binding activity of the antibody of A or B disclosed herein will change according to the ion concentration conditions. Specifically for heavy and / or light chains The CDR sequence and / or the FR sequence may include at least 1 flexible residue. For example, if the ion concentration is a 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. . Similarly, suitable flexible residues can be introduced into all or part of the light chain variable region and / or heavy chain variable region, such as an ion concentration-dependent antibody or an ion concentration-free antibody, in which the antibody can be exposed. At least one amino acid residue on the surface is modified to increase the isoelectric point value.

(Positions are displayed according to Kabat numbering.)

(Positions are displayed 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 in order to produce a protein that is more than The humanization of chimeric antibodies without such modifications has a shorter plasma half-life revealing antibodies to A or B. Modification of amino acid residues of humanized antibodies that may be exposed on the surface can be humanized in this antibody Before or simultaneously. Or by using a humanized antibody as a starting material, amino acid residues that may be exposed on the surface can be modified to further modify the isoelectric point value of the humanized antibody.

Adams et al. (Cancer Immunol. Immunother. 55 (6): 717-727 (2006)) reported humanized antibodies, trastuzumab (antigen: HER2), bevacizumab (antigen: VEGF), and pertuzumab (antigen: HER2), using the same human The antibody FR sequence is humanized and has roughly comparable pharmacokinetics in plasma. Specifically, humanization can be performed using the same FR sequence, and understanding of plasma pharmacokinetics is roughly comparable. According to one embodiment of Disclosure A, in addition to the humanization step, by modifying the amino acid residues that can be exposed on the surface of the antibody to increase the isoelectric point value of the antibody, the concentration of this antigen in the plasma is reduced. In revealing an alternative embodiment of either A or B, a human antibody can be used. By modifying the amino acid residues of human antibodies made from the human antibody library that may be exposed on the surface, it can increase the human antibody-producing mice, recombinant cells, etc., and increase the isoelectric point value of the human antibody, and the original The ability of human antibodies to eliminate antigens from plasma.

In one embodiment, the antibody of A may include a modified sugar chain. Antibodies with modified sugar chains include, for example, modified glycated antibodies (WO99 / 54342), antibodies lacking fucose (WO00 / 61739; WO02 / 31140, WO2006 / 067847; WO2006 / 067913), and intermediates GlcNAc sugar chain antibody (WO02 / 79255).

In one embodiment, it is disclosed that the antibodies of A or B can be used, for example, in a technology that displays antitumor activity against cancer cells, or a technology that promotes elimination of biologically harmful antigens from plasma.

In an alternative embodiment, reveal whether A or B is related to an increase Ion concentration-dependent antigen-binding domains of isoelectric points or libraries of ion concentration-dependent antibodies with increased isoelectric points, as described above.

In an alternative embodiment, it is revealed that A or B is related to a nucleic acid (polynucleotide) related to 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 appropriate known methods. For specific embodiments, refer to, for example, WO2009 / 125825, WO2012 / 073992, WO2011 / 122011, WO2013 / 046722, WO2013 / 046704, WO2000 / 042072, WO2006 / 019447, WO2012 / 115241, WO2013 / 047752, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101, WO2013 / 081143, WO2007 / 114319, WO2009 / 041643, WO2014 / 145159, WO2012 / 016227, and WO2012 / 093704, each of which is incorporated by reference in its entirety.

In one embodiment, it is disclosed that the nucleic acid of A or B may be an isolated or purified nucleic acid. The nucleic acid encoding the antibody that discloses A or B for this purpose can be any gene, and can be DNA or RNA or other nucleic acid analogs.

In the disclosure A and B, if the amino acid of the antibody is modified, the amino acid sequence of the antibody may be a known sequence or a newly obtained antibody amino acid sequence before modification. For example, it can be obtained from an antibody library or by selecting a nucleic acid encoding an antibody derived from a fusion cell to produce a monoclonal antibody-producing B cell. The method for obtaining a nucleic acid encoded as an antibody from a fusion tumor can use the following techniques: using the antigen of interest or expressing cells of the antigen of interest as a sensitizing antigen to immunize by a conventional method of immunization; Known parental cells are fused using conventional cell fusion methods; use known screening Methods Screening for cells (fusion tumors) producing single antibodies; synthesizing cDNA of the variable region (V region) of this antibody using reverse transcriptase from the mRNA of the obtained fusion tumor; and linking this cDNA to the constant region encoding the antibody of interest ( Region C).

The sensory antigens used to obtain the nucleic acids encoding the above heavy and light chains include, but are not limited to, complete antigens that are immunogenic; and incomplete antigens, including haptens that are not immunogenic. For example, the entire protein of interest, or a part of the peptide of this protein can be used. Also included are substances composed of polysaccharides, nucleic acids, lipids, and other compositions with known antigenic potential. Therefore, in some embodiments, the antigen of the A or B antibody disclosed herein is not particularly limited. This antigen can be produced by, for example, a baculovirus-based method (see, for example, WO98 / 46777). Fusion tumors can be generated according to the methods of G. Kohler and C. Milstein, Methods Enzymol. 73: 3-46 (1981)). When the antigen is low in immunogenicity, immunization can be carried out by linking the antigen with a large immunogenic molecule such as albumin. Alternatively, if desired, soluble antigens can be prepared by linking this antigen to other molecules. When a transmembrane molecule such as a membrane antigen (such as a receptor) is used as the antigen, a part of the extracellular region of the membrane antigen may be used as a fragment, or a cell expressing the transmembrane molecule on the surface may be used as an immunogen.

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

Animal immunization can be performed, for example, by appropriately diluting and suspending the antigen as a phosphate buffered saline (PBS), physiological saline or other, and mixing with an adjuvant as needed for emulsification; the animal is then injected intraperitoneally or subcutaneously. Then the sensory antigen, which has been mixed with Freud's incomplete adjuvant, should be given every 4 to 21 days, several times. The production of antibodies can be known, for example, by measuring the antibody titer of interest in animal serum.

Antibody-producing cells obtained from lymphocytes or animals immunized with the desired antigen can use conventional fusion agents (such as polyethylene glycol) (Goding, Antibodies: Principles and Practice, Academic Press, 1986, 59-103) and myeloma cells Fusion to produce a fusion tumor. If necessary, the fusion tumor can be cultured and expanded, and the binding specificity of the antibody produced by the fusion tumor can be evaluated by, for example, immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent analysis (ELISA). Then, if necessary, antibody-producing fusion tumors that have determined specificity, affinity, or activity of interest can be sub-selected using, for example, extreme dilution.

Nucleic acids encoding selected antibodies can be obtained from fusion tumors or antibody-producing cells (feeling to lymphocytes, etc.) using probes that specifically bind to the antibody (e.g., oligonucleotides complementary to sequences encoding the constant region of the antibody). Breeding. Alternatively, this nucleic acid can be cloned from mRNA using RT-PCR. Used to produce revealing A or B The heavy and light chains of an antibody can be derived from, for example, antibodies belonging to any Ig antibody subclass and subclass, preferably IgG.

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

In one embodiment, in order to express antibodies or fragments thereof that reveal 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 promoters or signal sequences, phenotypic selection genes, origins of replication, and other elements.

Methods for introducing desired amino acid modifications into antibodies have been established in this technical field. For example, the library can be modified by introducing at least one amino acid residue that may be exposed on the surface of the antibody that reveals A or B and / or at least one amino acid that will change the antigen-binding activity of the antibody according to the ion concentration conditions To make. In addition, if desired, flexible residues can be added using the method of Kunkel et al. (Methods Enzymol. 154: 367-382 (1987)).

In an alternative embodiment, it is disclosed that A is a vector containing a nucleic acid encoding an ion concentration-dependent antigen-binding domain having an increased isoelectric point value, or an ion concentration-dependent antibody having an increased isoelectric point value. In one embodiment, the vector can be obtained from vectors described in, for example, WO2009 / 125825, WO2012 / 073992, WO2011 / 122011, WO2013 / 046722, WO2013 / 046704, WO2000 / 042072, WO2006 / 019447, WO2012 / 115241 , WO2013 / 047752, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101, WO2013 / 081143, WO2007 / 114319, WO2009 / 041643, WO2014 / 145159, WO2012 / 016227, or WO2012 / 093704, each of which is completed The entire reference is for reference.

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

In embodiments, the expression vector can be used as a vector containing a nucleic acid directed against disclosure A or B. Expression vectors can be used to allow the polypeptide to be expressed in a test tube, E. coli, in cultured cells, or in vivo. For example, pBEST vector (Promega) can be used for in-tube expression; pET vector (Invitrogen) is used for E.coli expression; pME18S-FL3 vector (GenBank Accession No. AB009864) is used for culture cell performance; 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 conventional methods, such as by a ligase reaction using a restriction enzyme site (see Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

In an alternative embodiment, it is disclosed that A is a host or a host cell comprising a vector containing a nucleic acid encoding an ion concentration-dependent antigen-binding domain having the above-mentioned increased isoelectric point value or an ion concentration-dependent that the above-mentioned isoelectric point value is increased Sexual antibodies. In one embodiment, the host or host cell can be prepared by a method described in the following documents: for example, WO2009 / 125825, WO2012 / 073992, WO2011 / 122011, WO2013 / 046722, WO2013 / 046704, WO2000 / 042072, WO2006 / 019447, WO2012 / 115241, WO2013 / 047752, WO2013 / 125667, WO2014 / 030728, WO2014 / 163101, WO2013 / 081143, WO2007 / 114319, WO2009 / 041643, WO2014 / 145159, WO2012 / 016227 or WO2012 / 093704, each of which is fully incorporated by reference.

It is revealed that the type of the host cell of A or B is not particularly limited, and the host cell includes, for example, a bacterial cell such as E. coli and various animal cells. Host cells can be used appropriately as a production line for producing and expressing 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; amphiphilic 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 a cationic microlipid DOTAP (Boehringer-Mannheim), the electroporation method, and the lipid staining method.

Plant cells known as protein-producing lines include, for example, cells derived from Nicotiana tabacum and cells derived from Lemna minor. Healing tissue can be cultured from these cells to make antibodies that reveal A or B. 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 Cells of the genus Pleurotus are, for example, black fungus. 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.

In order to use the host cell to produce an antibody that reveals A or B, the host cell is transformed with a nucleic acid containing an antibody that encodes the antibody that reveals A or B, and cultured to express the nucleic acid. For example, when animal cells are used as a host, the culture medium may include, for example, DMEM, MEM, RPMI1640, and IMDM, a serum supplement such as FBS or fetal bovine serum (FCS) may be appropriately used in combination, or the cells may be cultured in serum-free.

On the other hand, an animal or plant can be used in an in vivo production system for the production of an antibody that reveals A or B. For example, a nucleic acid encoding the antibody that reveals A or B can be introduced into such an animal or plant to produce the antibody in vivo, This antibody can then be collected from the animal or plant.

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

In one example, a nucleic acid encoding an antibody that reveals A or B is prepared and a fusion gene encoding a gene specifically included in a polypeptide such as goat Beta-casein in milk, and a polynucleotide fragment containing the fusion gene is injected Goat embryos are transferred to female goats. Antibodies of interest can be obtained from milk secreted by the transgenic goat, which was born from or derived from a goat receiving the embryo. Hormones can be appropriately administered to the transgenic goat to increase the amount of antibody-containing milk produced by the goat (Ebert et al., Bio / Technology 12: 699-702 (1994)).

Insects used to produce antibodies revealing A or B include, for example, silkworms. When silkworms are used, they are inserted with a polynucleotide encoding an antibody of interest in a viral group. Infected silkworm with baculovirus. Antibodies of interest can be obtained from the body fluids of infected silkworms (Susumu et al., Nature 315: 592-594 (1985)).

When plants are used to produce antibodies that reveal 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 resistance system is obtained from the leaves of the infected tobacco. Such modified bacteria can also be used to infect duckweed (Lemna minor), and the expected antibodies can be obtained from the colony cells of infected duckweed (Cox et al. Nat. Biotechnol. 24 (12): 1591-1597 (2006) ).

In order for the antibodies expressed in the host cells to be secreted into the lumen, periplasmic space or extracellular environment of the endoplasmic reticulum, a stable secretion signal can be encapsulated 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.

The antibodies of A or B disclosed as described above can be isolated from the host cells or inside or outside the host (such as culture medium and milk) and refined into substantially pure and homogeneous antibodies. This antibody can be appropriately isolated and purified, for example, by appropriately selecting and combining chromatography into a 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, hydrophobic chromatography, gel filtration chromatography, reverse chromatography, and adsorption chromatography. Such chromatography can be performed, for example, 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 tube Columns include, for example, Hyper D, POROS, Sepharose F.F. (Pharmacia).

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

In an alternative embodiment, a method for producing an antibody containing an antigen-binding domain whose antigen-binding activity changes according to ionic concentration conditions is disclosed, which may include culturing a host cell or feeding a host, from which a cell culture, host secretion , Or other methods known in the art to collect antibodies.

In one embodiment, it is disclosed that A is related to a production method, which may include one or more steps selected from the group consisting of: (a) selecting an antibody that promotes elimination of 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 at neutral pH conditions; (d) Select antibodies with enhanced FcγRIIb binding activity at neutral pH conditions (E) selecting an antibody that has maintained or enhanced FcγRIIb binding activity and has reduced binding activity to one or more activated FcγRs, the activated FcγR is selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc , FcγRIIIa, FcγRIIIb, and FcγRIIa; (f) select antibodies with enhanced FcRn binding activity at neutral pH conditions; (g) select antibodies with pI; (h) confirm the isoelectric point (pI) of the collected antibodies, and then Select antibodies with increased isoelectric point (pI); and (i) select antibodies with antibodies whose antigen-binding activity changes or increases according to ionic concentration conditions.

Reference antibodies here include, but are not limited to, natural antibodies (such as natural Ig antibodies, preferably natural IgG antibodies) and before modification of antibodies (antibody library construction (Before or during construction, for example, an ion concentration-dependent antibody before an isoelectric point value is increased or an antibody having an increased isoelectric point value before an ion concentration-dependent antigen-binding domain is given).

After the antibody that reveals A is generated, the antibody can be obtained using antibody pharmacokinetic analysis using, for example, mouse, rat, rabbit, dog, monkey, and human plasma to select antibodies that have improved antigen elimination from plasma compared to reference antibodies .

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

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

Alternatively, after making an antibody that reveals A, the obtained antibody and the reference antibody can be compared with 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 may be a human FcRn.

Alternatively, after manufacturing an antibody that reveals A, the obtained antibody can be evaluated by isoelectric focusing or other methods for its isoelectric point to select antibodies with an increased isoelectric point value compared to the reference antibody. In this case, the optional isoelectric point value has been increased to Less than 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 value increased by at least 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 or more or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 or more or 3.0 or more antibodies.

Alternatively, after manufacturing the antibody that reveals A, the obtained antibody can be compared with the reference antibody under low and high ion concentration conditions for the binding activity of the desired antigen using BIACORE or other methods to select antibodies whose antigen binding activity changes or increases according to the ion concentration conditions. The ion concentration may be, for example, a hydrogen ion concentration or a metal ion concentration. When the ion concentration is a metal ion concentration, it may be, for example, a calcium ion concentration. Whether or not the binding activity is altered or increased can be assessed based on the presence of, for example: (a) altered or increased antigen uptake by the cell; (b) altered or increased ability to bind to different antigen molecules multiple times; (c) reduce the antigen concentration in the plasma Changes or enhancements; or (d) changes in the plasma retention of antibodies. Alternatively, any two or more of these selection methods may be optionally combined.

In an alternative embodiment, it is disclosed that A is a method for manufacturing or screening an antibody, which contains an antigen-binding domain, the antigen-binding activity of which is changed according to ion concentration conditions, and its pI may be exposed to the surface of the antibody by modification of at least 1 The number of amino acid residues is increased ("an ion concentration-dependent antibody with an increased isoelectric point value"). The production method may, for example, optionally combine the embodiments described in the scope of disclosure A, such as the embodiments of the aforementioned method for producing or screening antibodies with an increased isoelectric point value, and for producing or screening calcium ion concentration An embodiment of a method that depends on an antigen-binding domain or a calcium-ion-dependent antibody or the aforementioned library, wherein the antigen-binding activity is higher at high calcium ion concentration conditions than at low calcium ion concentration conditions, and / or for manufacturing or screening pH-dependent conditions Antigen binding domain or pH In an embodiment of the method of dependent antibodies or libraries, the antigen-binding activity is higher at neutral pH conditions than at acidic pH conditions.

In an alternative embodiment, it is disclosed that A provides a method for making or screening an antibody, the antibody comprising an antigen-binding domain, and its extracellular matrix-binding activity is increased, wherein its antigen-binding activity is changed according to ion concentration conditions, and pI is determined by The modification may increase by exposure to at least one amino acid residue on the surface of this antibody ("Ion concentration-dependent antibody with increased isoelectric point value"). An increase in the isoelectric point value of an ion concentration-dependent antibody can be considered in this method. Such a method can be carried out, for example, by appropriately and optionally combining relevant embodiments recorded in the scope of disclosure A, such as an embodiment of a method for manufacturing or screening the aforementioned isoelectric point-increasing antibody, and for manufacturing or screening An embodiment of a method for screening a calcium ion concentration-dependent antigen-binding domain or a calcium ion concentration-dependent antibody or a library thereof, the antigen-binding activity of the antigen-binding activity is higher at high calcium ion concentration conditions than at low calcium ion concentration conditions, and / or for manufacturing or In an embodiment of the method of screening a pH-dependent antigen-binding domain or a pH-dependent antibody or a library thereof, its antigen-binding activity is higher at neutral pH conditions than at acidic pH conditions. For example, the obtained antibody can be compared with a reference antibody for extracellular matrix binding capacity using electrochemical electroluminescence or other known techniques to select antibodies with increased extracellular matrix binding.

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

In an alternative embodiment, A is disclosed for a method for making an antibody, the antibody comprising an antibody whose antigen-binding activity varies according to ionic concentration conditions. The original binding domain, this method includes the steps of modifying at least one amino acid residue that may be exposed on the surface of the antibody to increase the isoelectric point value (pI). In some embodiments, the amino acid residue modification includes a modification selected from the group consisting of: (a) replacing a negatively charged amino acid residue with an uncharged amino acid residue; (b) a substitution band Negatively charged amino acid residues are positively charged amino acid residues; and (c) replacing uncharged amino acid residues into positively charged amino acid residues. In some embodiments, at least one modified amino acid residue is substituted with histamine. In a further embodiment, the antibody includes a variable region and / or a constant region, and the amino acid residue is modified in the variable region and / or the constant region. In a further embodiment, at least one amino acid residue modified according to this method is in a position in the CDR or FR selected from one of the group consisting of: (a) the conformation in the FR of the heavy chain variable region Kabat numbering 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) Kabat numbering in the CDRs of the heavy chain variable regions 31, 61, 62, 63, 64, 65, and 97; (c) FR of the variable region of the light chain in accordance with Kabat numbering 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) the CDRs of the light chain variable regions in accordance with Kabat numbering 24, 25, 26, 27, 52, 53, 54 , 55, and 56. In another embodiment, at least one amino acid residue modified according to this method is in a position selected from the group consisting of CDR or FR: (a) 8 of FR of the heavy chain variable region , 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, Positions 84, 85, and 105; (b) positions 31, 61, 62, 63, 64, 65, and 97 of the CDRs of the heavy chain variable region; (c) 16, 18 of the FRs of the light chain variable region , 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107; and (d) 24, 25, 26, 27, 52, 53, 54 of the CDRs of the light chain variable region , 55, and 56. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of the antibody produced by 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 includes selecting an antibody that has a KD (acidic pH range (e.g., pH 5.8)) / KD (neutral pH range (e.g., pH 7.4)) of 2 or higher against the antigen. In some embodiments, the method further comprises comparing the antigen-binding activity of the antibody produced by the method under high ion concentration (such as hydrogen ion or calcium ion concentration) with low ion concentration conditions. In a further embodiment, the method further comprises selecting an antibody with a higher ionic concentration (eg, 2 times) that has a higher antigen-binding activity than a low ionic concentration antibody. In some embodiments, if the ion concentration is a calcium ion concentration, the high calcium ion concentration may be selected between 100 μM and 10 mM, 200 μM and 5 mM, 400 μM and 3 mM, 200 μM and 2 mM, or 400 μM and 1 mM. . It is also preferably selected from a concentration between 500 μM and 2.5 mM, which is close to the plasma (blood) concentration of calcium ions in the living body. In some embodiments, the low calcium ion concentration can be selected between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, or between 1 μM and 5 μM or between 2 μM and 4 μM. It is also preferable to choose a concentration between 1 μM and 5 μM, which is close to the calcium ion concentration of the early endosomes in vivo. In some embodiments, the lower limit value of KD (low calcium ion concentration condition) / KD (high calcium ion concentration condition) (e.g., KD (3 μM Ca) / KD (2mM Ca)) is 2 or more, 10 or more More or 40 or more, high limit is 400 or Less, 1000 or less, or 10,000 or less. In some alternative embodiments, the lower limit of kd (low calcium ion concentration conditions) / kd (high calcium ion concentration conditions) (e.g., kd (3μM Ca) / kd (2mM Ca)) has a value of 2 or more, 5 or more, 10 or more or 30 or more, and the high limit is 50 or less, 100 or less or 200 or less. In some embodiments, if the ion concentration is a hydrogen ion concentration, the low hydrogen ion concentration (neutral pH range) may be selected from pH 6.7 to pH 10.0, from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0 range. The low hydrogen ion concentration is preferably pH 7.4, which is close to the pH in plasma (blood) in vivo, but for convenience of measurement, for example, pH 7.0 can be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) may 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 is connected to the living hydrogen ion concentration in the early endosome, but for convenience of measurement, for example, pH 6.0 can be used. In some embodiments, the lower limit of KD (acid pH range) / KD (neutral pH range) (e.g., KD (pH 5.8) / KD (pH 7.4)) is 2 or more, 10 or more, or 40 or More, the high limit is 400 or less, 1000 or less, or 10,000 or less. In some embodiments, the method further comprises comparing the elimination of the antigen from the plasma before administration of the antibody produced by this method and administration of the antibody from a plasma that does not include the modification of the reference antibody introduced by this method to a different reference antibody. In a further embodiment, the method further comprises selecting an antibody made in this way and an antibody that does not contain a modification introduced in this way, in order to promote elimination of the antigen (eg, 2-fold) from the plasma. In some embodiments, the method further comprises comparing the antibodies produced according to this method to the binding of the antibody to the extracellular matrix with and without the modified antibodies introduced by this method to different antibodies. In a further embodiment, this method further includes selecting antibodies made in this way, which are different than those that do not include modifications introduced in this way. Antibodies have increased extracellular matrix binding (for example, double when bound to antigens). In a further embodiment, the antibody produced by this method is compared to the antibody before modification or the modification of at least one amino acid residue before the increase of pI (natural antibody (such as natural Ig antibody, preferably natural). IgG antibodies) or reference antibodies (such as antibodies before or before or during library construction)), (substantially) retain this antigen-binding activity. In this case, "(substantially) retain this antigen-binding activity" may mean that compared to the binding activity of the antibody before modification or modification, it is at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and more preferably 80%, 85%, 90% or 95% or more activity.

In an additional embodiment, a method for manufacturing an antibody is disclosed. The antibody includes an antigen-binding domain whose antigen-binding activity changes according to ionic concentration conditions, wherein the method includes modifying at least one antibody that may be exposed on the surface of the constant region of the antibody. Amino acid residues to increase the isoelectric point (pI). In some embodiments, the amino acid residue modification includes a modification selected from the group consisting of: (a) replacing a negatively charged amino acid residue with an uncharged amino acid residue; (b) replacing a negatively charged amino acid The amino acid residue 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 histamine. In a further embodiment, the antibody includes a variable region and / or a constant region, and the amino acid residue is modified in the variable region and / or the constant region. In a further embodiment, at least one amino acid residue modified in accordance with this method is located in the constant region and is selected from one of 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, at least one amino acid residue modified in accordance with this method is located in the constant region and is selected from one of 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. In another embodiment, at least one amino acid residue modified according to this method is in the constant region selected from one of the group consisting of: 282, 309, 311, 315, 342 according to EU numbering , 343, 384, 399, 401. In some embodiments, the method further comprises comparing the antigen-binding activity of the antibodies made in this way under conditions of high ion concentration (such as hydrogen ion or calcium ion concentration) and low ion concentration conditions. In a further embodiment, the method further comprises selecting an antibody that has a higher antigen-binding activity at a higher ion concentration than an antibody at a lower ion concentration. In some embodiments, the method further comprises comparing the elimination of the antigen from the plasma before administration of the antibody produced by this method and administration of the antibody from a plasma that does not include the modification of the reference antibody introduced by this method to a different reference antibody. In a further embodiment, the method further comprises selecting an antibody made in this way and an antibody that does not contain a modification introduced in this way, in order to promote elimination of the antigen (eg, 2-fold) from the plasma. In some embodiments, the method further comprises comparing the antibodies produced according to this method to the binding of the antibody to the extracellular matrix with and without the modified antibodies introduced by this method to different antibodies. In a further embodiment, the method further includes selecting antibodies made according to this method, which has increased extracellular matrix binding (e.g., 2 when bound to an antigen, compared to only antibodies that do not include modifications introduced in this way). Times). In some embodiments, this method includes antibodies made in accordance with this method and includes natural IgG The reference antibody for the constant region of the antibody is directed to the Fc gamma receptor (FcγR) binding activity at a neutral pH (eg, pH 7.4). In a further embodiment, the method includes the step of selecting an antibody produced according to the method, which antibody has an enhanced FcγR binding activity at a neutral pH (eg, pH 7.4) compared to a reference antibody including a constant region of a natural IgG. In some embodiments, the antibodies produced according to this method are selected to have enhanced FcyRIIb binding activity at neutral pH. In some embodiments, the selected antibodies produced according to this method have binding activity to one or more activating FcγR (preferably selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa) and FcγRIIb Alternatively, the FcγRIIb binding activity is maintained or increased, and the binding activity to the activated FcγR is reduced, compared to a reference antibody that differs only in that the constant region is the constant region of the natural IgG. In some embodiments, the method further comprises comparing the FcRn-binding activity of the antibody produced according to this method with a reference antibody that differs only in the constant region of the constant region of the native IgG, at neutral pH conditions. In a further embodiment, the method further comprises selecting an antibody produced according to the method, which antibody has an FcRn-binding activity at neutral pH compared to a reference antibody that differs only in that the constant region is the constant region of a natural IgG. Increase (for example 2 times). In some embodiments, the antibody produced by this method is compared to the modification of the antibody or the modification of at least one amino acid residue before the increase of pI (natural antibody (such as natural Ig antibody, preferably natural IgG). Antibody) or a reference antibody (such as an antibody prior to antibody modification, or before or during library construction), and (substantially) retain this antigen-binding activity. In this case, "(substantially) retain this antigen-binding activity" may mean at least 50% or more, preferably 60% or more, and more preferably 70 compared to the binding activity of the antibody before modification or modification. % Or 75% or more, and preferably 80%, 85%, 90% or 95% or more.

In an additional embodiment, the method includes 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 constant regions disclosed above. In a further embodiment, at least one amino acid residue modified according to this method is located at one of the variable regions disclosed above. In a further embodiment, at least one amino acid residue modified according to this method is located at one of the constant regions disclosed above and at least one amino acid residue modified according to this method is located at above disclosed 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 the antibody produced by the method to its corresponding antigen at an acidic pH (eg, pH 5.8) and a neutral pH (eg, pH 7.4). In a further embodiment, the method includes selecting an antibody having 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 (such as hydrogen ion or calcium ion concentration) and low ion concentration conditions. In a further embodiment, the method further comprises selecting an antibody that has higher antigen-binding activity at high ion concentrations than at low ion concentrations. In some embodiments, if the ion concentration is a calcium ion concentration, the high calcium ion concentration may be selected from between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM, or between 400 μM and 1 mM. Of the concentration. It is also desirable to select a concentration between 500 μM and 2.5 mM. In some embodiments, the low calcium ion concentration may 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, it is selected from a concentration between 1 μM and 5 μM. In some implementations The lower limit of the KD (low calcium ion concentration condition) / KD (high calcium ion concentration condition) (e.g., KD (3μM Ca) / KD (2mM Ca)) value is 2 or more, 10 or more, or 40 Or more, the high limit is 400 or less, 1000 or less, or 10,000 or less. In some alternative embodiments, the lower limit of kd (low calcium ion concentration conditions) / kd (high calcium ion concentration conditions) (e.g., kd (3μM Ca) / kd (2mM Ca)) is 2 or more, 5 or More, 10 or more, or 30 or more, and the high limit is 50 or less, 100 or less, or 200 or less. In some embodiments, if the ion concentration is a hydrogen ion concentration, the low hydrogen ion concentration (neutral pH range) may be 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 selection. The low hydrogen ion concentration is preferably pH 7.4, which is close to the (blood) pH of plasma in vivo. For convenience of measurement, for example, pH 7.0 can be used. In some embodiments, the high hydrogen ion concentration (acidic pH range) can be selected from pH 4.0 to pH 6.5, from pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The acidic pH range can be pH 5.8 or pH 6.0. In some embodiments, the lower limit of KD (acid pH range) / KD (neutral pH range) (e.g., KD (pH 5.8) / KD (pH 7.4)) is 2 or more, 10 or more, or 40 or More, the high limit is 400 or less, 1000 or less, or 10,000 or less.

In some embodiments, the method further comprises comparing the elimination of the antigen from the plasma before administration of the antibody produced by this method and administration of the antibody from a plasma that does not include the modification of the reference antibody introduced by this method to a different reference antibody. In a further embodiment, the method further comprises selecting an antibody made in this way and an antibody that does not contain a modification introduced in this way, in order to promote elimination of the antigen (eg, 2-fold) from the plasma. In some embodiments, the method further includes comparing the antibodies produced by this method with only and excluding the modifications introduced by this method into different antibodies directed against the extracellular of the antibody. Matrix binding. In a further embodiment, the method further includes selecting antibodies made in this way, which has increased extracellular matrix binding (e.g., 5 when complexed with an antigen, compared to only antibodies that do not include modifications introduced in this way). Times). In some embodiments, the method further comprises comparing the Fc gamma receptor (FcγR) binding activity of the antibody produced by this method with a reference antibody including the constant region of natural IgG at a neutral pH (eg, pH 7.4). In a further embodiment, the method includes selecting an antibody made according to this method, which has an increased FcγR binding activity (eg, pH 7.4) at neutral pH compared to an antibody that includes the constant region of a control native IgG. In some embodiments, selected antibodies produced according to this method have enhanced FcγRIIb binding activity at neutral pH. In some embodiments, the selected antibodies produced according to this method have binding activity to one or more activated FcγR (preferably selected from the group consisting of: FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa), and FcγRIIb, And optionally, the FcγRIIb binding activity is maintained or increased, and the binding activity to the activated FcγR is reduced. In some embodiments, the method further includes comparing the antibody produced according to this method with the constant region of the IgG only in the constant region. The reference antibody differs in its FcRn binding activity at neutral pH. In a further embodiment, the method further comprises selecting an antibody produced according to the method, which antibody has an FcRn-binding activity at neutral pH compared to a reference antibody that differs only in that the constant region is the constant region of a natural IgG. Increase (for example 2 times). Compared to the antibody produced by this method, the pI (natural antibody (such as natural Ig antibody, preferably natural IgG antibody) or reference antibody) is increased before pI (natural antibody (for example, natural Ig antibody) is preferred) before modification of the antibody or modification of at least one amino acid residue. (E.g., antibodies before antibody modification or library construction or during construction)), (substantially) retain this antigen-binding activity. In this case, "(substantially) retain this antigen-binding activity" may Compared with the binding activity before modification or modification of this antibody, it means that the binding activity is at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and still more preferably 80%, 85 %, 90% or 95% or more activity.

In an alternative embodiment, revealing A is about an antibody obtained by the above method of revealing A's manufacturing 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 A can 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 administration of A is disclosed A pharmaceutical composition (administered to) the subject (preferably in vivo)). The pharmaceutical composition of Disclosure A may optionally contain a pharmaceutically acceptable carrier. A pharmaceutical composition as used herein generally refers to an agent used to treat, prevent, diagnose or examine a disease.

It is disclosed that the composition or pharmaceutical composition of A can be appropriately formulated. In some embodiments, it can be used parenterally, for example in the form of a sterile solution or suspension, for injection into water or any other pharmaceutically acceptable liquid. The composition can be appropriately formulated into a generally acceptable pharmaceutical practice unit dose by appropriately formulating with a pharmaceutically acceptable carrier or vehicle. Such pharmaceutically acceptable carriers or vehicles include, but are not limited to, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, carriers, preservatives and binding agents. Agent. The amount of the active ingredient in the composition can be adjusted so that the dose falls within a suitable preset range.

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

In some embodiments, the present disclosure provides antibodies that increase pI by modifying at least one amino acid residue that may be exposed on the surface (antibody with increased isoelectric point value); a method of producing such antibodies; or The use of the antibody to enhance the elimination of the antigen from the plasma (when the antibody is administered to a subject's living body). The scope of the disclosure A described herein and the content described in the examples can be understood, and 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 ("antibody with reduced isoelectric point value of an antibody"); a method of producing the antibody; Or the use of these antibodies to improve plasma retention (when the antibody system is administered to a subject in vivo). The inventors have discovered that the cellular internalization of antibodies can be enhanced by introducing specific amino acid mutations into specific positions of the amino acid sequence of the constant region to increase the isoelectric point value. Those with ordinary knowledge in the technical field can understand that by introducing amino acids with different side chain charges to the above sites due to the decrease in isoelectric point value, the cellular internalization of the antibody is suppressed, and the plasma retention of the antibody can be prolonged. It can be understood that the scope of A and the contents recorded in the paired examples can be appropriately applied to such an implementation.

In one embodiment, the present disclosure provides a method for producing a modified antibody. The antibody has a prolonged or reduced half-life in plasma compared to an antibody before modification. The method includes: (a) modifying a nucleic acid encoding the antibody before modification to modify the antibody. The charge of at least one amino acid residue in a position selected from one of 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; (b) culture host cells The antibody is produced by expressing the modified nucleic acid; and (c) collecting the produced antibody from the host cell culture.

An additional embodiment provides a method of extending or reducing the half-life of an antibody in plasma, comprising modifying at least one amino acid residue at a position 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.

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

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

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

Reveal B

In a non-limiting embodiment, it is disclosed that B is about Fc region variants, their uses And its manufacturing method.

Within the scope of the disclosures A and B described herein, "Fc region variant" may refer to, for example, modification of the Fc region by modifying at least one amino acid of the Fc region of a natural IgG antibody with another amino acid. Or, it may refer to an Fc region, which 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 an 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, it is disclosed that B is about an Fc region variant that contains an FcRn-binding domain that contains Ala at position 434; any one of Glu, Arg, Ser, and Lys at position 438; and Glu, Asp And Gln at position 440 (the category of Revelation B disclosed here, such Fc region variants are also referred to as "novel Fc region variants" for record purposes), in accordance with EU numbering.

In practice, it is revealed that the Fc region variant of B, regardless of the type of target antigen, can be introduced into almost any antibody (for example, a multispecific antibody such as a bispecific antibody). For example, anti-Factor IXa / Factor X bispecific antibodies (e.g., F8M-F1847mv (F8M-F1847mv1 (SEQ ID NO: 323) and F8M-F1847mv2 (SEQ ID NO) can be produced using the Fc region variants shown in Example 20 : 324) as heavy chain and F8ML (SEQ ID NO: 325) as light chain]; F8M-F1868mv [F8M-F1868mv1 (SEQ ID NO: 326) and F8M-F1868mv2 (SEQ ID NO: 327) as heavy chain and F8ML (SEQ ID NO: 325) as the light chain]; and F8M-F1927mv [F8M-F1927mv1 (SEQ ID NO: 328) and F8M-F1927mv2 (SEQ ID NO: 329) as the heavy chain and F8ML (SEQ ID NO: 325) As light chain]).

As mentioned above, WO2013 / 046704 reports: Added acid strips have been introduced The FcRn binding mutations and specific mutations (representative examples are double residue mutations, in accordance with EU numbering method Q438R / S440E) of the Fc region variants showed a significant reduction in binding to rheumatoid factors. However, WO2013 / 046704 does not record that the Fc region variant that reduces rheumatoid factor binding due to Q438R / S440E modification has better retention in plasma than antibodies with natural Fc region. Therefore, safe and more favorable Fc region variants that improve plasma retention but do not bind to pre-existing ADA are 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 was revealed for the first time that unexpectedly, the combination included mutations of amino acid residues (substitution of amino acids at position 434 of the EU numbering system to Ala (A)) and specific double residue mutations (representative examples are Q438R / S440E) is an Fc region variant suitable for prolonging retention in antibody plasma and maintaining significantly reduced binding to rheumatoid factors.

Therefore, the novel Fc region variant of Reveal B disclosed herein provides beneficial and amazing improvements to the Fc region variant described in WO2013 / 046704, which is incorporated herein by reference in its entirety.

In one embodiment, it is disclosed that B provides a novel amino acid substitution combination of the FcRn binding domain that increases the antibody's FcRn binding activity in an acidic pH range and a neutral pH range, especially in an acidic pH range.

In one embodiment, it is disclosed that the Fc region variant of B contains Ala at position 434; any one of Glu, Arg, Ser, and Lys at position 438; and any one of Glu, Asp, and Gln at position 440, in accordance with EU Numbering; and more preferably Ala at 434; Arg or Lys at 438; and Glu or Asp at 440, according to EU numbering. Ideally, it is revealed that the Fc region variant of B additionally contains Ile or Leu at position 428, and / or any of Ile, Leu, Val, Thr, and Phe at position 436, depending on 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 disclosed that the Fc region variant of B may be a Fc region variant of a natural Ig antibody, and more preferably a Fc region variant of a natural IgG (IgG1, IgG2, IgG3 or IgG4) antibody. The natural Fc region is partly described in the categories that reveal A and B. More specifically, in the disclosure B, the natural Fc region may refer to an unmodified or naturally occurring Fc region, preferably an unmodified or naturally occurring Fc region of a natural Ig antibody, the Fc region of which is an amino group. The acid residues remain unmodified. The antibody source of the Fc region may be Ig, such as IgM or IgG, such as human IgG1, IgG2, IgG3 or IgG4. In one embodiment, it may be human IgG1. A (control) antibody that includes a native Fc region can refer to an antibody that contains an unmodified or naturally occurring Fc region.

The Fc regions of 428, 434, 438, and 440 are common to all natural human IgG1, IgG2, IgG3, and IgG4 antibodies. But at position 436 of the Fc region, the natural human IgG1, IgG2 and IgG4 antibodies are all Tyr (Y), and the natural human IgG3 antibody is Phe (F). On the other hand, Stapleton et al. (Nature Comm. 599 (2011) reported that human IgG3 subtype containing amino acid substitution R435H according to the EU numbering method has comparable plasma half-life with IgG1. Therefore, the inventors also envisage 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 in accordance with the EU numbering method. When it is combined with FcRn that can increase in acidic conditions, it will Significantly reduced rheumatoid factor binding.

Therefore, in a preferred embodiment, the Fc region variant of B is disclosed. The FcRn binding domain may include a combination of substituted amino acid positions selected from the group consisting of: (a) N434A / Q438R / S440E according to EU numbering; (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 / S44 () D; (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 / S 440E; (ad) M428L / N434A / Y436V / Q438R / S440D; (ae) M428L / N434A / Y436V / Q438K / S440E; (af) M428L / N434A / Y436V / Q438K / S440D; (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (ah) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E.

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

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

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 natural FcRn binding domain (binding affinity). Sex) has increased. In this case, the KD (natural Fc region) / KD (revealing the Fc region variant of B) representing the difference in binding activity (binding affinity) may be at least 1.5 times, 2 times, 3 times, 4 times, 5 times , 10 times, 15 times, 20 times, 50 times, 70 times, 80 times, 100 times, 500 times or 1000 times. Such an increase may occur in the acidic pH range and / or in the neutral pH range; but for revealing the mechanism of action of B, it is desirable to increase in the acidic pH range.

In some embodiments, it is revealed that the Fc region variant of B has an increased FcRn binding activity (e.g., at pH 6.0 and 25 ° C) in the acidic pH range, which is, for example, 1.5 times, 2 times, 3 times greater than the Fc region of natural 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 increase in FcRn-binding activity of the Fc region variant in the acidic pH range may be at least 5 or 10 times greater than the FcRn-binding activity of the Fc region of the native IgG.

Manipulating FcRn-binding domains by introducing amino acid substitutions occasionally reduces antibody stability (WO2007 / 092772). Poorly stabilized proteins tend to aggregate easily during storage, and the stability of pharmaceutical proteins is very important in the manufacture of pharmaceutical agents. Therefore, the decrease in stability caused by the substitution of the Fc region may make it difficult to develop stable antibody preparations (WO2007 / 092772).

The purity of pharmaceutical proteins in the form of units and high molecular weight forms is also important for the development of pharmaceutical agents. After purification with Protein A, wild-type IgG1 does not contain a significant amount of high-molecular-weight compounds, and manipulation of the FcRn-binding domain by introduction of substitutions may produce a large number of high-molecular-weight compounds. In this case, such a high molecular weight substance must be removed from the drug using a refining step.

Amino acid substitutions of antibodies can cause negative effects, such as increasing the immunogenicity of therapeutic antibodies, which can cause cytokines to storm and / or produce anti-drug antibodies (ADAs). The clinical use and efficacy of therapeutic antibodies may be limited by ADAs because they affect the pharmacokinetics of therapeutic antibodies and sometimes cause serious 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. Similarly, the presence of pre-existing antibodies against therapeutic antibodies is also a problem. Such pre-existing antibodies are, for example, rheumatoid factor (RF), which are autoantibodies (antibodies against autoproteins) against the Fc portion of an antibody (ie, IgG). Rheumatoid factors are particularly found in patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis. In arthritic patients, RF combines with IgG to form an immune complex that contributes to disease progression. Recently, humanized anti-CD4 IgG1 antibodies with Asn434His mutations have been reported to cause 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 multiple autoantibody against human IgG. The RF epitope of the human IgG sequence will be different based on each selected colony; however, the RF epitope appears to be located in the CH2 / CH3 junction region and the CH3 domain, and it will overlap with the FcRn-binding epitope. Therefore, mutations that increase FcRn binding activity at neutral pH may also increase binding activity to specific RF clones.

In the context of Revealing B, the term "anti-drug antibody" or "ADA" may refer to an endogenous antibody that has binding activity to an epitope of a therapeutic antibody, and thus can bind to a therapeutic antibody. The terms "pre-existing anti-drug antibody" or "pre-existing ADA" may refer to an anti-drug antibody that is present and detectable in the patient's blood before administration of the therapeutic antibody to the patient. In some embodiments, the pre-existing ADA is a human antibody. In a further embodiment, ADA is pre-existing as 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 neutral pH. The ECL analysis method is described in, for example, Moxness et al. (Clin Chem. 51: 1983-1985 (2005)) and Example 6. The analysis can be performed under conditions such as MES buffer and 37 ° C. The antigen-binding activity of an antibody can be determined by, for example, BIACORE (registered trademark) analysis.

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

In the category of disclosure B described here, the binding activity for (pre-existing) ADA is significantly increased or equivalent performance may refer to the Fc region variant of disclosure B or the (pre-existing) ADA of the antibody containing this variant. Binding activity (binding affinity) (i.e., KD) compared to the (pre-existing) ADA binding activity (binding affinity) of the reference antibody containing the reference Fc region variant measured (preexisting), for example, 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 an increase in binding activity for pre-existing ADA can be observed in individual patients or patient groups.

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

In one embodiment of Revealing B, the binding activity to pre-existing ADA is significantly increased in a patient, and can refer to the binding of pre-existing ADA to an Fc region-containing antibody (such as a therapeutic antibody) 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 reference antibody for the pre-existing ADA binding activity. Alternatively, it can be indicated that the ECL response of this antibody is preferably 250 or at least 500 or at least 1000 or at least 2000 or more. Ideally, this increase can be relative to ECL response is less than the increase of 500 or 250 reference antibodies. Specifically, between the binding activity of the reference antibody to the pre-existing ADA and the binding activity of the Fc region variant, the ECL response should preferably be less than 250 to at least 250, less than 250 to at least 500, and less than 500 and 500. A range of more or less, less than 500 to 1,000 or more or less than 500 to at least 2000, but not limited.

In one embodiment, the increased binding activity to pre-existing ADA may refer to a group of patients that includes (a) increased binding activity to FcRn at acidic pH and (b) binding activity to pre-existing ADA at neutral pH. Patients with an ECL response of at least 500 (preferably at least 250) with an increased Fc region variant have a measured patient segment compared with a patient score of at least 500 (preferably at least 250 or more) with an ECL response to a reference antibody It can be improved by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the patient segment with ECL response to the reference antibody.

In one embodiment of Revelation B, the decrease in binding activity to pre-existing ADA can refer to a decrease in the binding activity (ie, KD or ECL response) measured by an antibody containing an Fc region variant compared to a reference antibody. Such reductions can be observed in individual patients or patient groups. In each patient, the significant decrease in affinity of pre-existing ADA for antibodies containing Fc region variants at neutral pH can refer to the binding activity of pre-existing ADA at neutral pH measured in patients, as compared to The binding activity of the reference antibody obtained at pre-existing ADA at neutral pH is reduced, for example, by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

Alternatively, in individual patients, the significantly reduced binding activity of Fc region-containing antibodies to pre-existing ADA can mean that the original ECL response to the antibody was 500 or more compared to the ECL response to the reference antibody (preferably 1000 Or more or 2000 or more) becomes less than 500, preferably less than 250.

In a preferred embodiment, it is revealed that the Fc region variant and the Fc region-containing antibody of B have low binding activity to pre-existing ADA at neutral pH. Specifically, it is preferred that an antibody containing a variant of the Fc region of revealed B has a pre-existing ADA at neutral pH versus a reference antibody that has a lower binding activity than a reference antibody of the Fc region containing natural IgG at a neutral pH (e.g., pH 7.4). The pre-existing binding activity of ADA did not increase significantly. For pre-existing ADA, the binding activity (binding affinity) is low or the affinity site is at the baseline level, which can mean, but is not limited to, an ECL response in an individual patient. The low binding activity to pre-existing ADA in a group of patients may refer, for example, to 90%, preferably 95%, more preferably 98% less than 500 of the ECL response of this group of patients.

The Fc region variant or antibody containing it that reveals B should be chosen for its (pre-existing) ADA binding activity in plasma at neutral pH does not significantly increase and FcRn binding activity at neutral pH and / or acidic pH Increaser. Preferably, the FcRn-binding activity is increased at an acidic pH (for example, pH 5.8). In one embodiment, the Fc region variant should preferably have no significant increase in the binding activity for ADA at neutral pH conditions (eg, pH 7.4) compared to the Fc region of natural IgG. ADA may be pre-existing ADA, and is preferably rheumatoid Sexual 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 natural IgG, and as a result, the clearance rate (CL) in plasma is reduced, and retention in plasma is reduced. Prolonged time or prolonged 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 an increased FcRn binding activity under acidic pH conditions compared to the Fc region of natural IgG, but at The ADA-binding activity at neutral pH did not increase significantly. As a result, the clearance rate (CL) in plasma was reduced, the residence time in plasma was extended, or the half-life (t1 / 2) in plasma was extended. ADA may be pre-existing ADA, preferably rheumatoid factor (RF).

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

Examples 5 to 7 compare 2 Fc region variants: Fc region variant F1718 (Fc region, mutations introduced at 4 sites: N434Y / Y436V / Q438R / S440E), and novel Fc region variants described in WO2013 / 046704 The plasma retention of F1848m (introduced mutations in 4 sites: N434A / Y436V / Q438R / S440E). The difference between the amino acid mutations of the two Fc region variants is only that according to EU numbering at position 434, the introduced amino acid mutation is Y (tyrosine) for F1718, and A (alanine) for F1848m. When F1848m showed improved plasma retention compared to natural IgG1, F1718 did not show such improved plasma retention (see Example (7-2)). Therefore, it is revealed that the Fc region variant of B should preferably have improved plasma retention compared to the reference Fc region variant containing a combination of amino acid substitution N434Y / Y436V / Q438R / S440E according to the EU numbering scheme. The experimental results described in Examples (5-2) and (7-3) prove that the various Fc region variants, namely F1847m, F1886m, F1889m, and F1927m, have better retention retention in plasma than F1848m. Therefore, those with ordinary knowledge in this technical field can know that the variants of Fc region of disclosure B including F1847m, F1886m, F1889m or E1927m and F1848m are compared with the reference Fc region variants that include the replacement N434Y / Y436V / Q438R / S440E. Better plasma retention improvement.

Binding to FcyR or alpha complement proteins may also have adverse effects (e.g., inappropriate platelet activation). Variants of the Fc region that do not bind to the effector receptor, such as the FcyRIIa receptor, are safer and / or more advantageous. In some embodiments, it is revealed that the Fc region variant of B has only weak effector receptor binding activity or does not bind to the effector receptor. Examples of effector receptors include activated FcyR, particularly FcyRI, FcyRII, and FcyRIII. FcyRI includes FcyRIa, FcyRIb, and FcyRIc, and their subtypes. FcyRII includes FcyRIIa (there are two subtypes: R131 and H131) and FcyRIIb. FcγRIII includes FcγRIIIa (there are two subtypes: V158 and F158) and FcγRIIIb (there are two subtypes: FcγRIIIb-NA1 and FcγRIIIb-NA2). Antibodies that have only weak effector receptor binding activity or do not bind to the effector receptor include, for example, antibodies containing silent Fc regions and antibodies without Fc regions (e.g. Fab, F (ab) ' ₂ , scFv, sc (Fv ) ₂ and dimer antibodies).

Fc regions that have only weak effector receptor binding activity or that do not bind to the effector receptor 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 silent Fc regions, which are used to silence their effector function (or suppress immunity) by operating the Fc region (IgG1-L234A / L235A, IgG1-H268Q / A330S / P331S, IgG1 -C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-L234F / L235E / P331S, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, and IgG4 -L236E). WO2008 / 092117 describes an antibody containing a silent Fc region, which contains substitutions G236R / L328R, L235G / G236R, N325A / L328R, or N325L / L328R according to EU numbering. WO2000 / 042072 describes antibodies containing silent Fc regions, which include substitutions at one or more of the following positions: EU233 (position 233 in accordance with EU numbering), EU234, EU235, and EU237. WO2009 / 011941 An antibody containing a silent Fc region lacks residues EU231 to EU238. Davis et al. (J. Rheum. 34 (11): 2204-2210 (2007)) describe antibodies with silent Fc regions, including substitutions C220S / C226S / C229S / P238S. Shields et al. (J. Biol. Chem. 276 (9): 6591-6604 (2001)) describes 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 variants that reveal B.

Expression as "weak binding to effector receptors" may refer to effector receptor binding activity, which is less than, for example, 95% or less, preferably 90%, compared to natural IgG or antibodies containing a natural IgG Fc region. Or less, 85% or less, 80% or less, 75% or less, more preferably 70% or less, 65% or less, 60% or less, 55% or less, 50 % Or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or Less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less Or 1% or less.

A "silent Fc region" is a variant of an Fc region that includes one or more amino acid substitutions, insertions, additions, deletions, etc., and has reduced binding to effector receptors compared to the natural Fc region. Because the effector receptor binding activity can be significantly reduced, such a silent Fc region will no longer bind to the effector receptor. The silent Fc region may include, for example, an Fc region containing an amino acid substitution at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269 , EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332. These amino acid position modifications can also be appropriately introduced into Fc region variants that reveal B.

In a further embodiment, the silent Fc region is substituted at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271 , EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332, preferably selected from the group consisting of: EU235, EU237, EU238, EU239, EU270, EU298, EU325, and EU329 A group wherein it is substituted with an amino acid residue selected from:

The amino acid at position 234 of EU 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 position 236 of EU 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 position 238 in EU is preferably substituted with an amino acid selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, and Arg.

The amino acid at position 239 of EU is preferably substituted with a group selected from the group consisting of Amino acids in the group: Gln, 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 the EU267 position 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, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position 270 of EU 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 position 296 of EU 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 with Ala.

The amino acid at EU298 is preferably substituted with a group selected from the group consisting of Amino acids in the group: Arg, Gly, Lys, Pro, Trp and Tyr.

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

The amino acid at position 324 of EU is preferably substituted with Lys or Pro.

The amino acid at position 325 of EU is preferably substituted with an amino acid selected from the group consisting of Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, and Val.

The amino acid at position 327 of EU is preferably substituted with an amino acid selected from the group consisting of Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val .

The amino acid at 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 EU 330 should be 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: Lys or Arg in EU235, Lys or Arg in EU237, Lys or Arg in EU238, Lys or Arg in EU239, Phe in EU270, or Gly in EU298 , Gly in EU325, or Lys or Arg in EU329. More preferably, the silent Fc region may include substitution with arginine in EU235 or lysine in EU239. More preferably, the silent Fc region may include a L235R / S239K substitution. Modifications of these amino acid residues can also be appropriately introduced into the Fc region variants that reveal B.

In one embodiment, an antibody comprising a variant of the Fc region of B is disclosed that has only weak complement protein binding activity or does not bind to complement protein. 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 a native IgG or an antibody that includes a native IgG Fc region. More or 100 times or more. The complement protein binding activity of the Fc region can be reduced using amino acid modifications such as amino acid substitutions, insertions, additions or deletions.

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

In one embodiment, a method of modifying the constant region of an antibody to produce variants of the Fc region revealing B can be based on, for example, evaluating the structural isotypes of several constant regions (IgG1, IgG2, IgG3, and IgG4) to select antigens in the acidic pH range. Structural isoforms with reduced binding activity and / or increased dissociation rates in the acidic pH range. Alternative methods may be based on the introduction of amino acids in place of the amino acid sequence of the native IgG construction isoform 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 sequence of the hinge region of the constant region of the antibody is greatly different in different structural isotypes (IgG1, IgG2, IgG3, and IgG4). The difference in amino acid sequence of the hinge region significantly affects the antigen-binding activity. Therefore, structural isoforms with reduced antigen-binding activity in the acidic pH range and / or increased dissociation rates in the acidic pH range can be selected by the type of antigen or epitope. The structure is the same type to choose. And because the amino acid sequence difference in the hinge region has a significant effect on the antigen-binding activity, the amino acid substitution of the natural structural homology amino sequence can be located in the hinge region.

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

The term "ability to eliminate antigen from plasma" or equivalent can mean the ability to eliminate antigen from plasma when the antibody is administered or secreted in vivo. Therefore, the "increased ability of this antibody to eliminate antigen from plasma" can mean that if the antibody is administered, the rate of elimination of antigen from plasma is increased compared to before the modification of its FcRn binding domain. The increased activity of the antibody in eliminating plasma antigen can be assessed by, for example, administering a soluble antigen and the antibody in vivo, and measuring the concentration of the soluble antigen in plasma after administration. The soluble antigen may be a bound antibody or an antibody without binding, and the concentration may be "the concentration of the antigen bound by the antibody in the plasma" and "the concentration of the antigen bound by the antibody in the 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 the bound antibody and the antigen concentration of the unbound antibody.

In an alternative embodiment, disclosure B provides an extended disclosure The method of showing the plasma retention time of the antibody of the Fc region variant of B. Natural human IgG can bind to FcRn from non-human animals. For example, because natural human IgG binds more strongly to mouse FcRn than human FcRn (Ober et al., Intl. Immunol. 13 (12): 1551-1559 (2001)), this antibody is administered to mice for evaluation Quantify the properties of this antibody. Alternatively, for example, a mouse (Roopenian et al., Meth. Mol. Biol. 602: 93-104 (2010)) in which the FcRn gene itself is disrupted but which replaces and expresses the human FcRn gene as a transgenic gene is also suitable for evaluating this antibody.

Within the scope of the disclosures A and B described herein, the plasma concentration of free antigen that is not bound to the antibody, or the ratio of the free antigen concentration to the total antigen concentration can be determined (for example, Ng et al., Pharm. Res. 23 (1): 95-103 (2006)). Or if the antigen exhibits a specific function in vivo, whether the antigen is bound to an antibody (antagonistic molecule) that will neutralize the function of the antigen can be evaluated by testing whether the antigen function is neutralized. Whether this antigen function is neutralized can be assessed by measuring specific in vivo markers that reflect this antigen function. Whether or not the antigen is bound to an antibody (a potent molecule) that activates the antigen function can be evaluated by measuring a specific in vivo label that reflects this antigen function.

There are no special restrictions on the measurement, such as determining the concentration of free antigen in plasma, determining the ratio of free antigen in plasma to the total amount of antigen in plasma, and in vivo marker measurement; however, the measurement should be performed within a certain period after antibody administration After implementation. In the context of revealing B, "after a certain period after antibody administration" is not particularly limited, and this period may be appropriately determined by those with ordinary knowledge in the technical field based on the nature of the antibody administered, including, for example, 1 Days, 3 days, 7 days, 14 days or 28 days. The term "plasma antigen concentration" may refer to "total antigen concentration in plasma", which is the sum of the antigen concentration of the bound antibody and the antigen concentration of the unbound antibody, Or refers to "free antigen concentration in plasma", which is the antigen concentration without binding antibodies.

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

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

In some aspects, when the antibody of Rev. B is administered, the Molar ratio of the antigen / antibody is reduced by two, five, or ten times compared to a reference antibody that includes a natural human IgG Fc region as a human FcRn binding domain. , 20 times, 50 times, 100 times, 200 times, 500 times, or 1,000 times or more.

The reduction of the total antigen concentration or the molar ratio of the antigen / antibody in the plasma can be evaluated using methods known in the art, such as the methods described in Examples 6, 8, and 13 of WO2011 / 122011. More specifically, if the antibody of interest B is not cross-reacted with the mouse paired antigen, the mouse FcRn gene can be used to transfect mouse strain 32 or 276 according to the steady-state antigen in the antigen-antibody co-injection model or fusion model. Jackson Laboratories, Methods Mol. Biol. 602: 93-104 (2010)). When this antibody and mouse paired antigen cross-react, this antibody can be simply injected into human FcRn gene transgenic mouse strains 32 or 276 (Jackson Laboratories) for evaluation. In a co-injection model, a mixture of antibodies and antigens is administered to mice. For the steady-state antigen of the fusion model, a fusion pump filled with an antigen solution is transplanted into a mouse to achieve a certain antigen concentration in plasma, and then the antibody is injected into the mouse. All test antibodies were administered at the same dose. Total antigen concentration in plasma, free antigen concentration in plasma, and blood The antibody concentration in the slurry can be measured at the appropriate time.

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

In order to evaluate the short-term effects of the antibody revealed by B, the total or free antigen concentration or the antigen / antibody concentration 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, in order to assess the properties of this antibody, the total or free antigen concentration or the mole of the antigen / antibody in the plasma can be measured 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration. The ratio determines the antigen concentration in the plasma over a short period of time. When it is difficult to determine human plasma retention, it can be predicted from mouse plasma retention (such as normal mice, human antigen-expressing gene transgenic mice or human FcRn-expressing gene transgenic mice) or monkeys (such as Malay monkey).

In an alternative embodiment, disclosure B is about an antibody comprising an Fc region variant of disclosure B as described above. Various embodiments of this antibody are within the scope of disclosures A and B described herein, unless they are inconsistent with the context, and can be applied without violating general technical knowledge in the technical field.

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

In one embodiment, antibodies that include variants of the Fc region of B may have modified sugar chains. Antibodies having modified sugar chains may include, for example, antibodies having modified glycosylation (WO99 / 54342), antibodies lacking fucose (WO00 / 61739, WO02 / 31140, WO2006 / 067847, WO2006 / 067913), and Antibodies to the sugar chains of GlcNAc (WO02 / 79255). In one embodiment, the antibody may be deglycosylated. In some embodiments, the antibody includes, for example, a mutation at a glycosylation site of a heavy chain to inhibit glycosylation at this site, as described in WO2005 / 03175. Such an unglycosylated antibody can be produced by modifying a heavy chain glycation site, that is, introducing N297Q or N297A substitution in accordance with EU numbering, and expressing the protein in an appropriate host cell.

In an alternative embodiment, it is disclosed that B is about a composition or a pharmaceutical composition that includes an antibody comprising such an Fc region variant. Various embodiments of the composition or the pharmaceutical composition described within the scope of disclosure A and B may be applied without violating the general technical knowledge in the technical field unless they are inconsistent with the context. Such a composition can be used to enhance plasma retention (within a subject, when the antibody system of the present disclosure B is administered (administered) to the subject).

In an alternative embodiment, it is disclosed that B is about a nucleic acid that encodes an Fc region variant or an antibody comprising the Fc region variant. Various embodiments of nucleic acids within the scope of disclosure A and B described herein can be applied without violating the general technical knowledge in the technical field unless they are inconsistent with the context. Or reveal that B is about the vector containing this nucleic acid. Various embodiments within the scope of the disclosures A and B described herein, unless inconsistent with the context, may not violate the general technology in the technical field. Application of technical knowledge. Or reveal that B is about the host or host cell containing the antibody. Various embodiments within the scope of disclosure A and B described herein can be applied without violating the general technical knowledge in the technical field unless they are inconsistent with the context.

In an alternative embodiment, it is disclosed that B is a method for manufacturing an Fc region variant comprising an FcRn binding domain or an antibody comprising the Fc region variant, which comprises culturing or growing the above-mentioned host cell, from the cell culture, the host, The secreted material collects the Fc region variant or an antibody containing the Fc region variant. In this case, the disclosure B may include a manufacturing method, which optionally further includes one or more of the following steps: (a) selecting an Fc region with an increased FcRn binding activity under acidic pH conditions compared to the Fc region of a natural IgG (B) selection of Fc region variants, which have no significant increase in (pre-existing) ADA binding activity at neutral pH compared to the Fc region of natural IgG; (c) selection of Fc region variants, Increased plasma retention compared to the Fc region of native IgG; and (d) selection of antibodies, which include an Fc region variant that promotes elimination of antigen from plasma, can improve plasma from the plasma compared to reference antibodies that include the Fc region of natural IgG Eliminate the antigen.

From the viewpoint of evaluating the plasma retention of the Fc region variant that reveals B, it is not limited, and it is preferred that the antibody containing the Fc region variant and the "reference antibody containing the Fc region containing the natural IgG" made by B are identical to each other, only The exception is the Fc region for comparison. The FcRn may be a human FcRn.

For example, after manufacturing an antibody including a variant of the Fc region revealing B, this antibody and a reference antibody including a natural IgG Fc region can be used for the FcRn binding activity (e.g., pH 5.8) of acidic pH conditions using BIACORE (registered trademark) Or compare with other known techniques to select an Fc region variant or an antibody containing the Fc region variant that has increased FcRn binding activity under acidic pH conditions.

Or, for example, after manufacturing an antibody including a variant of the Fc region revealing B, this antibody and a reference antibody including a natural IgG Fc region can be subjected to electrochemiluminescence (ECL) for ADA binding activity at neutral pH conditions. Or compared with known techniques, an Fc region variant or an antibody containing the Fc region variant that does not significantly increase ADA binding activity at neutral pH conditions is selected.

Alternatively, for example, after producing an antibody including a variant of the Fc region of B, the antibody and the reference antibody including the native IgG Fc region can be borrowed using plasma from, for example, mouse, rat, rabbit, dog, monkey, or human. An antibody pharmacokinetic test is performed to select an Fc region variant or an antibody containing the Fc region variant that has been shown to improve plasma retention in a subject.

Alternatively, for example, antibodies can be produced that include variants of the Fc region revealing B. The antibodies and reference antibodies including the natural IgG Fc region can be produced by using plasma from, for example, mouse, rat, rabbit, dog, monkey, or human. An antibody pharmacokinetic test is performed to select an Fc region variant or an antibody containing the Fc region variant that is enhanced from plasma elimination antigen.

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

In one embodiment, it is disclosed that B is a method for producing a variant of an Fc region, an antibody comprising an FcRn binding domain, or an antibody comprising the variant. The method comprises substituting an amino acid to obtain an obtained Fc region variant or This antibody for 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 includes substituting an amino acid to obtain an obtained Fc region variant or an antibody comprising the antibody, further comprising Ie or Leu at position 428 and / or Ile, Leu, Val, Thr or Phe are at 436. In another embodiment, according to this method The amino acid substitution with the obtained Fc region variant or the antibody containing the variant further includes Leu at position 428 and / or Val or Thr at position 436 according to EU numbering.

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

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

In an alternative embodiment, Rev. B is related to an Fc region variant or an antibody comprising the same, and is obtained by any of the methods disclosed above for Rev. B.

In an alternative embodiment, Rev. B provides a method for reducing Fc region variants with increased FcRn binding activity at acidic pH (e.g., pH 5.8). For (pre-existing) ADA-binding activity; and a method for making an Fc region variant with increased FcRn-binding activity at acidic pH and reduced pre-existing ADA-binding activity, including: (a) providing an antibody comprising The FcRn-binding activity of pH is increased compared to the reference antibody in the Fc region (variant); and (b) introduced into the Fc region according to the EU numbering method; (i) the amino acid is replaced with Ala at position 434; (ii) An amino acid substitution at position 438 to any of Glu, Arg, Ser, and Lys; and (iii) an amino acid substitution at position 440 to any of Glu, Asp, and Gln; (iv) optionally , The amino acid at position 428 is replaced with Ile or Leu; and / or (v) optionally, the amino acid at position 436 is replaced with any 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). In addition, in order to increase the FcRn binding activity at an acidic pH and decrease the (pre-existing) ADA binding activity in a neutral pH range (for example, pH 7.4), the Fc region (variant) should contain a group selected from the group consisting of Combination of amino acid substitution: (a) N434A / Q438R / S440E; (b) N434A / Q438R / S440D; (c) N434A / Q438K / S440E; (d) N434A / Q438K / S440D; (e) N434A / Y436T / Q438R / S440E; (f) N434A / Y436T / Q438R / S440D; (g) N434A / Y436T / Q438K / S440E; (h) N434A / Y436T / Q438K / S440D; (i) N434A / Y436V / Q438R / S440E; (j ) N434A / Y436V / Q438R / S440D; (k) N434A / Y436V / Q438K / S440E; (l) N434A / Y436V / Q438K / S440D; (m) N434A / R435H / F436T / Q438R / S440E; (n) N434A / R435H / F436T / Q438R / S440D; (o) N434A / R435H / F436T / Q438K / S440E; (p) N434A / R435H / F436T / Q438K / S440D; (q) N434A / R435H / F436V / Q438R / S440E; (r) N434A / R435H / F436V / Q438R / S440D; (s) N434A / R435H / F436V / Q438K / S440E; (t) N434A / R435H / F436V / Q438K / S440D; (u) M428L / N434A / Q438R / S440E; (v) M428L / N434A / Q438R / S440D; (w) M428L / N434A / Q438K / S440E; (x) M428L / N434A / Q438K / S440D; (y) M428L / N434A / Y436T / Q438R / S440E; (z) M428L / N434A / Y436T / Q438R / S440D; (aa) M428L / N434A / Y436T / Q438K / S440E; (ab) M428L / N434A / Y436T / Q438K / S440D; (ac) M428L / N434A / Y436V / Q438R / S440E; (ad) M428L / N434A / Y436V / Q438R / S440D; (ae) M428L / N434A / Y436V / Q438K / S440E; (af) M428L / N434A / Y436V / Q438K / S440D; (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (ah) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E .

This method may optionally further include: (c) assessing whether the Fc region variant includes an antibody that has a reduced binding activity compared to a reference antibody, and the (pre-existing) ADA binding activity of the antibody is reduced.

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

Reveal C

Disclosure C is also related to anti-IL-8 antibodies, nucleic acids encoded by the antibodies, pharmaceutical compositions containing the antibodies, methods of producing the antibodies, and the use of the antibodies to treat diseases associated with IL-8, as detailed below Described. The meanings of the terms given below apply to revealing all of C, and are not inconsistent with the general technical knowledge of those with ordinary knowledge in this technical field and the embodiments known to those with ordinary knowledge in this technical field.

I. Reveal the definition within the scope of C

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

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

The term "IL-8" used in Revealing C refers to any vertebrate, primate (e.g., human, Malay, Rhesus), and other mammals (e.g., dogs and rabbits) unless otherwise specified Coming of natural IL-8. The term "IL-8" includes full-length IL-8, untreated IL-8, and any form of IL-8 obtained as a result of processing in cells. The term "IL-8" also includes derivatives of natural IL-8, such as cleavage 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 "IL-8-binding antibody" refer to antibodies capable of binding IL-8 with sufficient affinity, and this antibody is useful as a diagnostic and / or therapeutic agent for target IL-8 .

In one embodiment, anti-IL-8 antibodies White binding is, for example, less than about 10% of the binding of this antibody to IL-8.

The term "affinity" used in the context of revealing C generally refers to the total intensity 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 revealing C refers to inherent binding affinity, reflecting the 1: 1 interaction of members of the binding pair (such as antibodies and antigens). The affinity of a molecule X for its partner Y is generally represented by the dissociation constant (KD). Binding affinity can be measured using methods known in the art, including narrative writers that disclose the scope of C.

In certain embodiments, the dissociation constant (KD) of an antibody that binds to IL-8 can be, for example, 1000nM, 100nM, 10nM, 1nM, 0.01nM or 0.001 nM (for example, 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 category of revealing C is used in a broad sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments, as long as they show the desired antigen-binding activity. Just fine.

"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, reference antibody blocking This antibody binds to its antigen by, for example, 50%, 60%, 70%, or 80% or more. An exemplary competitive analysis method can be used here without limitation.

A "chimeric antibody" refers to a portion of the heavy and / or light chain of an antibody from a particular source or species and the other portion from a different source or species.

A "humanized" anti-system refers to a chimeric antibody that includes amino acid residues from non-human HVR and amino acid residues from human FR. In certain embodiments, A humanized antibody may include substantially at least one, and usually two variable regions, where all (or substantially all) HVRs (eg, CDRs) correspond to non-human antibodies, and all (or substantially all) of the FRs Corresponds to human antibodies. A humanized antibody may optionally include at least a portion of an antibody constant region from a human antibody.

The term "monoclonal antibody" described in the category of disclosure C refers to antibodies obtained from a group of substantially homogeneous antibodies, that is, the individual antibodies constituting the group are the same and / or bound to the same epitope, unless it is possible to change Somatic antibodies, such as those that contain naturally occurring mutations or variants due to the manufacture of monoclonal antibodies, are generally present in trace amounts. In contrast, multiple antibody preparations generally include different antibodies directed to different determinants (antigenics), and each single antibody in a single antibody preparation is directed to a single determinant on the antigen. The modifier "single strain" therefore represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and is not to be construed as requiring the production of this antibody by a particular method. For example, the monoclonal antibodies used according to Rev. C can be produced using various techniques, including but not limited to fusion tumor methods, recombinant DNA methods, phage presentation methods, and methods of transgenic animals that include all or part of the human immunoglobulin locus. Such methods and other methods for making monoclonal antibodies will be described here.

In the context of disclosure C, "natural antibodies" refer to immunoglobulin molecules with various naturally occurring structures. In one embodiment, the natural IgG antibody is, for example, a heterotetrameric glycoprotein of about 150,000 daltons, which is formed by two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three certain domains (CH1, CH2, and CH3). ). Similarly, from the N-to the C-terminus, each light chain has a variable region (VL), called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Antibody light chain The amino acid sequence is designated as one of two types, namely kappa (κ) and lambda (λ). Such invariant domains used by the present disclosure C include any reported subtype (dual type) or any subclass / structural isotype. Heavy chain constant regions include, but are not limited to, constant region antibodies (IgG1, IgG2, IgG3, and IgG4) of native IgG. Known IgG1 dual genes include, for example, IGHG1 * 01, IGHG1 * 02, IGHG1 * 03, IGHG1 * 04, and IGHG1 * 05 (see imgt.org), any of which can be used as natural human IgG1 sequences. The invariant domain sequence may be from a single dual gene or subclass / tissue homotype or from multiple dual genes or subclass / tissue isotypes. Specifically, such antibodies include, but are not limited to, antibodies with CH1 from IGHG1 * 01, and CH2 and CH3 from IGHG1 * 02 and IGHG1 * 01.

The "effector function" recorded in the category of "C" refers to the biological activity attributable to the Fc region of an antibody, which changes with the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (such as B-cell receptors) ) Is down-regulated; and B cell activation, but is not limited thereto.

The term "Fc region" described in the category 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 natural Fc regions and variant Fc regions. The native Fc region refers to the Fc region of a natural antibody.

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

The "framework" or "FR" described in the category C is a variable domain residue other than a high variable region (HVR) residue. 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" recorded in the category C is the framework of the amino acid residues most commonly found in the selected human immunoglobulin VL or VH framework sequences. Generally, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Generally, the subgroups of the sequence follow 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 the subgroup κI, as described in Kabat et al. In one embodiment, the subgroup for VH is subgroup III, as described in Kabat et al.

The "recipient human framework" used to disclose the scope of C is a framework that includes amino acid sequences from the VL or VH framework of the human immunoglobulin framework or the human common framework. An acceptor human framework "from" a human immunoglobulin framework or a human common framework may include its same 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 receptor human framework and VL human The immunoglobulin framework sequence or human common framework sequence is the same.

In the context of the disclosure of C, the terms "variable region" or "constant domain" refer to the domain of the heavy or light chain of an antibody involved in binding this antibody to an antigen. The heavy and light variable regions of natural antibodies (VH and VL each) generally have similar structures, and each domain includes four conservative framework regions (FR) and three highly variable regions (HVRs) (see Kindt et al., Kuby Immunology, 6 ^th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain is sufficient but not limited to this. In addition, antibodies that bind to a specific antigen can use VH or VL domains from the antibody to screen complementary VL or VH domains for isolation, see, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

The term "highly variable region" or "HVR" used in the context of the disclosure of C means that the sequence has high variability ("complementarity determining region" or "CDR") and / or forms a structurally defined loop (" Highly variable loops ") and / or regions of antibody variable domains containing antigen contact residues (" antigen contact points "). In general, antibodies include six highly variable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

Not limited to this, here, for example, HVR: (a) Highly variable loops, in which the amino acid residues are 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)); (b) CDRs, wherein the amino acid residue is 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contact point, in which the amino acid residue is 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) a combination of (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, the residues of HVR and other variable regions (such as FR residues) are numbered according to Kabat et al.

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

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

In the context of the disclosure of C, "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of the natural environment. An isolated nucleic acid includes a nucleic acid molecule that is generally contained in a cell containing the nucleic acid molecule, but that is a nucleic acid molecule that is present extrachromosomally or on a chromosome different from the natural chromosomal location.

"Isolated nucleic acid encoding an anti-IL-8 antibody" within the scope of the disclosure of C refers to one or more nucleic acid molecules encoding an anti-IL-8 antibody heavy and light chain (or a fragment thereof). , Including nucleic acids in a single or isolated vector, and A cell that is present in one or more host cells.

Within the scope of the disclosure of 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 descendants of such cells . Host cells include "transformants" and "transformed cells", including primary cells to be transformed and their descendants regardless of their generation number. Progeny and their parent cells are not necessarily identical in nucleic acid content and may include mutations. This also includes the selection or selection of mutant progeny that have the same function or biological activity as the original transformed cells.

In the context of the disclosure of C, the term "carrier" refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. The term includes vectors that self-replicate a nucleic acid structure, and vectors that are introduced into a host cell and encapsulated in their genome. Certain vectors are capable of directing the performance of nucleic acids to which they are operatively linked. Such a carrier is called a "expression carrier".

Within the scope of the disclosure of C, the term "package insert" is used to refer to a manual that is custom-made to include a commercially available package of a medicinal product, which contains indications, usage, dosage, administration, combination Information on treatments, contraindications, and / or warnings about the use of such medicinal products.

Within the scope of the disclosure of C, the "percent amino acid sequence identity (%)" of the control polypeptide sequence is defined as the amino acid residue in the candidate sequence and the amino acid residue in the control polypeptide sequence. The percentage of sequence after alignment is the same. In order to achieve the maximum percentage of sequence identity, gaps can be introduced as needed, and any conservative substitutions are not considered as part of sequence identity. In order to determine the alignment of the amino acid sequence identity percentage, it can be achieved in various ways within the ability of those with ordinary knowledge in the technical field, such as by using publicly available Computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for arranging sequences, including arbitrary algorithms that need to achieve the maximum alignment of the full length of the alignment sequences. For this purpose, values of% amino acid sequence identity are generated, for example, using the comparison computer program ALIGN-2. ALIGN-2 is produced by Genentech, Inc., the source code and user files have been submitted to U.S. Copyright Office, Washington D.C., 20559, and U.S. Copyright Registration No. TXU510087 has been registered. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California or can be compiled from source code. The ALIGN-2 program should use UNIX (registered trademark) operating system codes, including digital UNIX (registered trademark) V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

When using ALIGN-2 for amino acid sequence comparison, a given amino acid sequence A versus a given amino acid sequence B (also can be said that a given amino acid sequence A has or includes for a given amino acid sequence B Amino acid sequence identity%) of amino acid sequence identity% is calculated in the following way: the fraction X / Y is 100 times, where X is the same match when the sequence arrangement program ALIGN-2 is used to arrange A and B Amino acid residue fraction, Y is the total number of amino acid residues in B. It will be understood that if the length of the amino acid sequence A is not equal to the length of the 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 are obtained using the ALIGN-2 computer program as evidenced by the scope of the disclosure in C.

Within the scope of disclosure C, "pharmaceutical composition" generally refers to a medicament for the treatment, prevention, inspection, or diagnosis of a disease. "Medically acceptable "Carrier" refers to ingredients other than the active ingredients in the pharmaceutical composition and is non-toxic to the subject. Such pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, and preservatives.

"Treatment" (and its grammatical changes, such as "treat" or "treating") used in the context of the disclosure of C refers to clinical intervention to change the natural course of the individual to be treated. Such clinical interventions may be performed prophylactically or during a clinical lesion. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or reducing the disease state, and reducing or improving the prognosis. In one embodiment, antibodies that disclose C can be used to slow the progression of a disease or disorder.

In the context of the disclosure of C, an "effective amount" of an antibody or a pharmaceutical composition refers to an amount that is effective if used at the dosage and for the period of time required to achieve the desired therapeutic or prophylactic result.

II. Compositions and methods

In one embodiment, it is disclosed that C is based on the use of a pH-dependent affinity anti-IL-8 antibody against IL-8 as a pharmaceutical composition. Antibodies of C have been found useful, for example, in the diagnosis or treatment of a large number of diseases in which IL-8 is present.

A. Examples of anti-IL-8 antibodies

In one embodiment, it is disclosed that C provides an anti-IL-8 antibody that has a pH-dependent affinity for IL-8.

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

In another embodiment, it is disclosed that C provides an anti-IL-8 antibody that has a pH-dependent affinity for IL-8 and includes at least one amino acid substitution in at least one amino acid sequence: (a) HVR-H1 , Including amino acid sequence of SEQ ID NO: 67; (b) HVR-H2, including amino acid sequence of SEQ ID NO: 68; (c) HVR-H3, including amino acid sequence of SEQ ID NO: 69 (D) HVR-L1, including the amino acid sequence of SEQ ID NO: 70; (e) HVR-L2, including the amino acid sequence of SEQ ID NO: 71; and (f) HVR-L3, including SEQ ID NO amino acid sequence: 72.

Unless otherwise specified, the amino acid may be substituted with any other amino acid. In one embodiment, the anti-IL-8 antibody of C is disclosed to include one or more amino acid substitutions at a position selected from the group consisting of: (a) one of positions 1 of the sequence of SEQ ID NO: 67 Aspartic acid; (b) tyrosine at position 2 in the sequence of SEQ ID NO: 67; (c) tyrosine at position 3 of the sequence of SEQ ID NO: 67; (d) at SEQ ID NO : Leucine at position 4 of the sequence of 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) the isoleucine at position 2 of the sequence of SEQ ID NO: 68; (h) the arginine at position 3 of the sequence of SEQ ID NO: 68; (i) the sequence at SEQ ID NO: 68 Aspartic acid at position 4; (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; (1) Aspartic acid at position 7 in the sequence of SEQ ID NO: 68; (m) in SEQ ID Glycine at position 8 of the sequence of NO: 68; (n) tyrosine at position 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) glutamic acid at position 12 of the sequence of SEQ ID NO: 68; (r) sequence of SEQ ID NO: 68 Tyrosine at position 13; (s) serine at position 14 in the sequence of SEQ ID NO: 68; (t) alanine at position 15 in the sequence of SEQ ID NO: 68; (u) in SEQ Serine at position 16 of the sequence of ID NO: 68; (v) Valine at position 17 of the sequence of SEQ ID NO: 68; (w) Limine at position 18 of the sequence of SEQ ID NO: 68 Acid; (x) glycine at position 19 in the sequence of SEQ ID NO: 68; (y) glutamate at position 1 in the sequence of SEQ ID NO: 69; (z) in SEQ ID NO: 69 Aspartic acid at position 2 of the sequence; (aa) Tyrosine at position 3 of the sequence of SEQ ID NO: 69; (ab) Arginine at position 4 of the sequence of SEQ ID NO: 69; (ab) ac) tyrosine at position 5 of the sequence of SEQ ID NO: 69; (ad) aspartic acid at position 6 of the sequence of SEQ ID NO: 69; (ae) Valine at position 7; (af) in SEQ ID NO: 6 Glutamic acid at position 8 of the sequence of 9; (ag) leucine at position 9 of the sequence of SEQ ID NO: 69; (ah) alanine at position 10 of the sequence of SEQ ID NO: 69; (ai) ) Tyrosine 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) at position 2 of the sequence of SEQ ID NO: 70 (Al) serine at position 3 in the sequence of SEQ ID NO: 70; (am) glutamate at position 4 in the sequence of SEQ ID NO: 70; (an) in SEQ ID NO: Isoleucine at position 5 of the sequence of 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) serine at position 8 of the sequence of SEQ ID NO: 70; (ar) at SEQ ID Tyrosine at position 9 of the sequence of NO: 70; (as) leucine at position 10 of the sequence of SEQ ID NO: 70; (at) alanine at position 11 of the sequence of SEQ ID NO: 70; (au) aspartic acid at position 1 of the sequence of SEQ ID NO: 71; (av) alanine at position 2 of the sequence of SEQ ID NO: 71; (aw) sequence at SEQ ID NO: 71 Lysine at position 3; (ax) threonine at position 4 of the sequence of SEQ ID NO: 71; (ay) leucine at position 5 of the sequence of SEQ ID NO: 71; (az) at 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) Bran at position 1 of the sequence of SEQ ID NO: 72 (Bc) Histidine at position 2 in the sequence of SEQ ID NO: 72; (bd) Histidine at position 3 of the sequence of SEQ ID NO: 72; (be) in SEQ ID NO: Phenylalanine at position 4 of the sequence of 72; (bf) glycine at position 5 of the sequence of SEQ ID NO: 72; (bg) 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) Position 9 of the sequence of SEQ ID NO: 72 Of threonine.

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

In one embodiment, the anti-IL-8 antibody of C is disclosed to include a combination of amino acid substitutions at positions selected from the group consisting of: (a) alanine at position 6 of the sequence of SEQ ID NO: 68 ; (B) the sequence of SEQ ID NO: 68 Glycine at position 8; (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) at Tyrosine at position 3 of the sequence of SEQ ID NO: 69.

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes an amino acid substitution at: (a) 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, it is disclosed that the anti-IL-8 antibody of C includes an amino acid substitution 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; (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 of C is disclosed: (a) at position 6 of the sequence of SEQ ID NO: 68, replacing alanine as aspartic acid; (b) from SEQ ID NO: 68 At position 11 of the sequence, the substituted arginine is proline; and (c) at position 3 of the sequence of SEQ ID NO: 69, the substituted tyrosine is histidine.

In one embodiment, the anti-IL-8 antibody of C is disclosed: (a) at position 8 of the sequence of SEQ ID NO: 68, substituting glycine for tyrosine; and (b) for SEQ ID NO: 68 At position 9 of the sequence, tyrosine is replaced by histidine.

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

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

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

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

In one embodiment, the anti-IL-8 antibody of C is disclosed to include one or more amino acid substitutions at a position selected from the group consisting of: (a) a 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 SEQ ID No. 72 sequence of glutamic acid. In yet another embodiment, the anti-IL-8 antibody includes a combination of any two, three, or all four of these substitutions.

In one embodiment, the anti-IL-8 antibody of C is disclosed to include 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 Acid; (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 SEQ ID NO Glutamic acid at position 1 of the sequence of 72. In yet another embodiment, the anti-IL-8 antibody includes a combination of any two, three, or all four of these substitutions.

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

In one embodiment, it is disclosed that the anti-IL-8 antibody of C includes an amino acid substitution at: (a) a serine at position 8 of the sequence of SEQ ID NO: 70; (b) at SEQ ID NO: Aspartic acid at position 1 of sequence 71; (c) Leucine at position 5 of sequence of SEQ ID NO: 71; and (d) Bran at position 1 of sequence of SEQ ID NO: 72 Amino acid.

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

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

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

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

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

In one embodiment, the anti-IL-8 antibody of C is disclosed to include an amino acid substitution at: (a) alanine at position 55 in the sequence of SEQ ID NO: 77; (b) at SEQ ID NO: 77 Glycine at position 57 of the sequence; (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 in the sequence of SEQ ID NO: 77; (f) serine at position 87 in the sequence of SEQ ID NO: 77; and (g) 103 position tyrosine.

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

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

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

In one embodiment, the disclosed anti-IL-8 antibody of C includes: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78; and a light chain variable region comprising the amino group of SEQ ID NO: 79 Acid sequence. Including 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 The anti-IL-8 antibody may be an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. This anti-IL-8 antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 78 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 79 can be stably located An anti-IL-8 antibody that maintains IL-8 neutralizing activity in vivo (eg, in plasma). The 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 have low immunity Antibodies.

In an alternative aspect, the disclosed anti-IL-8 antibodies of C also include those that have a pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least one 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 : The amino acid sequence of 104; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 105; (e) HVR-L2, which contains 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, the disclosed anti-IL-8 antibodies of C also include those that have a pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least one 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 : Amino acid sequence of 110; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 111; (e) HVR-L2, which contains the amino acid sequence of SEQ NO ID: 112; and (f) HVR-L3, containing the amino acid sequence of SEQ ID NO: 113.

In an alternative aspect, it is revealed that the anti-IL-8 antibody of C also includes Those having a pH-dependent affinity for IL-8 and containing at least one amino acid substitution in at least one of the following amino acid sequences: (a) HVR-H1, which contains the amino acid of SEQ ID NO: 114 Sequence; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO: 115; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: 116; (d) HVR-L1, containing SEQ ID (E) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 118; and (f) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 119.

In an alternative aspect, the disclosed anti-IL-8 antibodies of C also include those that have a pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least one 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 : The amino acid sequence of 122; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 123; (e) HVR-L2, which contains 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, the disclosed anti-IL-8 antibodies of C also include those that have a pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least one 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 : Amino acid sequence of 128; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2, which contains 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, the disclosed anti-IL-8 antibodies of C also include those that have a pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least one of the following amino acid sequences : (A) HVR-H1, containing the amino acid sequence of SEQ ID NO: 132; (b) HVR-H2, containing the amino acid sequence of SEQ ID NO: 133; (c) HVR-H3, containing the amino acid sequence of SEQ ID NO: 134; (d) HVR-L1, containing the amino acid sequence of SEQ ID NO: 135; (e) HVR-L2, containing the amine of SEQ ID NO: 136 Amino acid sequence; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 137.

In an alternative aspect, the disclosed anti-IL-8 antibodies of C also include those that have a pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least one 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 : The amino acid sequence of 140; (d) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 141; (e) HVR-L2, which contains 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, an anti-IL-8 antibody of C is disclosed to have IL-8 neutralizing activity. IL-8 neutralizing activity refers to an activity that inhibits the biological activity of IL-8, or may refer to an activity that inhibits the binding of IL-8 receptors.

In an alternative aspect, it is revealed that the anti-IL-8 antibody of C is an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. In the context of Revealing 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 at neutral pH The binding affinity of -8 is reduced. For example, pH-dependent anti-IL-8 antibodies include antibodies against IL-8 that have a higher affinity at neutral pH than at acidic pH. In one embodiment, it is revealed that the anti-IL-8 antibody of C has an IL-8 affinity at neutral pH that is at least 2 times, 3 times, 5 times, 10 times, 15 times greater than that 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, 10,000 times or more. Binding affinity can be measured using, but not limited to, the surface plasmon resonance method (such as BIACORE (registered trademark)). The combination rate constant (kon) and dissociation rate constant (koff) can be calculated using BIACORE (registered trademark) T200 Evaluation Software (GE Healthcare) by fitting the combination and dissociation induction maps simultaneously based on a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) is calculated as the ratio of koff / kon. In order to screen antibodies that depend on pH-changing binding affinity, there is no particular limitation, and ELISA, KinExA ^™ , and the like, as well as surface plasmon resonance methods (such as BIACORE (registered trademark)) can be used. The pH-dependent IL-8 binding ability 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 evaluated according to the method described in WO2009 / 125825.

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

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

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

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

In one embodiment, it is desirable to disclose 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, it is disclosed that the dissociation constant ratio of the anti-IL-8 antibody of C between acidic pH and neutral pH, ie [KD (acidic pH) / KD (neutral pH)], is, for example, 30 or more But it is not limited to this. In one embodiment, it is disclosed that the dissociation constant ratio of the anti-IL-8 antibody of C between acidic pH and neutral pH, ie [KD (acidic pH) / KD (neutral pH)], is, for example, 100 or more , Such as 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500 but is not limited thereto.

In one embodiment, it is revealed that the dissociation constant ratio of the anti-IL-8 antibody of C 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, it is disclosed that the dissociation constant ratio of the anti-IL-8 antibody of C between pH 5.8 and pH 7.4, that is, [KD (pH 5.8) / KD (pH 7.4)] is, for example, 100 or more, such as 200 , 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500 But it is not limited to this.

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

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

In one embodiment, the anti-IL-8 antibodies of C disclosed herein should stably maintain IL-8 neutralizing activity in a solution (eg, PBS). Is it stable in solution The maintenance activity can be evaluated by measuring the IL-8 neutralizing activity of the antibody of Revelation C added to the solution before and after storage at a specific temperature for a specific period. In one embodiment, the storage period is, for example, 1, 2, 3, or 4 weeks, but is not limited thereto. In one embodiment, the storage temperature is, for example, but not limited to 25 ° C, 30 ° C, 35 ° C, 40 ° C, or 50 ° C. In one embodiment, the storage temperature is, for example, 40 ° C. but is not limited thereto; and the storage period is, for example, 2 weeks but is not limited thereto. In one embodiment, the storage temperature is, for example, 50 ° C. but is not limited thereto; and the storage period is, for example, 1 week but is not limited thereto.

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

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

In one embodiment, it is desirable to predict the immunogenicity of the anti-IL-8 antibody revealed by C, that is, to predict the immunogenicity of a human host, to reduce it. "Low immunogenicity" can mean, but is not limited to, for example, when an adequate amount of anti-IL-8 antibody is administered for a sufficient period of time to achieve a therapeutic effect in vivo of at least half or more of the individuals, no immune response is induced. Inducing an immune response may include the production of anti-drug antibodies. "Low resistance to antibody production" and "Low immunity "Engogenicity" is used interchangeably. Human immunogenicity levels are evaluated using T cell epitope prediction programs. Such T cell epitope prediction programs include Epibase (Lonza), iTope / TCED (Antitope), EpiMatrix (EpiVax) EpiMatrix is a system for predicting the immunogenicity of the sequence of the protein of interest, which uses the amino acid sequence of the protein to be analyzed to cut the amino acid sequence of the peptide to automatically design the sequence of the peptide fragment. Ability of 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 modified amino acid sequence of the amino acid sequence of the anti-IL-8 antibody can be analyzed using the above T cell epitope prediction program to design a reduction Immunogenic sequences. Preferred sites for amino acid modification that reduce the immunogenicity of the anti-IL-8 antibodies of this disclosure include, but are not limited to, the weight of the anti-IL-8 antibodies shown in SEQ ID NO: 78 In the chain sequence, amino acids at positions 81 and / or 82b according to Kabat numbering are used.

In one embodiment, Reveal C provides a method for enhancing the elimination of IL-8 from an individual compared to using a reference antibody, comprising administering to the individual an anti-IL-8 antibody to Rev C. In one embodiment, it is disclosed that the anti-IL-8 antibody of C is used to enhance the elimination of IL-8 from an individual compared to the use of a reference antibody. In one embodiment, disclosure C is an anti-IL-8 antibody related to disclosure C compared to the use of a reference antibody for the purpose of enhancing the elimination of IL-8 from an individual. In one embodiment, the disclosure of C is related to the use of the 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 compared to the use of a reference antibody. In one embodiment, Reveal C is a pharmaceutical composition comprising an anti-IL-8 antibody that promotes the elimination of IL-8 from a living body compared to the use of a reference antibody. In one embodiment, C is disclosed It relates to a method for enhancing the elimination of IL-8 from a living body compared to the use of a reference antibody, including administering an anti-IL-8 antibody of Rev. C to a subject. In the embodiment of Rev. C, the reference anti-system refers to: before the modification of the anti-IL-8 antibody of the Rev. C antibody, or an antibody with strong IL-8 binding affinity at both acidic and neutral pH. The reference antibody may be an antibody comprising the amino acid sequence of SEQ ID NO: 83 and 84 or SEQ ID NO: 89 and 87.

In one embodiment, disclosed C provides a pharmaceutical composition comprising the anti-IL-8 antibody of disclosed C, which is characterized in that the anti-IL-8 antibody of disclosed C is bound to IL-8 and then to the extracellular matrix. In one embodiment, disclosure C relates to the use of an anti-IL-8 antibody of disclosure C to manufacture a pharmaceutical composition, which is characterized in that 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 may be a humanized antibody.

In one aspect, the antibody of this disclosure C includes the heavy chain variable region of any of the above embodiments and the light chain variable region of any of the above embodiments. In one embodiment, the antibody of this disclosure 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 its sequence.

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

1. Chimeric antibodies and humanized antibodies

In certain embodiments, the antibodies provided in Disclosure C may be chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984). In one example, a chimeric antibody can include a non-human variable region (eg, a variable region from a mouse, rat, hamster, rabbit, or non-human primate monkey), and a human constant region.

In certain embodiments, the chimeric antibody is a humanized antibody. Generally, non-human anti-human systems are humanized to reduce human immunogenicity, while retaining the specificity and affinity of parental non-human antibodies. Generally, a humanized antibody includes one or more variable regions, where HVRs, such as CDRs (or portions thereof), are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody may be substituted with residues of a corresponding non-human antibody (eg, an antibody derived from its HVR residue) to, for example, maintain or improve antibody specificity or affinity.

Humanized antibodies and production methods are reviewed in, for example, 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) (SDR) transplantation); Padlan, Mol. Immunol. 28: 489-498 (1991) (documented "resurfacing"); Dall'Acqua et al., Methods 36: 43-60 (2005) (documented "FR Drag"); and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer 83: 252-260 (2000) (documenting the choice of FR drag "guidance" "law).

Human frame regions that can be used for humanization include, but are not limited to, frame regions selected using the "best-fit" method (see, eg, Sims et al., J. Immunol. 151: 2296 (1993)); Framework regions derived from human antibody common sequences of specific subgroups of the heavy chain variable region (see, for example, 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 or antibody fragments provided by C are disclosed. Antibody fragments include, but are not limited to, Fab, Fab ', Fab'-SH, F (ab') ₂ , Fv and scFv fragments, and other fragments described below. For a review of antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For comments on scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

Bifunctional antibodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993 ). Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

Single domain antibodies are antibody fragments that include all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody may be a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Patent 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 (such as E. coli or phage), as described in the scope of the disclosure of C.

3.Human antibodies

In certain embodiments, the antibodies provided in Disclosure C may be human antibodies. Human antibodies can be prepared using a variety of techniques known in the art. Generic terms for human antibodies described in Van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008). Human antibodies can be used to administer immunogens to modified genetically modified animals to produce intact human antibodies that respond to antigenic challenges or intact antibodies with human variable regions. Such animals generally contain all or part of the human immunoglobulin gene locus, which is the replacement of the animal (non-human) immunoglobulin gene locus, which is located extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, the immunoglobulin loci of animals (non-humans) 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 ^TM Technology; US Patent Nos. 5,770,429 for HUMAB ^TM Technology; US Patent No. 7,041,870 for KM MOUSE ^TM Technology; and US Patent Application Publication No. US 2007/0061900 for VELOCIMOUSE ^TM Technology .

Human variable regions of intact antibodies produced by such animals can be further modified by combining different human constant regions. Human antibodies can also be prepared using fusion tumor-based methods. Human myeloma and mouse-human 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 A method for manufacturing a single human IgM antibody from a fusion tumor cell line, as described in U.S. Patent No. 7,189,826, and a human-human fusion tumor described in, for example, Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) Method. 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 individually isolating Fv selectable variable domain sequences selected from human-derived phage presentation libraries. Such a variable domain sequence can be combined with the desired human invariant domain. Techniques for selecting human antibodies from an antibody library are as follows.

4. Antibodies derived from libraries

Antibodies of C can be isolated by screening combinatorial libraries with the desired activity against the antibodies. For example, various methods have been developed to generate phage display libraries and to screen libraries of antibodies possessing the desired binding characteristics. Such a method is described in Hoogenboom et al., In Meth. Mol. Biol. 178: 1-37 (O'Brien et al., Ed., Human Press, Totowa, NJ, 2001), and, for example, McCafferty et al., Nature 348: 552-554 (1990); Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Meth Mol. Biol. 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al. J. Immunol. Meth. 284 (1-2): 119-132 (2004).

In some phage presentation methods, VH and VL coding sequences can be The stocks were individually selected using polymerase chain reaction (PCR) and reorganized randomly in the phage bank. The obtained phage library was screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). Bacteriophages generally present antibody fragments in the form of single-chain Fv (scFv) fragments or Fab fragments.

Alternatively, unaltered stocks can be selected (e.g., from humans) to provide antibodies against a wide range of non-self and single antigens without any immunization, such as Griffiths et al., EMBO J. 12: 725-734 (1993 ).

Finally, it is also possible to clone unrearranged V-gene segments from stem cells, use PCR primers with random sequences to encode highly variable CDR3 regions, and complete rearrangement in a test tube to synthesize unaltered structures (See below and Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992); patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373; and US Application Publication No. 2005 / 0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. Here, antibodies or antibody fragments isolated from the human antibody library are considered human antibodies Or human antibody fragments.

5. Multispecific antibodies

In certain embodiments, the antibodies provided in accordance with disclosure C may be, for example, multispecific antibodies, such as bispecific antibodies. The multispecific antibody system is directed against a single antibody with binding specificity in 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, a bispecific antibody can bind to two different epitopes on IL-8. Bispecific antibodies can also be used for performance Cell-localizing cytotoxic agent of 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 exhibit 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 Patent No. 5,731,168). Multispecific antibodies can be made by cross-linking 2 or more antibodies or fragments using an electrostatic steering effect to prepare Fc-heterodimer molecules (WO 2009 / 089004A1) (US Patent No. 4,676,980; and Brennan et al. ., Science 229: 81 (1985)), by using a leucine zipper to produce a bispecific antibody (Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992)), by using "Bifunctional antibody" technology to make bispecific antibody fragments (Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), by using a single chain Fv (scFv) binary (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 here (see, for example, US 2006/0025576).

Within the context of the disclosure of C, this antibody or antibody fragment also includes a "double-acting Fab" or "DAF", including an antigen-binding site that binds to IL-8 and one of different antigens (see, for example, US 2008/0069820) .

6. Antibody variants

The amino acid sequence variants of the antibody can be encoded as The nucleotide sequence of this antibody may be prepared by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. The final construct may include any combination of deletion, insertion, and substitution. The principle is that the final construct is an antibody with the desired properties that reveal the context of C.

In one embodiment, it is disclosed that C provides an antibody variant that is substituted with one or more amino acids. Such a substitution site may be any position in the antibody. Conservatively substituted amino acids are shown in Table 10 under the heading "Conservative substitutions". General substituted amino acids that cause more substantial changes are shown in Table 10, titled "Generally Substituted" and further referenced in the amino acid side chain category.

Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: white Amino acid, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) alkaline: His, Lys, Arg; (5) residues affecting chain alignment: Gly, Pro; and (6) aromaticity: Trp, Tyr, Phe.

Non-conservative substitutions will cause one member of these categories to become another.

Amino acid insertions include fusions comprising 1, 2, or 3 to 100 or more residues at the N- and / or C-terminus of the polypeptide, and insertion of one or more amino acid residues into a sequence. Antibodies having such terminal insertions include, for example, antibodies with N-terminal methionamine residues. Other antibody molecule insertion variants include those derived from N- or C-terminally fused antibodies to enzymes (such as ADEPT) or polypeptides that increase the plasma half-life of the antibody.

7. Glycated variants

In one embodiment, the antibody provided in accordance with Disclosure C may be a glycated antibody. Glycosylation sites can be created or removed by adding or deleting antibodies to alter the amino acid sequence.

When the antibody includes an Fc region, additional sugar chains can be altered. Unchanged antibodies produced by animal cells generally contain branched and dibranched 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, a GlcNAc attached to the "stem" of the dibranched oligosaccharide structure. In one embodiment, it is disclosed that the oligosaccharide of the antibody of C is modified to create an antibody variant with specific improved properties.

8. Fc region variants

In one embodiment, one or more amino acid modifications are introduced The Fc region of the antibody is provided to produce an Fc region variant. Fc region variants include those that have 1, 2, 3, or more amino acid modifications (eg, substitutions) in the native human Fc region sequence (eg, the Fc region of human IgG1, IgG2, IgG3, or IgG4).

It is revealed that the anti-IL-8 antibody of C may include an Fc region having at least one of the following five properties, but is not limited to: (a) the binding affinity of FcRn relative to the natural Fc region at an acidic pH. The binding affinity of FcRn to the Fc region is increased; (b) the binding affinity of the Fc region to the existing ADA is reduced relative to the native Fc region; (c) the binding affinity to the existing ADA is reduced; The plasma half-life of the Fc region is increased; (d) the plasma clearance of the Fc region is reduced relative to the natural Fc region; and (e) the effector receptor is targeted relative to the natural Fc region The binding affinity of the Fc region to the effector receptor is reduced. In some embodiments, the Fc region has two, three, or four of the properties listed above. In one embodiment, Fc region variants include those with increased FcRn binding affinity at an acidic pH. Fc region variants with increased FcRn binding affinity include, but are not limited to, FcRn binding affinity increased up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold compared to the FcRn-binding affinity of antibodies containing natural IgG Fc regions -Fold, 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 C, the term "ADA" refers to an endogenous antibody that has a binding affinity for an epitope on a therapeutic antibody. As used in the context of Revealing C, the term "existing ADA" refers to a detectable anti-drug antibody present in a patient's blood before the patient is administered a therapeutic antibody. Pre-existing ADA includes rheumatoid factors. Fc region variants with low binding affinity for existing ADA include but are not Limited to ADA binding affinity reduced to 1/10 or less, 1/50 or less, or 1/100 or less Fc region variants compared to the ADA binding affinity of antibodies containing natural IgG Fc regions .

In one embodiment, Fc region variants include Fc region variants that have low or no binding affinity for complement proteins. Complement proteins include C1q. Fc region variants with low binding affinity for complement proteins include, but are not limited to, reduced binding affinity for complement proteins to 1/10 or less compared to complement protein binding affinity for antibodies containing natural IgG Fc regions , 1/50 or less, or 1/100 or less.

In one embodiment, Fc region variants include Fc region variants that have low binding affinity for the effector receptor or have no binding affinity for the 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 the following types. FcγRII includes but is not limited to FcγRIIa (there are two subtypes: R131 and H131) and FcγRIIb. FcyRIII includes, but is not limited to, FcyRIIIa (there are two subtypes: V158 and F158) and FcyRIIIb (there are two subtypes: FcyRIIIb-NA1 and FcyRIIIb-NA2). Variants of Fc regions with low binding affinity for effector receptors include, but are not limited to, reduced binding affinity for effector receptors to at least 1 / compared to the binding affinity of antibodies containing natural IgG Fc regions 10 or less, 1/50 or less or 1/100 or less Fc region variants.

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

In one embodiment, Fc region variants include those substituted with one or more amino acids at positions 235, 236, 239, 428, 434, 436, 438, and 440 compared to the native Fc region, as compared to the native Fc region. Fc region.

In one embodiment, Fc region variants include one or more of positions 235, 236, 327, 330, 331, 428, 434, 436, 438, and 440 as compared to natural Fc regions. Amino acid substituted Fc region.

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

In one embodiment, the Fc region variant includes an Fc region comprising an amino acid substitution of M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the Fc region variant includes an amino acid-containing Fc region of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E.

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

In one embodiment, the anti-IL-8 antibody of C is disclosed including 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 be in vivo (e.g., plasma) Internal stability maintains IL-8 neutralizing activity. The anti-IL-8 antibody including the amino acid sequence of SEQ ID NO: 80 and / or the amino acid sequence of SEQ ID NO: 82 may be an antibody with low immunogenicity. 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 include the FcRn binding affinity at an acidic pH compared to the native Fc region , An FcRn with an acidic pH (eg, pH 5.8) binds to an Fc region 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 include a binding affinity compared to the native Fc region against existing ADA, Fc region with reduced binding affinity for existing ADA. 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 include an Fc region with an increased half-life in plasma compared to the native Fc region. 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 include a binding affinity for an effector receptor compared to a native Fc region Sexually reduced Fc region. In yet another embodiment, this anti-IL-8 antibody includes any of the properties of any of 2, 3, 4, 5, 6 in combination of all 7 properties.

In one embodiment, the anti-IL-8 antibody of C is disclosed including 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 including the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 can stably maintain IL-8 neutralizing activity in vivo (e.g., in plasma) . This anti-IL-8 antibody including the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may be a low immunogenic antibody. This includes the amino acid sequence of SEQ ID NO: 81 and / or The anti-IL-8 antibody of the amino acid sequence of SEQ ID NO: 82 may include an Fc region with increased FcRn binding affinity at an acidic pH compared to the FcRn binding affinity of the native Fc region. This anti-IL-8 antibody including the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may include binding affinity compared to the native Fc region against existing ADA, Fc region with reduced binding affinity for existing ADA. This anti-IL-8 antibody including the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may include an Fc region with an increased half-life in plasma compared to the native Fc region. This anti-IL-8 antibody including the amino acid sequence of SEQ ID NO: 81 and / or the amino acid sequence of SEQ ID NO: 82 may include a binding affinity for an effector receptor compared to a natural Fc region Sexually reduced Fc region. In yet another embodiment, this anti-IL-8 antibody includes any of the properties of any of 2, 3, 4, 5, 6 in combination of all 7 properties.

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

Primary cultured cells targeting intermediate ADCC and NK cells only show FcγRIII, while mononuclear spheres show FcγRI, FcγRII and FcγRIII. FcRs expressed on heme-producing cells are summarized in Table 3 on page 464 of Ravetch et al., Annu. Rev. Immunol. 9: 457-492 (1991). A non-limiting example of in-tube analysis of the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom. Et al., Proc. Natl Acad. Sci. USA 83: 7059-7063 (1986), Hellstrom et al. al., Proc. Natl Acad. Sci. USA 82: 1499-1502 (1985); U.S. Patent No. 5,821,337 and Bruggemann et al., J. Exp. Med. 166: 1351-1361 (1987)). Or you can use non-radioactive isotope analysis to evaluate effector cell function (see, for example, ACTI (TM) nonradioactive cytotoxic assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ^TM nonradioactive cytotoxic assay (Promega, Madison, WI)). Effector cells useful in such analyses include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.

Alternatively or additionally, the ADCC activity of the antibody variant of interest can be assessed in vivo, for example in an animal model, such as Clynes et al., Proc. Natl. Acad. Sci. USA 95: 652-656 (1998) Revealed. A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs described in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC analysis can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Meth. 202: 163 (1996); Cragg et al., Blood 101: 1045-1052 (2003); and Cragg et al., Blood 103: 2738-2743 (2004)). The determination of FcRn binding and in vivo clearance / half-life can be performed using methods known in the art (see, for example, Petkova et al., Intl. Immunol. 18 (12): 1759-1769 (2006)).

Antibodies with reduced effector function include one or more substitutions of one or more Fc region residues at positions 238, 265, 269, 270, 297, 327, or 329 (US Patent No. 6,737,056). Such Fc region variants are included at 265, 269, 270, 297 or Fc region variants with 2 or more substitutions at residue 327, including "DANA" Fc region variants with residues at positions 265 and 297 being alanine (US Patent No. 7,332,581).

Antibody variants that bind to improved or weakened FcR groups are described below. (See, for example, U.S. Patent No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001)).

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

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

9. Antibody derivatives

In certain embodiments, the antibodies provided by Disclosure C can be further modified to contain additional non-proteinaceous structures known and readily available in the art. Suitable structures for deriving this antibody 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, polyvinyl pyrrolidone, poly-1 , 3-dioxolane, poly-1,3,6-tri [lipoxane] ane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), poly (n- Vinylpyrrolidone) polyethylene Alcohols, propylene propylene glycol homopolymers, prolylpropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in industrialization because it is stable in water.

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

In another embodiment, a conjugate of an anti-IL-8 antibody revealing C and a non-protein structure can be provided, which can be selectively heated by exposure to radiation. In one embodiment, the non-protein structure is, for example, a carbon nanotube (see, for example, Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be at any wavelength, including but not limited to a temperature that is not harmful to humans but can heat the non-protein structure to a temperature that will kill cells close to the antibody-non-protein structure.

B. Recombination methods and compositions

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

In one embodiment, the host is a eukaryotic cell (eg, Chinese Hamster Ovary (CHO) cells or lymphocytes (eg, Y0, NSO, SP20 cells)).

In one embodiment, a method of manufacturing an anti-IL-8 antibody disclosed by C is provided, the method comprising: culturing a host cell comprising a nucleic acid encoded as the anti-IL-8 antibody as described above under suitable conditions for expressing the antibody, and This antibody is optionally recovered (eg, from a host cell or a host cell culture medium).

In order to recombinantly make an anti-IL-8 antibody, for example, as described above, a nucleic acid encoding an antibody is isolated and inserted into one or more vectors to further colonize and / or express host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that specifically bind to nucleic acids encoding the heavy and light chains of this antibody).

Host cells suitable for colonization or expression of a vector encoding an antibody include prokaryotic or eukaryotic cells recorded within the scope of the disclosure of C. For example, when saccharification and Fc effector functions are not required, antibodies can be produced in bacteria. For bacterial expression antibody fragments and polypeptides, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254, directed against expressing antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste of the soluble fraction and further purified.

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable hosts for colonization or expression of vectors encoding antibodies, including fungi and yeast strains whose saccharification pathways have been "humanized". Fully human glycated style antibody. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

Host cells suitable for expressing glycated antibodies can also be derived from multicellular organisms (invertebrate and spinal organisms). Examples of invertebrate cells include plant and insect cells. There is no 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 used as hosts. See U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (documented with the 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 such as the monkey kidney CV1 strain, which is transformed by SV40 (COS-7); human embryonic kidney cell line (293 cells, documented 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 rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); Mouse breast tumors (MMT 060562); TRI cells, described in Mather et al., Annals NYAcad. Sci. 383: 44-68 (1982); MRC5 cells; and FS4 cells.

Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines , Such as Y0, NS0, and Sp20, but it is not limited. 8AD6 for other mammalian host cell lines suitable for antibody production, see Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

The antibody of Rev. C produced by culturing a host cell carrying a nucleic acid encoding the antibody as described above under conditions suitable for the expression of the antibody can be isolated from inside or outside the host cell (medium, milk) and refined into essentially Pure homogeneous antibody. The isolation / refinement method generally used for purifying polypeptides can be suitably used to isolate and purify the antibody; however, this method is not limited to the above examples. Antibodies can be appropriately selected and combined with column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, And recrystallization for proper isolation and purification, but it is not limited. Chromatography includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography. Such chromatography can be performed using liquid chromatography such as HPLC and FPLC. Columns used in affinity chromatography include, but are not limited to, Protein A columns and Protein G columns. Protein A columns include, but are not limited to, Hyper D, POROS, Sepharose F.F. (Pharmacia), and the like.

Aiming at the characteristics of the anti-IL-8 antibody, such as the above-mentioned increase in extracellular matrix binding activity and enhancement of cellular uptake complexes, it was revealed that C provides a method for selecting antibodies with increased extracellular matrix binding activity and enhanced antibodies in cell uptake complexes. In one embodiment, it is disclosed that C provides a method for producing an anti-IL-8 antibody that includes a variable region in a pH-dependent manner for the binding activity of IL-8, including the following steps: (a) assessing anti-IL-8 Binding of the antibody to the extracellular matrix; (b) selection of an anti-IL-8 antibody that strongly binds to the extracellular matrix; (c) culturing a host including a vector carrying a nucleic acid encoding this antibody; and (d) selection from a culture medium Off this antibody.

Any combination of extracellular matrix can be used without restriction, only It should be known to those skilled in the art. For example, an ELISA system can be used to detect the binding between the antibody and the extracellular matrix, wherein the anti-system is added to the extracellular matrix immobilized plate, and a labeled antibody against the antibody is added. Alternatively, for example, electrochemiluminescence (ECL) can be used for the analysis, in which a mixture of the antibody and a ruthenium antibody is added to an extracellular matrix immobilized plate, and the binding of the antibody to the extracellular matrix is evaluated based on the electrochemiluminescence of ruthenium. .

The anti-IL-8 antibody used to evaluate the extracellular matrix binder in step (a) may be the antibody itself or contacted with IL-8. The "selection of an anti-IL-8 antibody that strongly binds to the extracellular matrix" in step (b) refers to the selected anti-IL-8 antibody. -IL-8 antibody binding value is higher than the value representing the binding of extracellular matrix to the reference antibody, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times or more; but the ratio is not particularly limited to the above example. Conditions other than the presence of IL-8 should be the same as those used to evaluate the binding between the anti-IL-8 antibody and the extracellular matrix. The control anti-IL-8 antibody used to compare several modified anti-IL-8 antibodies may be an unmodified anti-IL-8 antibody. In this case, conditions other than IL-8 should be the same. Specifically, in one embodiment, it is disclosed that C includes selecting from several anti-IL-8 antibodies that have not been in contact with IL-8 antibodies that have a higher value for extracellular matrix binding. In another embodiment, revealing C includes selecting from several anti-IL-8 antibodies that are in contact with IL-8 antibodies that have a higher value for extracellular matrix binding. In an alternative embodiment, "selecting an anti-IL-8 antibody that binds strongly to the extracellular matrix" in step (b) means that when assessing extracellular matrix binding, it can be based on the presence of IL-8, The antibody is selected by binding it differently to the extracellular matrix. Represents extracellular matrix binding of anti-IL-8 antibodies in contact with IL-8 relative to anti-IL-8 antibodies The value of the extracellular matrix binding ratio of the -IL-8 antibody is, for example, 2 to 1,000. 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 context of the disclosure of C can be identified, screened, or characterized for their physical / chemical properties or biological activity using various methods known in the art.

1. Combination analysis and other analysis methods

In one aspect, this revealed that the antibody of C can utilize known methods for its antigen-binding activity, such as ELISA, Western blotting, kinetic exclusion analysis (KinExA ^TM ), and surface plasmon resonance, using, for example, BIACORE (GE Healthcare) Device evaluation.

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

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

2. Activity analysis method

In one aspect, analysis is 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. The present 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 ^TM CHO-K1 CXCR2 Beta-Arrestin Cell Line (DiscoveRx, Cat. # 93-0202C2) is an artificial cell line that creates a human CXCR2 that expresses the known human IL-8 receptor and uses human IL-8 When receiving a signal, it emits chemical electroluminescence. When human IL-8 is added to the cell's culture medium, the cell emits chemiluminescence in a manner that depends on the concentration of human IL-8 added. When the combination of human IL-8 and anti-human IL-8 antibody was added to the culture medium, the chemical electroluminescence of the cells was reduced or failed to be detected compared to the absence of this antibody, because the anti-human IL-8 antibody could block IL-8 signal transfer. Specifically, the stronger the human IL-8 neutralizing activity of this antibody, the weaker the level of chemiluminescence; and the weaker the human IL-8 neutralizing activity 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-mentioned differences.

D. Pharmaceutical formula

Pharmaceutical formulations including anti-IL-8 antibodies within the scope of disclosure C can be frozen by mixing anti-IL-8 antibodies to a desired degree of purity with one or more optionally pharmaceutically acceptable carriers. Prepared as a dry or aqueous formulation (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, histidines, and other organic acids; antioxidants, including Ascorbic acid and methionine; Preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalconium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol Phenyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight ( Less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamate, Asparagine, histamine, 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; relative salt formation Such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactant such as TWEEN ^TM, PLURONICS ^TM or polyethylene glycol (PEG).

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

An example of a lyophilized antibody formulation is described in US Patent No. 6,267,958. Aqueous antibody formulations US Patent No. 6,171,586 and those described in WO2006 / 044908; WO2006 / 044908 formulations include a histidine-acetic acid buffer.

Formulas in the category of C revealed that they may also include more than one active ingredient necessary for the treatment of a specific indication, and should be complementary to each other without adverse authors. Such active ingredients are suitably present in combination in an amount effective for the purpose.

The active ingredients can be encapsulated in microcapsules, for example, prepared by coacervation technology or interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, in colloidal drug delivery systems (such as microlipids) Body, albumin microspheres, microemulsifiers, nano particles and nano capsules) or macro emulsifiers. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

It can be prepared as a sustained release preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the anti-IL8 antibody of the present disclosure C, where this matrix is in the form of a shaped article, such as a film or microcapsule.

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

E. Treatment methods and compositions

In some embodiments, it is disclosed that the anti-IL-8 antibody system provided by C is used in treatment method.

In one aspect, anti-IL-8 antibodies for use in pharmaceutical compositions are provided. In an alternative aspect, anti-IL-8 antibodies are provided to treat 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 providing a subject for use in treating a disease in which IL-8 is present in excess (e.g., a disease due to the presence of excess IL-8), comprising administering to the individual an effective amount of anti-IL-8 antibody. In another embodiment, it is disclosed that C provides an anti-IL-8 antibody for use in such a method. In one embodiment, it is disclosed that C is 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, the use of C is disclosed for the use of an anti-IL-8 antibody to manufacture a pharmaceutical composition for treating a disease in which IL-8 is present in excess. In one embodiment, the use of 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 disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; inflammatory liver disease 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; vocal cord inflammation; artificial organs / prosthetic blood vessels Malignant tumors such as ovarian cancer, lung cancer, prostate cancer, gastric cancer, breast cancer, melanoma, head and neck cancer, and kidney cancer; sepsis caused by infection; cystic fibrosis; pulmonary fibrosis; and acute lung injury.

In an alternative embodiment, it is disclosed that C provides an anti-IL-8 antibody for inhibiting the accumulation of biologically active IL-8. "Inhibition of accumulated IL-8" can be achieved by preventing an increase in the amount of existing IL-8 in the living body or reducing the amount of existing IL-8 in the living body. In one embodiment, the present 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 mean IL-8 complexed with an anti-IL-8 antibody or free IL-8; or, total IL-8. "Living in vivo" herein can mean "secreting out of cells in vivo". In one embodiment, it is disclosed that C provides a method for inhibiting the accumulation of biologically active IL-8, including the step of administering an effective amount of an anti-IL-8 antibody to one individual. In one embodiment, it is disclosed that C is a pharmaceutical composition for inhibiting the accumulation of biologically active IL-8, including an effective amount of an anti-IL-8 antibody. In one embodiment, it is disclosed that C is about using an anti-IL-8 antibody to produce a pharmaceutical composition for inhibiting the accumulation of biologically active IL-8. In one embodiment, it is disclosed that C is about using an effective amount of an anti-IL-8 antibody to inhibit the accumulation of biologically active IL-8. In one embodiment, it is disclosed that an anti-IL-8 antibody of C inhibits IL-8 accumulation compared to an anti-IL-8 antibody that does not have pH-dependent binding activity. In the above embodiments, the "individual" is preferably a human.

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

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

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

Antibodies of C may be used alone or in combination with other agents in therapy. For example, it is revealed that the antibody of C can be co-administered with at least one additional therapeutic agent.

It is revealed that the antibody of C (and any additional therapy) can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal. If local treatment is desired, focal administration can be performed. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, such as injection, such as intravenous or subcutaneous, depending in part on whether it is administered short-term or long-term. each Such administration procedures include, but are not limited to, single or multiple administrations at various time points, administration of bolus, and intermittent infusion.

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 specific condition to be treated, the specific mammal to be treated, the clinical conditions of the individual patient, the cause of the disease, the delivery site of the pharmaceutical composition, the administration procedure, and other medical prescription personnel Cognition factor. This antibody does not necessarily have to be formulated with one or more agents currently used in the prevention or treatment of the condition 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. It is usually administered at the same dose and the same route as described in the disclosure of C, or from 1 to 99% of the scope of the disclosure in C, or any dose that is experimentally / clinically deemed appropriate And arbitrary paths.

In order to prevent or treat a disease, the appropriate dosage of the antibody of C (when alone or in combination with one or more other additional therapeutic agents) is disclosed, depending on the type of disease to be treated, the type of antibody, the severity and progress of the disease, whether or not The antibody transformation system is administered for preventive or therapeutic purposes, the previous treatment, the patient's clinical history and the response to this antibody, and the judgment of the attending physician. This antibody is 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 can be selected from about 1 μg / kg to 15 mg / kg (for example, 0.1 mg / kg to 10 mg / kg) antibodies, such as single administration or several separate administrations or continuous Infusion. Depending on the above factors, the daily dose is usually about 1 mg / kg to 100 mg / kg or more. When repeated administration is performed for several days or longer, depending on the conditions, the treatment is generally continued until the desired effect of suppressing the symptoms of the disease is exhibited. A typical dose of the antibody is, for example, about 0.05 A range of mg / kg to about 10 mg / kg. Thus, for example, one or more doses of about 0.5 mg / kg, such as 2.0 mg / kg, such as 4.0 mg / kg, or, for example, 10 mg / kg (or any combination) may be administered to a patient. Such administration may be intermittent, such as every week or every three weeks (for example, the patient receives about 2 to about 20 doses or about 6 doses of antibody in this manner). Higher loading doses 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 analysis.

F. Articles of manufacture

In another aspect of Revelation C, the present disclosure provides articles of manufacture, including materials useful in treating, preventing, and / or diagnosing the aforementioned conditions. Such articles of manufacture include containers, labels, or imitations of medicines on or associated with the containers. Suitable containers include, for example, bottles, vials, and intravenous solution bags. The container can be made of various materials, such as glass or plastic. Such container-retaining compositions are, by themselves or other compositions useful for treating, preventing and / or diagnosing conditions, and may also have sterile access openings (e.g. the container may be an intravenous solution bag or may be pierced by a hypodermic needle Glass bottle with closed cap). At least one active ingredient in the composition is an antibody that reveals C. The label or drug product alone represents the use of the composition in the treatment of a selected condition.

In addition, the article of manufacture may include: (a) a first container including a composition containing an antibody that discloses C; and (b) a second container including a composition containing an additional cytotoxic agent or a different therapeutic agent. The article of manufacture of the disclosed embodiment of C may further include a pharmaceutical copy, indicating that the composition can be used to treat a specific condition. Alternatively or in addition, the article of manufacture further includes, for example, a second (or third) container, which includes a pharmaceutically acceptable buffer solution, such as sterilized water for injection (BWFI), phosphate buffered saline, Ringer's Liquid solution and glucose solution. This manufactured item may further include other sales or Materials desired by the user, including other buffers, filters, needles and syringes.

Those with ordinary knowledge in the technical field may know based on the general technical knowledge in the technical field: The present disclosure C includes a combination of one or more of all or all 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 combinations of "and / or" before and after, including combinations of terms appropriately linked by this word.

Although various elements described herein are labeled as, for example, first, second, third, fourth, etc., it should be understood that this element is not limited to these terms. These terms are only used to distinguish elements, and it should be understood that without deviating from the categories of A, B, and C, for example, the first element may be referred to as the second element, and similarly, the second element may be referred to as the first element.

Unless explicitly stated or unless inconsistent with the context, any singular form expressed herein also includes the plural form, and any plural form expressed here also includes the singular form.

The terminology herein is used for the purpose of describing particular embodiments and is not intended to limit the present disclosure. Unless otherwise specified, all terms (including technical and scientific terms) used herein are to be interpreted with the meaning of the common understanding of those who have ordinary knowledge in the technical field to reveal the common understanding of A, B and C, and are not ideal or excessive. Formal idea explanation.

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

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

Unless there is inconsistency in the context, the numerical values used herein are understood as a specific range understood by those with ordinary technical knowledge in the technical field. For example, the expression "1mg" is understood as "about 1mg" with some deviations. For example, the expression "1 to 5 items" is understood to be specific and individually "1 item, 2 items, 3 items, 4 items, and 5 items" unless they are inconsistent with the context.

Hereinafter, Examples 1 to 4 and 21 to 23, Examples 5 to 7, 19 and 20, and Examples 8 to 19 will be specifically used to disclose A, B, and C, but it is not intended to be limited thereto. It should be understood that within the general description given above, various other embodiments may be implemented.

[Example]

[Example 1]

Manufacture of pH-dependent human IL-6 receptor-binding human antibodies with increased isoelectric point

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

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

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

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

WO2011 / 122011 reveals that 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) enters cells by substitution of amino acids into Fv4-IgG1 The Fc region is enhanced and FcRn-binding ability is conferred at neutral pH conditions. also WO2013 / 125667 reveals that Fv4-IgG1-F1180 (hereinafter referred to as Low_pI-F1180) its FcγR-mediated uptake into cells is enhanced by the introduction of amino acids into the Fc region of Fv4-IgG1 to enhance the neutral pH conditions. FcγR binding capacity. At the same time, amino acid modification to increase the FcRn binding of endosomes to increase the plasma retention of this antibody was introduced into Fv4-IgG1-F1180. The anti-systems shown below are made by increasing the isoelectric point value of antibodies containing these novel Fc region variants.

Specifically, VH3-IgG1-F11 (SEQ ID NO: 30) and VH3-IgG1-F939 (SEQ ID NO: 26) of WO2011 / 122011 and VH3-IgG1-F1180 (SEQ ID NO: 28) According to Kabat numbering method, the glutamic acid at position 16 is replaced by glutamic acid, the glutamic acid at position 43 is replaced by arginine, the glutamic acid at position 64 is replaced by lysine, and the position 105 Glutamic acid was replaced with glutamic acid to manufacture VH3 (High_pI) -F11 (SEQ ID NO: 31), VH3 (High_pI) -F939 (SEQ ID NO: 27), and VH3 (High_pI) -F1180 (SEQ ID NO: 29), as a heavy chain with an increased isoelectric point value.

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

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

[Example 2]

Antigen elimination effect of antibodies showing an increase in isoelectric point value of pH-dependent binding

(2-1) In vivo analysis of isoelectric point-adjusted pH-dependent human IL-6 receptor binding antibody

As described below, in vivo assays were performed using various pH-dependent human IL-6 receptor binding antibodies produced in Example 1: Low_pI-IgG1, High_pI-IgG1, Low_pI-F939, Middle_pI-F939, High_pI-F939, Low_pI-F1180 , Middle_pI-F1180, and High_pI-F1180.

Soluble human IL-6 receptor (also referred to as "hsIL-6R"), anti-human IL-6 receptor antibody, and human immunoglobulin preparation Sanglopor prepared according to the method of Reference Example 3 were simultaneously administered to human FcRn Transgenic mice (B6.mFcRn-/-. HFcRn Tg line 32 + / + mice, Jackson Laboratories; Method Mol. Biol. 602: 93-104 (2010)), and then evaluated this soluble human IL-6 receptor In vivo in vivo dynamics. A mixed solution (concentrations of 5 μg / mL, 0.1 mg / mL, and 100 mg / mL each) containing soluble human IL-6 receptor, anti-human IL-6 receptor antibody, and Sanglopor was administered through the tail vein at 10 mL / kg. I 1 time. Since the amount of the anti-human IL-6 receptor antibody is sufficient in excess of the soluble human IL-6 receptor, it is assumed that almost all soluble human IL-6 receptors have bound to this antibody. Blood was collected 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. The separated plasma is frozen at -20 ° C or lower until measurement.

(2-2) Measurement of soluble human IL-6 receptor concentration in plasma by electrochemiluminescence

The soluble human IL-6 receptor concentration in mouse plasma was measured by electrochemiluminescence. A sample of soluble human IL-6 receptor was adjusted to a concentration of 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg / mL for the preparation of a calibration curve, and a mouse plasma analysis method was diluted 50 times or more, respectively sample. Samples and 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 human IL-6 Tocilizumab (CAS No .: 375823-41-9) was mixed with the receptor-binding antibody, and then reacted at 37 ° C overnight. The final Tocilizumab concentration was adjusted to 333 μg / mL. The reaction solution was then distributed to Streptavidin Gold Multi-ARRAY Board (Meso Scale Discovery). After reacting for another hour at room temperature, the reaction solution was washed. Read Buffer T (x4) (Meso Scale Discovery) was then immediately allocated to the board and measurements were performed using SECTOR Imager 2400 (Meso Scale Discovery). The soluble human IL-6 receptor concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

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

In all cases, it has been shown that by increasing the isoelectric point value of the antibody, the rate of pH-dependent binding antibody elimination of the antigen can be accelerated. It has also been shown that by increasing the binding capacity to FcRn or FcγR by further imparting neutral pH conditions, the rate of antigen elimination can be further accelerated compared to when isoelectric point values are increased only with pH-dependent binding antibodies (compared to Figure 1 And Figures 2 and 3).

(2-3) pH-dependent human IL-6 receptor binding antibody in vivo infusion analysis with adjusted isoelectric point

Using various pH-dependent human IL-6 receptor binding antibodies prepared in Example 1, the following in vivo assays were performed: Low_pI-IgG1, High_pI-IgG1, Low_pI-F11, and High_pI-F11.

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

Specifically, a single anti-mouse CD4 antibody obtained according to a known method was injected into the tail vein at a single dose of 20 mg / kg to suppress a neutralizing antibody against soluble human IL-6 receptor that may be produced by the mouse itself . Then, an infusion pump containing 92.8 μg / ml soluble human IL-6 receptor was subcutaneously transplanted on the back of the mouse. Three days after the graft infusion pump, an anti-human IL-6 receptor antibody was administered to the tail vein at a single dose of 1 mg / kg. 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 On days, 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. The separated plasma is frozen at -20 ° C or lower until measurement.

(2-4) Measurement of plasma hsIL-6R concentration by electrochemiluminescence

The hsIL-6R concentration in mouse plasma was measured by electrochemiluminescence. The hsIL-6R sample was adjusted to a concentration of 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg / mL for the preparation of a calibration curve, and a mouse plasma analysis sample diluted 50 times or more was prepared. Samples and 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), mixed with Tocilizumab, and then The reaction was carried out at 37 ° C overnight. Adjust Tocilizumab final concentration to 333 μg / mL. The reaction solution was then distributed to a Streptavidin Gold Multi-ARRAY plate (Meso Scale Discovery). After reacting for another hour at room temperature, the reaction solution was washed. Read Buffer T (x4) (Meso Scale Discovery) was then immediately allocated to the board and measurements were performed using SECTOR Imager 2400 (Meso Scale Discovery). The hsIL-6R concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).

Changes in the measured human IL-6 receptor concentration are shown in Figure 4. Compared to the antibody whose Fc region belongs to natural IgG1 (High_pI-IgG1), and this antibody which contains a novel Fc region variant (High_pI-F11) with enhanced FcRn binding at neutral pH conditions, compared to the administration The low isoelectric point antibody (also referred to as "Low_pI"), when administered with the higher isoelectric point antibody (also referred to as "High_pI"), decreases the soluble human IL-6 receptor concentration in plasma.

Without being bound by a particular theory, the results obtained from these experiments can also be explained as follows: When the Fc region of the administered antibody is a natural IgG antibody, it is thought that the uptake into the cell is 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 (ie, the charge of the overall molecule is biased to be positively charged), the closer the complex is to the cell membrane, and the more non-specific uptake occurs. When the isoelectric point value of the antibody and antigen form a complex, the overall isoelectric point value of the complex also increases compared to the complex formed by the original antibody and the antigen; therefore, cell uptake can increase. Therefore, by increasing the isoelectric point value of the antibody showing pH-dependent antigen binding, the rate or rate of antigen elimination from plasma can be further accelerated, and the concentration of antigen in plasma can be maintained at a lower level.

In these examples, the isoelectric point of the antibody is increased by introducing a possible reduction in the number of negatively charged amino acids and / or increasing the number of positively charged amino acids. The amino acid substitution on the antibody molecule surface is in the variable region of the antibody. Those with ordinary knowledge in this technical field will understand that the effect obtained due to such an increase in isoelectricity does not depend mainly (or substantially) on the type of target antigen or the amino acid sequence that constitutes this antibody, but it is expected to depend on 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 main metabolic pathways do not involve renal excretion. IgG antibodies with Fc are known to be recovered via the FcRn expressed by cells including endothelial cells of blood vessels as a salvage pathway, and therefore have a long half-life, and IgG antibodies are considered 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, and molecules that cannot bind FcRn are metabolized. IgGs with reduced FcRn binding ability have a shorter half-life, and conversely, blood half-life can be extended by increasing their ability to bind 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 replacing 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 type of antigen, by modifying the isoelectric point of the variable region of the antibody, blood half-life can be controlled without modifying Fc. It is believed that the rate of non-specific uptake of IgG antibodies to endothelial cells depends on the physicochemical Coulomb interaction between the negatively charged cell surface and the IgG antibodies. Therefore, lowering (increasing) the isoelectric point value of IgG antibodies and thus reducing (increasing) Coulomb interactions are considered to reduce (increase) their non-specific uptake into endothelial cells, thus reducing (increasing) their metabolism in endothelial cells, thereby Can control plasma pharmacokinetics. Because the coulomb interaction between the endothelial cell and the negative surface of the cell is a physiochemical interaction, it is believed that this interaction does not mainly depend on this antibody group Into the amino acid sequence itself. Therefore, the methods for controlling plasma pharmacokinetics provided herein can be applied not only to specific antibodies, but also to any polypeptide containing variable regions of antibodies. A decrease (increase) in Coulomb interaction means a decrease (increase) in Coulomb force expressed as gravity, and / or an increase (decrease) in Coulomb force expressed as repulsion.

The amino acid substitution used to achieve the above may 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 multiple amino acid substitutions to a position exposed to 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 found that, for example, when replacing an amino acid that may be exposed to the surface of an antibody molecule with a positively charged amino acid (preferably arginine or lysine), or when using a pre-existing positively charged amino acid (preferably Arginine or lysine), it is advisable to further replace those amino acids in configuration 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 in which there is a local positive charge cluster in a position close to the configuration. The definition of "structurally close to the position" is not particularly limited, but may mean, for example, that a single amino acid substitution or a plurality of amino acid substitutions are introduced to each other within 20 angstroms, preferably 15 angstroms or more preferably within 10 angstroms. . Whether the amino acid substitution of interest is located at a position exposed to the surface of the antibody molecule, or whether the amino acid substitution is located at a close position can be determined by known methods such as X-ray crystallography.

In this way, note that the isoelectric point value is an indicator representing the overall charge of the molecule, and the charge buried in this antibody molecule is treated indistinguishably from the charge on the surface of this antibody molecule. The inventor also envisages Considering the effect of charge to produce antibody molecules, not only the isoelectric point but also the surface charge and local clustering of charges on the antibody molecule can further accelerate the elimination of antigen from plasma. And even maintain this antigen concentration in plasma at lower levels.

Receptors such as FcRn or FcγR are expressed on the cell membrane, and antibodies believed to have enhanced affinity for FcRn or FcγR at neutral pH conditions are mainly taken up into cells via these Fc receptors. The cell membrane is negatively charged, so when the isoelectric point is high (the overall charge of the molecule shifts to a positive charge), the administered antibody-antigen complex is easier to access the cell membrane, and the uptake through the Fc receptor is more likely to occur. Therefore, antibodies that have an increased affinity for FcRn or FcγR and an increased isoelectric point value at neutral pH conditions also show an increase when they are taken up into the cell via the Fc receptor when they form a complex with the antigen. Therefore, antibodies that bind to the antigen in a pH-dependent manner and have increased affinity for FcRn or FcγR at neutral pH conditions can eliminate antigens from plasma by increasing the isoelectric point, and the plasma antigen concentration can be increased. Keep it low.

[Example 3]

Assess extracellular matrix binding of pH-dependent binding antibodies with increased isoelectric point

(3-1) Evaluation of extracellular matrix binding capacity

The following experiments were performed to evaluate the effect of conferring pH-dependent antigen-binding properties of antibodies and further modifying the isoelectric point on the extracellular matrix binding capacity.

In a similar manner to Example 1, three types of antibodies with different isoelectric points were prepared as antibodies that showed pH-dependent binding to the IL-6 receptor: Low_pI-IgG1, Middle_pI-IgG1, and High_pI-IgG1. Manufactured according to the method of Reference Example 2: Low_pI (NPH) -IgG1, including H54 (SEQ ID NO: 34) and L28 (SEQ ID NO: 35); and High_pI (NPH) -IgG1, including H ( WT) (SEQ ID NO: 36) and L (WT) (SEQ ID NO: 37) as a general antibody that does not show pH-dependent binding to the IL-6 receptor.

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

(3-2) Evaluation of the binding of antibodies to extracellular matrix by electrochemiluminescence (ECL) method

The extracellular matrix (BD Matrigel Basement Membrane Matrix; manufactured by BD) was diluted to 2 mg / mL using TBS (Takara). The diluted extracellular matrix was distributed in a MULTI-ARRAY 96 well plate, high binding, bare (Bare) (Meso Scale Discovery: made by MSD), 5 μL per well, and fixed at 4 ° C. overnight. 20 mM ACES buffer at 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. The antibody to be evaluated was diluted to 30, 10, and 3 μg / mL using 20 mM ACES buffer (ACES-T buffer) containing 150 mM NaCl, 0.05% Tween 20, and 0.01% NaN ₃ pH 7.4, and then using 150 mM NaCl, 0.01% Tween 20, 0.1% BSA, and 0.01% NaN _{3 were} diluted in 20 mM ACES buffer (dilution buffer) at pH 7.4 to a final concentration of 10, 3.3, and 1 μg / mL. Add the diluted antibody solution to the plate from which the blocking solution has been removed, and shake at room temperature for 1 hour. This antibody solution was removed, and ACES-T buffer containing 0.25% glutaraldehyde was added. After standing for 10 minutes, the plate was washed with DPBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween 20. The ECL detection system was prepared using Sulfo-Tag NHS Ester (manufactured by MSD) using sulfo-labeled goat anti-human IgG (gamma) (manufactured by Zymed Laboratories). The antibody to be detected was diluted to 1 μg / mL in a dilution buffer, added to the plate, and then shaken at room temperature in the dark for 1 hour. The antibody to be detected was removed, and a two-fold dilution solution prepared with MSD Read Buffer T (4x) (manufactured by MSD) diluted with ultrapure water was added; and the luminescence was measured with SECTOR Imager 2400 (manufactured by MSD).

The results are shown in Figure 5. Antibodies that show pH-dependent binding and antibodies that do not show pH-dependent binding both increase their binding to the extracellular matrix by increasing their isoelectric point. In addition, unexpectedly, in an antibody that binds with a pH-dependent antigen, increasing the isoelectric point has a significant effect on improving extracellular matrix binding. In other words, it was found that antibodies that bind to the antigen in a pH-dependent manner and have high pI (High_pI-IgG1) have the strongest affinity for the extracellular matrix.

Without being limited to a particular theory, the results obtained from such experiments can also be explained as follows. The introduction of histidine modifications into antibody variable regions is known as a method of conferring antibody pH-dependent antigen binding (see, for example, WO2009 / 125825). Histidine has an imidazolyl group in the side chain and is not charged at neutral pH to alkaline pH, but it is known to be positively charged at acidic pH conditions. Using this property of histidine, by introducing histidine into the variable region of the antibody, especially the CDR near the site where it interacts with the antigen, the charge environment at the site where the neutral and acidic pH conditions interact with the antigen can be changed And configuration environment. Such antibodies can be expected in a pH-dependent manner Altered antigen affinity. Those of ordinary skill in the art will understand that the effect obtained by such introduction of histidine does not mainly (or essentially) depend on the type of target antigen or the amino acid sequence of the antibody, but rather on histamine Number of acid introduction sites or histamine residues introduced.

WO2009 / 041643 is described in general terms as follows: protein-protein interactions are composed of hydrophobic interactions, electrostatic interactions, and hydrogen bonds. The strength of this type of binding can usually be determined using binding constants (affinity) or appearance. Binding constant (avidity) is expressed. pH-dependent binding, the change in binding strength between neutral pH conditions (e.g., pH 7.4) and acidic pH conditions (e.g., pH 5.5 to pH 6.0) depends on naturally occurring protein-protein interactions. For example, the binding between the aforementioned IgG molecule and FcRn, which is a rescue receptor of the IgG molecule, 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 change 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 changes between neutral and acidic pH conditions. More specifically, histidine residues are uncharged and neutral at neutral pH conditions, and act as acceptors of hydrogen atoms; while histidine residues are positively charged at acid pH conditions, and act as donors of hydrogen atoms. Also, as described above for the IgG molecule-FcRn interaction, 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 impart pH dependence to protein-protein interactions. A similar approach has been used for protein-protein interactions between antibodies and antigens; others have introduced histidine to the CDRs of anti-protein lysozyme antibodies The sequence successfully obtained antibody mutants with reduced antigen affinity under acidic pH conditions (Ito et al., FEBS Lett. 309 (1): 85-88 (1992)). In addition, an antibody has been reported because histidine is introduced into a CDR sequence, which specifically binds to an antigen at a low pH of cancer tissue, and weakly binds to a corresponding antigen under a neutral pH condition (WO2003 / 105757).

The amino acid residue introduced to increase the isoelectric point value is preferably an lysine, arginine or histidine having a positively charged side chain. The 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 theory of acid-base balance known in the art, these pKa values refer to that in a solution of pH 10.5, 50% of the lysine side chains are positively charged and the remaining 50% are uncharged. As the pH of the solution increases, the proportion of positively charged side chains of lysine decreases. In a solution of pH 11.5 with a pH higher than the pKa value of lysine, the positively charged ratio becomes about 9%. On the other hand, as the pH of the solution decreases, the proportion of positive charge increases. In a solution of pH 9.5 at a pH value lower than the pKa value of the lysine, the positive charge ratio becomes about 91%. This theory applies equally to arginine and histidine. More specifically, in a neutral pH solution (eg, pH 7.0), almost 100% of the lysine or arginine is positively charged, and about 9% of the histidine is positively charged. Therefore, although histidine at neutral pH is positively charged, it is less than lysine or arginine, so lysine and arginine are considered to be more suitable for being introduced to increase the isoelectric point. Value of amino acids. According to Holash et al. (Proc. Natl. Acad. Sci. 99 (17): 11393-11398 (2002), although the introduction of modifications that increase the isoelectric point is considered effective for increasing extracellular matrix binding, but for the reasons mentioned above, Substitutions that introduce lysine or arginine are considered more suitable than substitutions that introduce histamine.

As previously revealed, by combining the introduction of histidine to confer pH-dependent antigen-binding properties and modifying the isoelectric point of this antibody, it is possible to have a surprisingly synergistic increase in the affinity of the antibody for the extracellular matrix. 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 is significantly higher. This display of affinity for the extracellular matrix may not necessarily be explained only by the isoelectric point level, and it can be said that a synergistic effect is exhibited by introducing a combination of an increase in isoelectric point and a histidine modification. This result is unexpected and unexpected.

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

[Example 4]

Use an amino acid substitution in the constant region to increase the isoelectric point

A method of increasing the isoelectric point value of an antibody that binds an antigen in a pH-dependent manner by introducing an amino acid instead of an antibody variable region has been described. In addition, the method of increasing the isoelectric point value of the antibody can also be implemented by as little as 1 in the constant region of the antibody. Amino acid substitutions are made.

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

Without limitation, the position where the amino acid substitution is introduced into the constant region is preferably a position where the amino acid side chain may be exposed on the surface of the antibody molecule. Ideal examples include a method of introducing a combination of multiple amino acid substitutions at a position that can be exposed on the surface of the antibody molecule. Alternatively, the positions of the plurality of amino acid substitution introductions should be such that they are structurally close to each other. In addition, it is not limited, but the plurality of amino acid substitutions introduced should preferably be replaced with positively charged amino acids, so that in some cases they cause a plurality of positively charged to be in a state where the configuration is close. The "confornationally close site" is not particularly limited, but may mean, for example, that one or more amino acid substitutions are introduced within each other, such as within 20 angstroms, preferably within 15 angstroms, and more preferably within 10 angstroms. Whether the amino acid substitution site of interest is a position exposed on the surface of the antibody molecule or whether a plurality of amino acid substitution positions are close can be evaluated by known methods such as X-ray crystallography.

In addition, a method of giving a plurality of positively-closed positions in the configuration includes, in addition to the above-mentioned method, a method of using an amino acid that is 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 in accordance with EU numbering; and (b) 121 in accordance with 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 structurally approaching these positively charged amino acids, substitution with positively charged amino acids may be performed, and multiple positive charges may be imparted to the structurally close positions.

(4-1) Production of pH-dependent anti-IgE binding antibody

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

(4-2) Assess the 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. The kinetic analysis of human IgE and Ab1 was performed using BIACORE T100 (GE Healthcare). The measurement system uses the following two buffers as running buffers: (1) 1.2mM CaCl ₂ /0.05% tween 20, 20mM ACES, 150mM NaCl, pH 7.4; and (2) 1.2mM CaCl ₂ /0.05% tween 20, 20mM ACES, 150 mM NaCl, pH 5.8.

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

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

A biotinylated peptide derived from human glycican 3 (also known as GPC3) (amino acid sequence is (VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO: 44)) will be used to add biotin to the chemical synthesis An appropriate amount of the peptide obtained from the C-terminal Lys was added to Sensor chip SA (GE Healthcare) and fixed on the wafer by using the affinity between streptavidin and biotin. The appropriate concentration was injected. Immobilized hIgE and immobilized on the wafer by capturing this biotinylated GPC3 peptide. Inject an appropriate concentration of anti-hIgE antibody as an analyte and interact with hIgE on the induction wafer. Then regenerate the induction wafer and inject 10 mM glycine-HCl, pH 1.5 \. All measurements were performed at 37 ° C. The combination of the rate constant ka (1 / Ms) and the dissociation rate constant kd (1 / s) was performed by using BIACORE T200 Evaluation Software (GE Healthcare) The curve fit was calculated by analyzing the measurement results, and the dissociation constant KD (M) was calculated based on the above values.

The results are presented in Table 5. All antibodies, Ab1, Ab2 and Ab3 bind to human IgE in a pH-dependent manner, and their affinity at acidic pH (pH 5.8) is significantly higher than their affinity at neutral pH (pH 7.4). weak. Therefore, administration of such antibodies to living animals is expected to accelerate the elimination effect of human IgE that is an antigen.

The theoretical isoelectric point values of Ab1-Ab3 calculated by a method similar to that in Example 1 are shown in 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 uses the Fc region of Ab1H, which is modified to the heavy chain of Ab1, by replacing proline at position 238 with aspartic acid according to EU numbering, and serine at position 298 with EU numbering. The acid was replaced with alanine for preparation. In addition, various Fc variant systems were manufactured according to the method of Reference Example 2, and were substituted into the Fc region of Ab1H-P600 by introducing various single amino acids indicated in Tables 7-1 and 7-2, respectively. For all Fc variants, Ab1L (SEQ ID NO: 39) was used as the light chain. The affinity of these antibodies for hFcγRII2b is comparable to that of the P600 variant (data not shown).

(4-4) Human FcγRIIb binding assay using novel Fc region variant-containing antibodies using BIACORE

A binding analysis method of an antibody containing an Fc region variant between a soluble human FcγRIIb (also referred to as "hFcγRIIb") and an antigen-antibody complex was performed using BIACORE (registered trademark) T200 (GE Healthcare). A method known in the art is used to make a His-tagged molecular form of soluble hFcγRIIb. A His capture kit (GE Healthcare) was used to fix an appropriate amount of anti-His antibody to Sensor chip CM5 (GE Healthcare) by amine coupling method to capture hFcγRIIb. The antibody-antigen complex is then injected with a running buffer (as a reference solution) to interact with hFcγRIIb captured on the sensor wafer. 20 mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl ₂ and 0.05% (w / v) Tween 20 at pH 7.4 were used as running buffers, and separate buffers were used. Solution to dilute soluble hFcγRIIb. To regenerate the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were performed at 25 ° C. The analysis is based on the calculated binding (RU) based on the sensorgram obtained from 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. Several Fc variants have shown improved affinity for hFcγRIIb immobilized on a BIACORE (registered trademark) sensor chip.

Although not limited to a specific theory, this result can be explained as follows. It is known that a BIACORE (registered trademark) induction wafer 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 a negatively charged BIACORE sensor chip is speculated to be similar to the case where this antigen-antibody complex binds to hFcγRIIb existing on the surface of a negatively charged cell membrane.

Antibody system prepared by introducing modification with increased isoelectric point into the Fc region. The Fc region (constant region) is more positively charged than the antibody before the modification. Therefore, the Coulomb interaction between the Fc region (positive charge) and the surface of the induction wafer (negative charge) can be considered to be enhanced by the modification of these electric points. It is also expected that this effect will also occur on the negatively charged cell membrane surface; therefore, it can also be expected to show the effect of accelerating the rate or rate of uptake into cells in vivo.

From the above results, it is believed that compared to hFcγRIIb bound to Ab1H-P600, the ratio of hFcγRIIb bound to the variant is about 1.2 times or more, showing that the antibody has a strong charge effect on the binding of hFcγRIIb on the sensor chip. Therefore, modifications that are expected to produce a charge effect include, for example, 196, 282, 285, 309, 311, 315, 345, 356, 358, 359, 361, 362, 382, 384, 385, 386, 387, 389, etc. , 399, 415, 418, 419, 421, 424, or 443. 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 that can be expected to have such a charge effect include, for example, glutamic acid at position 430 according to EU numbering. The more ideal amino acid introduced at position 430 is substituted with positively charged arginine or lysine, or glycine or threonine in the uncharged residue.

(4-5) Uptake of antibodies containing Fc region variants by hFcγRIIb-expressing cells

In order to evaluate the rate of intracellular uptake into hFcγRIIb-expressing cell lines using the produced novel Fc region-containing antibodies, the following analysis was performed.

A known method is used to produce a MDCK (Madin-Darby canine kidney) cell line that expresses hFcγRIIb consistently. Using these cells, intracellular uptake of the antigen-antibody complex was evaluated. Specifically, pHrodoRed (Life Technologies) was used to calibrate human IgE (antigen) according to established experimental procedures, and an antigen-antibody complex was formed in a culture solution with an antibody concentration of 10.8 mg / mL and an antigen concentration of 12.5 mg / mL. . The culture solution containing the antigen-antibody complex was added to the aforementioned culture plate of MDCK cells expressing hFcγRIIb, and the cells were incubated for 1 hour, and then the fluorescence intensity of the antigen absorbed into the cells was quantified using InCell Analyzer 6000 (GE healthcare). The amount of antigen taken up is represented by the value relative to P600, and the value of P600 is 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 was observed in cells in some Fc variants.

Although not limited to a specific theory, this result can be explained as follows: The antigen and antibody added to the cell culture fluid form an antigen-antibody complex in the culture fluid. Via the antibody Fc region, this antigen-antibody complex binds to hFcγRIIb expressed on the cell membrane and is taken up into the cell in a receptor-dependent manner. Ab1 used in this experiment is an antibody that binds to the antigen in a pH-dependent manner; therefore, the antibody can be dissociated from the antigen. Because the dissociated antigen is calibrated with pHrodoRed as described earlier, it fluoresces in the endosome. Therefore, if the fluorescence intensity in the cell is stronger than that in the control group, it is considered that the faster or more frequent the ingestion of the antigen-antibody complex into the cell occurs.

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

[Example 5]

Fc variants with enhanced FcRn binding produced at acidic pH conditions to improve retention in plasma

In the endosome under acidic pH conditions, it is known that IgG antibodies that have entered the cell will return to the plasma by binding to FcRn. Therefore, IgG antibodies generally have longer plasma half-lives than proteins that do not bind to FcRn. By utilizing this property, a method for improving the plasma retention of the antibody by introducing amino acid modification into the Fc region of the antibody to increase the affinity of FcRn under acidic pH conditions is known. Specifically, a method for improving the plasma retention of antibodies by increasing the affinity of FcRn under acidic pH conditions by amino acid modification is 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 mentioned above, Fc variants known to have increased FcRn affinity under acidic pH conditions will show unwanted affinity for rheumatoid factor (RF) (WO2013 / 046704). Therefore, the following tests were performed to make Fc variants that can improve plasma retention with reduced or substantially no binding to rheumatoid factors.

(5-1) Production of novel antibodies containing Fc region variants

Fc variants with increased FcRn affinity under acidic pH conditions include, for example, known modifications, YTE, LS or N434H, and some novel Fc variants (F1847m, F1848m, F1886m, F1889m, F1927m, With F1168m).

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

(5-2) Kinetic analysis of human FcRn binding

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.

The kinetic analysis of human FcRn and each antibody was performed using BIACORE T100 (GE Healthcare). Use amine coupling method to fix appropriate amount Protein L (ACTIGEN) on Sensor chip CM4 (GE Healthcare) to capture the antibody of interest. The human FcRn and the antibody captured on the sensor chip are then interacted by injecting the diluted FcRn solution with a running buffer (as a reference solution). For the running buffer, 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 chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were performed at 25 ° C. The KD (M) of human FcRn of each antibody was calculated based on the kinetic parameters calculated from the measured induction map, combining the rate constant ka (1 / Ms) and the dissociation rate constant kd (1 / s). Each parameter was calculated using BIACORE T100 Evaluation Software (GE Healthcare).

The results are shown in Table 8.

[Example 6]

Assessing the affinity of Fc region variant-containing antibodies for rheumatoid factors with enhanced FcRn binding at acidic pH

Anti-drug antibodies (ADAs) affect the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes cause serious side effects; therefore, the clinical use and efficacy of therapeutic antibodies may be limited by the production of ADA. There are many factors that affect the immunogens of therapeutic antibodies Sex, and the appearance of effector T cell epitopes is one of the factors. In addition, ADA (also known as "pre-existing ADA") that is present in a patient prior to the administration of therapeutic antibodies may have similar problems. Specifically, for therapeutic antibodies against patients with autoimmune diseases such as rheumatoid arthritis (RA), rheumatoid factors (RF) that are anti-human IgG autoantibodies may cause " The problem of "ADA" exists. Recently, humanized anti-CD4 IgG1 antibodies with N434H (Asn434His) mutations have been 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, compared to parent human IgG1, increases the binding of rheumatoid factors such as the Fc region to antibodies.

Rheumatoid factors are multiple autoantibodies against human IgG. Their epitopes in human IgG vary depending on the colony, and they appear to be located in the CH2 / CH3 junction region. They may be determined by binding 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 specific colonies of rheumatoid factors.

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

On the other hand, some amino acid modifications that selectively inhibit affinity to rheumatoid factors without affecting affinity to FcRn are disclosed in WO2013 / 046704. Among them, a combination of two amino acid mutations is indicated, That is, Q438R / S440E, Q438R / S440D, Q438K / S440E, and Q438K / S440D. Therefore, it is revealed for the first time that Q438R / S440E is introduced to have a newly increased affinity under acidic pH conditions to check whether the binding to rheumatoid factors will decrease.

(6-1) Rheumatoid factor binding assays for antibodies containing Fc region variants

Individual serum (Proteogenex) from 30 RA patients was used to perform a combined analysis of rheumatoid factors with electrochemiluminescence (ECL) at pH 7.4. Each 50-fold diluted serum sample, biotinylated test antibody (1 μg / mL), and SULFO-TAG NHS Ester (Meso Scale Discovery) -calibrated test antibody (1 μg / mL) were mixed and incubated at room temperature for 1 hour. . Thereafter, this mixture was added to a MULTI-ARRAY 96 well plate (Meso Scale Discovery) coated with streptavidin, and the plate was incubated at room temperature for 2 hours and then washed. After adding Read Buffer T (x4) (Meso Scale Discovery) to each well, the plate was immediately placed in a SECTOR imager 2400 Reader (Meso Scale Discovery), and chemical electroluminescence was measured.

The analysis results are shown in FIGS. 8 to 17. Fv4-IgG1 with natural human IgG1 (Figure 8) shows only weak binding to rheumatoid factors, while existing Fc variants with increased FcRn binding, namely Fv4-YTE (Figure 9), Fv4 -LS (Figure 10) and Fv4-N434H (Figure 11) showed significant increases in rheumatoid factor binding in some donors. On the other hand, all novel Fc region variants with increased FcRn binding, namely Fv4-F1847m (Figure 12), Fv4-F1848m (Figure 13), Fv4-F1886m (Figure 14), Fv4-F1889m (Figure 15 (Figure), Fv4-F1927m (Figure 16), and Fv4-F1168m (Figure 17), showing only weak rheumatoid factor binding, which shows that rheumatoid factor binding caused by modification to increase FcRn binding is significantly inhibited .

Figure 18 shows the average of rheumatoid factor binding affinity in the serum of 30 RA patients for each variant. The six new variants all showed lower affinity than the three pre-existing variants (YTE, LS, and N434H), and they also showed lower affinity for rheumatoid factors than natural human IgG1. As such, when considering the development of therapeutic antibodies with improved affinity for FcRn for clinical development of autoimmune diseases such as rheumatoid arthritis, the Fc variants disclosed here for the first time may inhibit pre-existing Fc variants The risk associated with rheumatoid factors makes it safer to use than currently known Fc variants.

[Example 7]

PK evaluation of Fc variants with increased FcRn binding in acidic pH conditions in cynomolgus monkeys

In Example 7, the effect of improving plasma retention in Malay monkeys was assessed using a novel Fc region-containing antibody identified herein that inhibits binding to rheumatoid factors.

(7-1) Manufacturing novel antibodies containing Fc region variants

Manufacture the following anti-human IgE antibodies: (a) OHB-IgG1, including OHBH-IgG1 (SEQ ID NO: 56) as the heavy chain and OHBL-CK (SEQ ID NO: 57) as the light chain; (b) OHB-LS, Including OHBH-LS (SEQ ID NO: 58) as the heavy chain and OHBL-CK as the light chain; (c) OHB-N434A, including OHBH-N434A (SEQ ID NO: 59) as the heavy chain and OHBL-CK as the light chain (D) OHB-F1847m, including OHBH-F1847m (SEQ ID NO: 60) as the heavy chain and OHBL-CK as the light chain; (e) OHB-F1848m, including OHBH-F1848m (SEQ ID NO: 61) as the heavy chain Chain and OHBL-CK as 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 a heavy chain and OHBL-CK as a light chain.

(7-2) PK analysis for monkeys with novel antibodies containing Fc region variants

The in vivo kinetics of anti-human IgE antibodies in plasma after administration of anti-human IgE antibodies to Malay monkeys was evaluated. The anti-human IgE antibody solution was administered intravenously at 2 mg / kg once. 5 minutes, (2 hours), 7 hours, 1 day, 2 days, 3 days, (4 days), 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and Blood was collected for 56 days. The collected blood was immediately centrifuged at 15,000 rpm and 4 ° C for 5 minutes to obtain plasma. The separated plasma was frozen at -60 ° C or lower until measurement. Eight types of anti-human IgE antibodies were used, namely OHB-IgG1, OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m.

(7-3) Measurement of anti-human IgE antibody concentration in plasma by ELISA

The concentration of anti-human IgE antibodies in the plasma of Malay monkeys was measured by ELISA. First, the anti-human IgG kappa chain antibody (antibody solution) was distributed in Nunc-ImmunoPlate, MaxiSorp (Nalge Nunc International), and allowed to stand overnight at 4 ° C to produce an anti-human IgG kappa chain antibody immobilized plate. Prepare calibration curve samples with plasma concentrations of 640, 320, 160, 80, 40, 20, or 10 ng / mL and samples of Malay monkey plasma diluted 100 times or more. These calibration curve samples and plasma measurement samples were prepared so that the Malay monkey IgE (product prepared in the company) was added at a concentration of 1 μg / mL. Then, this sample was distributed to an anti-human IgG kappa chain antibody immobilization plate, and left at room temperature for 2 hours. HRP-anti-human IgG gamma Chain antibody (Southern Biotech), and allowed to stand at room temperature for 1 hour. Then, the color reaction was performed using TMB Chromogen Solution (Life Technologies) as a substrate, and the reaction was stopped by adding 1N sulfuric acid (Wako), and then the absorbance at 450 nm was measured with a microplate reader. The analysis software SOFTmax PRO (Molecular Devices) was used to calculate the anti-human IgE antibody concentration in monkey plasma from the calibration curve. The change in the anti-human IgE antibody concentration measured in monkey plasma is shown in FIG. 19. The change in anti-human IgE antibody concentration was measured from monkey plasma, and the 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 the concentration of anti-human IgE antibody and the clearance rate in monkey plasma, samples from individuals who showed a positive response to the anti-administrated antibodies in the plasma were excluded.

(7-4) Measurement of anti-administrative antibodies in plasma by electrochemiluminescence

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

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

(7-5) Mouse PK analysis of Fc variants

The following experiment is to compare the Fc variant F1718 described in WO2013 / 046704 and the newly found Fc variant F1848m as Fc variants with increased FcRn binding 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 prepared according to the method of Reference Example 1. Using these heavy chains, the following antibodies were made according to the method of Reference Example 2: (a) Fv4-IgG1, including VH3-IgG1 as the heavy chain and VL3-CK as the light chain; and (b) Fv4-F1718, including VH3- F1718 (SEQ ID NO: 65) was used as the heavy chain and VL3-CK was used as the light chain.

Human FcRn gene transgenic mice (B6.mFcRn-/-. HFcRn Tg line 32 + / + mice; Jackson Laboratories, Methods Mol. Biol. 602: 93-104 (2010) were administered at 1 mg / kg in the tail vein The 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, and 28 days after administration of the anti-human IL-6 receptor antibody. Blood was collected on days. The collected blood was immediately centrifuged at 15,000 rpm and 4 ° C for 15 minutes to obtain plasma. Plasma was stored frozen at -20 ° C or lower until taken.

(7-6) Measurement of anti-human IL-6 receptor antibody concentration in plasma by ELISA

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

The results are shown in Figure 20. F1718 is an Fc variant described in WO2013 / 046704 that increases FcRn binding at acidic pH, and does not show any effect on elongating antibody PK, but shows equivalent plasma retention as natural IgG1.

F1718 has four mutations, namely N434Y / Y436V / Q438R / S440E introduced into the Fc region. In contrast, F1848m, which was first revealed here, also introduced 4 mutations, namely N434A / Y436V / Q438R / S440E. The difference between the amino acid mutations introduced by these two types of Fc is only in F1718, where the amino acid mutation was introduced into Y (tyrosine) at position 434 according to EU numbering, and A (alanine) in F1848m. In Example (7-2), compared to natural IgG1, F1848m showed improved plasma retention, while F1718 did not show any improvement in plasma retention. Therefore, it is not limited, and this suggests that it is desirable that A (alanine) is introduced as an amino acid mutation at position 434 to improve plasma retention.

(7-7) Predicted immunogenicity score of Fc variant

The generation of anti-drug antibodies (ADA) will affect the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes bring serious side effects; therefore, the production of ADA may limit the clinical use and drug efficacy of therapeutic antibodies. The immunogenicity of therapeutic antibodies is known to be affected by many 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 simulation tools, T cell epitopes in each amino acid sequence can be predicted (Walle et al., Expert Opin. Biol. Ther. 7 (3): 405-418 (2007)), and can be Assess the potential immunogenicity of therapeutic antibodies.

EpiMatrix was used to calculate the immunogenicity score of the Fc variant to be evaluated. EpiMatrix is a system for predicting the immunogenicity of a protein of interest. It uses 9 amino acids to sequence the amino acid sequence of the protein to be predicted. The sequences of peptide fragments are automatically designed by cutting off, and then their binding ability to 8 major MHC class II dual genes is calculated (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 4 variants, namely F1718, F1848m, F1756 and F1847m, are calculated as described above and are shown in the "EpiMatrix score" column of Table 31. Regarding the EpiMatrix score, the immunogenicity score corrected for the Tregitope content is displayed in the "tReg Adjusted Epx Score" column. Tregitope is a peptide fragment sequence that is mainly abundant in naturally occurring antibody sequences, and is believed to be a sequence that suppresses immunogenicity by activating regulatory T cells (Treg).

Based on these results, both the "EpiMatrix score" and "tReg Adjusted Epx score" show that the immunogenicity scores of the N434A variant F1848m and F1847m are reduced compared to the N434Y variant. This indicates that it is desirable that A (alanine) is introduced as an amino acid mutation at position 434 to obtain a lower immunogenicity score.

[Example 8]

Make humanized anti-human IL-8 antibody

(8-1) Production of humanized anti-human IL-8 antibody hWS-4

U.S. Patent No. 6,245,894 discloses a humanized anti-IL-8 antibody that binds to human IL-8 (hIL-8) and blocks its physiological functions. Modified humanized anti-IL-8 antibodies can be produced by combining the variable region sequences of the heavy and light chains disclosed in US Patent No. 6,245,894 and the sequence of almost any of a variety of known human antibody constant regions. Therefore, the human antibody constant region sequence of these modified antibodies is not particularly limited. Natural human IgG1 sequence or natural human IgG4 sequence can be used as the heavy chain constant region, and natural human Kappa sequence can be used as the light chain constant region sequence.

From the humanized IL-8 antibody disclosed in U.S. Patent No. 6,245,894, the coding sequence of hWS4H-IgG1 (SEQ ID NO: 83) uses the heavy chain variable region RVHg in combination with the natural human anti-IgG1 sequence for the heavy chain The constant region was manufactured according to the method of Reference Example 1. In addition, the coding sequence of hWS4L-k0MT (SEQ ID NO: 84), which uses the light chain variable region RVLa and the natural human Kappa sequence for the light chain constant region, is manufactured according to the method of Reference Example 1. An antibody combining the above heavy chain and light chain was manufactured and named 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, a mixed sequence of VH3-23 and VH3-64 was used as the heavy chain FR1, a sequence found in VH3-15 and VH3-49 was used as FR2, and a sequence found in VH3-72 was used as FR3 (however, excluding those in accordance with Kabat numbering 82a), using the sequence found in JH1 as FR4. Link these sequences to the hWS-4 heavy chain CDR sequence to make 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 the heavy chain and hWS4L-k0MT as the light chain; and Hr9, with Hr9-IgG1 as the heavy chain and hWS4L-k0MT as the light chain. In the context of revealing C, when specifying a particular light chain, Hr9 is written as Hr9 / hWS4L. This antibody was expressed using FreeStyle 293F cells (Invitrogen) according to the product experimental procedure. The antibody was purified from the culture supernatant according to the method of Reference Example 2. As a result, the antibody amounts shown in Table 11 were obtained. Unexpectedly, the performance level of Hr9 is about 8 times the performance level of hWS-4.

(8-3) Human IL-8 binding activity of hWS-4 and Hr9

The binding affinity of hWS-4 and Hr9 to human IL-8 was measured using BIACORE T200 (GE Healthcare) in the following manner.

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 fixed to 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 the diluted human IL-8 solution with a running buffer (as a reference solution). For the running buffer, use the composition described above, and also use this buffer to dilute human IL-8. To regenerate the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were performed at 37 ° C. Calculated from the sensorgram obtained from the measurement The calculated kinetic parameters were combined with the rate constant kon (1 / Ms) and the dissociation rate constant koff (1 / s) to calculate the KD (M) of human IL-8 for each antibody. Each parameter was calculated using BIACORE T200 Evaluation Software (GE Healthcare).

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

In order to develop antibody medicine, the production level of antibody molecules is an important factor, and high production levels are generally desired. It may be particularly noticeable 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 impart pH dependence

The purpose of the study was to impart pH-dependent IL-8 affinity to the Hr9 antibody obtained in Example 8.

Although not limited to a specific theory, an antibody having a pH-dependent affinity for IL-8 may exhibit the following behaviors in vivo. This antibody administered to living organisms strongly binds to IL-8 and blocks its function while maintaining a neutral pH (such as in plasma). Some of these IL-8 / antibody complexes are taken up into cells by non-specific interaction with cell membranes (pinocytosis) (hereinafter referred to as non-specific uptake). Under acidic pH conditions in the endosome, The binding affinity of the antibody to IL-8 is weakened, so the antibody dissociates from IL-8. The antibodies dissociated from IL-8 can then be returned to the cell via FcRn. The above-mentioned antibodies returned to the extracellular (intra-plasma) in this way can re-bind to other IL-8 and block their function. Antibodies with a pH-dependent affinity for IL-8 are believed to bind to IL-8 multiple times by the mechanism described above.

Conversely, when the antibody does not have the properties of the above-mentioned antibody, the antibody molecule can only neutralize the antigen once, and cannot neutralize the antigen multiple times. Generally, because an IgG antibody has two Fabs, a single antibody molecule can neutralize two 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 body. For example, a single molecule of a pH-dependent IL-8 binding antibody is taken up into a cell 10 times after administration, and can neutralize up to 20 molecules of IL-8 until it is eliminated. Therefore, antibodies that can bind IL-8 multiple times have the advantage of neutralizing several IL-8 molecules even in small amounts. From another point of view, an antibody capable of binding IL-8 multiple times has the advantage of being able to maintain a state that can neutralize IL-8 for a longer period of time than when an equivalent amount of antibody having no such property is administered. From another point of view, an antibody capable of binding IL-8 multiple times has the advantage of being able to block the biological activity of IL-8 more strongly than when an equivalent amount of antibody without this property is administered.

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

For example, the symbol "Y97H" in Table 13 shows the mutation introduction position according to the Kabat numbering method, the amino acid before the mutation is introduced, and the amino acid after the mutation is introduced. Specifically, when "Y97H" is marked, the amine group at position 97 of the Kabat numbering system is displayed. The acid residue was replaced from Y (tyrosine) to H (histidine). When a plurality of amino acid substitutions are combined and introduced, it is described as, for example, "N50H / L54H".

(9-2) pH-dependent IL-8 affinity

The human IL-8 binding affinity of the antibody produced in Example 9-1 was measured using BIACORE T200 (GE Healthcare) according to the following method. The following two 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 a Sensor chip CM4 (GE Healthcare) by an 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 the diluted human IL-8 solution with a running buffer (as a reference solution). For the running buffer, any of the above solutions was used, and human IL-8 was diluted with each buffer. To regenerate the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were performed at 37 ° C. The KD (M) of human IL-8 of each antibody was calculated based on the kinetic parameters calculated from the measured induction map, combining the rate constant kon (1 / Ms) and the dissociation rate constant koff (1 / s). Each parameter was calculated using BIACORE T200 Evaluation Software (GE Healthcare).

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

(9-3) Manufacturing and evaluating modified antibodies to impart pH dependence

The combination of the promising modification and neoamino acid mutation found in 9-2 was evaluated, and the following combinations were found.

This variant was made according to the methods 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) with H89-IgG1 (SEQ ID NO: 86) as the heavy chain and L63-k0MT (SEQ ID NO: 87) as the light chain at neutral pH is equivalent to human IL-8 Hr9 and reduced human IL-8 binding affinity at acidic pH (pH 5.8). Specifically, H89 / L63 is at pH 5.8 Both koff (dissociation rate constant) and KD (dissociation constant) are higher than Hr9. This means that under the acidic pH conditions of the endosome, H89 / L63 has the property of easily releasing human IL-8.

Surprisingly, H89 / L118 with H89-IgG1 as the heavy chain and L118-k0MT (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 compared to Hr9, human IL-8 binding affinity (KD) is weaker at acidic pH conditions. It is not particularly limited. In general, if an antibody capable of binding to an antigen multiple times is used as a pharmaceutical product, the pH-dependent antigen-binding antibody should preferably have a strong binding affinity (small KD) so that it can be strongly neutralized at neutral pH conditions. This antigen (such as in plasma). On the other hand, the antibody should have a large off-rate constant (koff) and / or a weak binding affinity (large KD), so that the antigen can be quickly released under acidic pH conditions (such as in endosomes) . Compared to Hr9, H89 / L118 has obtained these advantageous properties at both neutral and acidic pH conditions.

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

Although not limited to a specific theory, it is thought that the use of a pH-dependent antigen-binding antibody as an important factor in medicine is whether the administered antibody can release the antigen in the endosome. In this regard, it is considered important to have weak enough binding (large dissociation constant (KD)) or fast enough dissociation rate (large dissociation rate constant (koff)) under acidic pH conditions. Therefore, the following experiment was performed to check whether the KD or koff of H89 / L118 obtained in the endosome in vivo was sufficient to dissociate the antigen with BIACORE.

[Example 10]

Manufacture of high affinity antibodies for mouse PK analysis

The method for confirming the effect of antibodies on the elimination rate of human IL-8 in mice is not particularly limited. For example, this method involves administering antibodies to mice after mixing with human IL-8 and 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 strong binding affinity at both neutral and acidic pH conditions. A search was then performed for modifications that would give Hr9 high affinity, creating H998 / L63 with H998-IgG1 (SEQ ID NO: 89) as the heavy chain and L63-k0MT as the light chain.

H998 / L63 was used in a similar manner to Example 9-2 to evaluate human IL-8 binding affinity. The obtained induction diagram is shown in FIG. 21.

Surprisingly, H998 / L63 showed a slow dissociation rate at both neutral and acidic pH conditions, and had a stronger IL-8 binding affinity than Hr9. However, it is known that due to the mechanical limitations of BIACORE, in the case of a low dissociation rate of a protein-protein interaction, the analytical values, such as the dissociation rate constant (koff) and dissociation constant (KD), cannot be calculated correctly. Since the correct analysis value cannot be obtained for H998 / L63, its analysis value is not shown here. However, the results of this experiment confirmed that H998 / L63 has a very strong binding affinity at both neutral and acidic pH, and is suitable as an antibody for comparison of mouse PK analysis.

[Example 11]

Mouse PK assay using pH-dependent IL-8 binding antibody H89 / L118

(11-1) Mouse PK analysis using H89 / L118

The elimination rate of human IL-8 in vivo was evaluated using H89 / L118 manufactured in Example 9 and H998 / L63 manufactured in Example 10.

After administering human IL-8 and anti-human IL-8 antibodies to mice (C57BL / 6J, Charles river) at the same time, the pharmacokinetics of human IL-8 were evaluated. A mixed solution of human IL-8 and anti-human IL-8 antibody (10 μg / mL and 200 μg / mL each) was administered to the tail vein at a single dose of 10 mL / kg. At this time, there was a sufficient excess of anti-human IL-8 antibody relative to human IL-8, so it is believed that almost all human IL-8 has bound to the antibody. Blood was collected 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. The separated plasma is frozen at -20 ° C or lower until measurement.

(11-2) Measure human IL-8 concentration in plasma

Determination of human IL-8 concentration in mouse plasma by electrochemiluminescence. First, an anti-human IL-8 antibody (made in the factory) including a mouse IgG constant region was distributed to a MULTI-ARRAY 96 well plate (Meso Scale Discovery), and allowed to stand at room temperature for 1 hour. Then anti-human IL-8 antibody-immobilized plates were prepared by blocking with PBS-Tween solution containing 5% BSA (w / v) at room temperature for 2 hours. Prepare calibration curve samples containing human IL-8 with plasma concentrations of 275, 91.7, 30.6, 10.2, 3.40, 1.13, or 0.377ng / mL, and mouse plasma measurement samples diluted 25-fold or more. Samples and hWS-4 were mixed and allowed to react overnight at 37 ° C. Then 50 μL of this mixed solution was distributed to each well of the anti-human IL-8 antibody immobilization plate, and the solution was stirred at room temperature for 1 hour. Adjust the final concentration 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, and assign Read Buffer T (x1 ) (Meso Scale Discovery) and immediately performed the measurement with SECTOR Imager 2400 (Meso Scale Discovery). Use analysis software SOFT Max PRO (Molecular Devices) calculates human IL-8 concentration based on the response of the calibration curve.

The data of human IL-8 concentration in plasma are shown in Figure 22, and the human IL-8 clearance rate (CL) values from mouse plasma are shown in Table 15.

It can be seen from FIG. 22 that the simultaneous administration of human IL-8 and H89 / L118 was unexpectedly eliminated from mouse plasma faster than the simultaneous administration of human IL-8 and H998 / L63. The CL value, which quantitatively represents the rate of elimination of human IL-8 from mouse plasma, indicates that the rate of elimination of human IL-8 by H89 / L118 is about 19 times higher than that of H998 / L63.

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

(11-3) Mouse PK Assay for Increasing H89 / L118 Dose

An experiment to confirm the effect of the dose change of H89 / L118 was then performed as follows. Mice (C57BL / 6J, Charles river) were administered human IL-8 and H89 / L118 (2 mg / kg or 8 mg / kg) at the same time, and then the pharmacokinetics of human IL-8 was evaluated. A single solution of 10 mL / kg was administered to the tail vein as a mixed solution of human IL-8 (2.5 μg / mL) and anti-human IL-8 antibody (200 μg / mL or 800 μg / mL). At this time, since there is a sufficient excess of anti-human IL-8 antibodies compared to human IL-8, almost all human IL-8 is believed to have bound to the antibodies. Blood was collected 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. The separated plasma is frozen at -20 ° C or lower until measurement.

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

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

The following inventors will describe the possible factors that may be brought about by the scientific background, but the content of the disclosure C is not limited to the following discussion.

Among antibodies that return to plasma via FcRn from the endosome, the ratio of antibodies that bind to human IL-8 is preferably low. Focusing on human IL-8 present in endosomes, it is desirable to have a high proportion of free rather than bound antibodies. When human IL-8 is administered with antibodies that do not have pH-dependent IL-8-affinity, most (almost 100%) of human IL-8 is considered to be present in the endosome in the form of antibody complexes, in small amounts (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 endosome as a free form. Hypothesis, the proportion of free form can be understood as follows: [proportion of free human IL-8 in endosome (%)] = [concentration of free human IL-8 in endosome] / [ Total human IL-8 concentration] x 100.

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

Therefore, there is a correlation between the above-mentioned ratio of free human IL-8 in the endosome and the binding affinity (KD) and / or off-rate constant (koff) for human IL-8 at acidic pH. That is, the weaker the binding affinity for human IL-8 at acidic pH and / or the larger the dissociation rate, the higher the proportion of free human IL-8 in the endosome. However, in the case of pH-dependent IL-8 binding antibodies, the proportion of free human IL-8 can be made close to 100% in the endosome, and further weakening the binding affinity and / or increasing the dissociation rate at acidic pH may not necessarily be effective Increase the proportion of free human IL-8. I can easily understand that, for example, even if the proportion of free human IL-8 is improved from 99.9% to 99.99%, the degree of improvement may not be significant.

According to the general chemical equilibrium theory, when the 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 proportion of free human IL-8 is reduced. On the other hand, when the antibody concentration is low, when the antigen concentration is low, or when the dissociation constant (KD) is large, it is not easy to form a complex, and the proportion of free human IL-8 increases.

In this experiment, when the dosage of H89 / L118 was 8 mg / kg, the rate of elimination of human IL-8 was slower than when the dosage of this antibody was 2 mg / kg. Therefore, this implies that the ratio of free human IL-8 to the dosage of the antibody is 8 mg / kg compared with when the dosage of the antibody is 2 mg / kg. The reason for the decrease may be to increase the antibody dose in the endosome up to 4 times the antibody concentration, thereby promoting the formation of the IL-8-antibody complex in the endosome. That is, in the group that increased the dose of antibody, the proportion of free human IL-8 in the endosome decreased, so the rate of elimination of human IL-8 decreased. This also implies that when the antibody dosage was 8 mg / kg, the dissociation constant (KD) of H89 / L118 under acidic pH conditions was not sufficient to achieve almost 100% of free human IL-8. More specifically, if the antibody has a large dissociation constant (KD) (weak binding) under acidic pH conditions, even when the antibody dosage is 8 mg / kg, almost 100% free IL-8 In the state, the elimination speed of human IL-8 is equivalent to when the antibody administration amount is 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 body, it is not particularly limited, we can verify whether there is room to increase the elimination of this antigen in vivo Degree of effect. For example comparing the use of novel pH-dependent IL-8 binding antibodies with the use of H89 / L118 A novel human IL-8 elimination rate method in which novel antibodies have weaker binding affinity at acidic pH and / or increased off-rate at acidic pH compared to H89 / L118. If the above-mentioned novel pH-dependent IL-8 binding antibody shows the same human IL-8 elimination rate as H89 / L118, this implies that the binding affinity and / or dissociation rate of H89 / L118 is sufficient to achieve Levels of almost 100% free human IL-8 in endosomes. On the other hand, when the above-mentioned novel pH-dependent IL-8 binding antibody shows a higher human IL-8 elimination rate, it suggests that there is room for improvement in the binding affinity and / or dissociation rate of H89 / L118 at acidic pH.

[Example 12]

Manufacturing and evaluation of pH-dependent IL-8 binding antibody H553 / L118

(12-1) Production of antibody H553 / L118 with pH-dependent IL-8 binding ability

The purpose of the inventors herein is to make antibodies that have weaker human IL-8 binding affinity and / or larger off-rates than H89 / L118 under acidic pH conditions.

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

Some results are shown in Table 17. Antibody H553 / L118 including H553-IgG1 (SEQ ID NO: 90) as the heavy chain and L118-k0MT as the light chain, and including H496-IgG1 (SEQ ID NO: 101) as the heavy chain and L118-k0MT as the light chain The antibody H496 / L118 was shown to be more pH-dependent 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, of which only R57P was introduced into the heavy chain of H89 / L118, compared with H89 / L118, its binding affinity for human IL-8 was increased at neutral pH, but at acidic pH, human IL- 8 Binding affinity is almost unchanged. More specifically, the R57P modification introduced into H89 / L118 improved human IL-8 binding affinity only at a neutral pH, but did not change the binding affinity at an acidic pH. In addition, H553 / L118, which is produced by the introduction of Y55H modification to the heavy chain of H496 / L118, has a binding affinity that is maintained or slightly improved at neutral pH, but on the other hand, it is more acidic than H89 / L118 The binding affinity decreases. That is, when two amino acid-modified combinations are introduced, namely, Y55H and R57P to H89 / L118, the binding affinity that is reduced at acidic pH can be further improved while the binding affinity that is maintained or slightly increased at neutral pH can be further improved.

(12-2) Mouse PK analysis using H553 / L118

The rate of human IL-8 elimination in mice was evaluated using a method similar to that of Example 11-2 using H553 / L118. The results of human IL-8 concentration in plasma are shown in Figure 24, and the human IL-8 clearance rate (CL) values from mouse plasma are shown in Table 18.

As a result, no significant difference was observed between H553 / L118 and H89 / L118 when comparing data for mice administered with 2 mg / kg antibody; however, when comparing data for mice administered with 8 mg / kg antibody, it was confirmed Compared with H89 / L118, H553 / L118 eliminates human IL-8 by 2.5 times or more. From another point of view, between 2 mg / kg and 8 mg / kg, the human IL-8 elimination rate did not show a difference in H553 / L118, and no decrease in antigen elimination rate caused by an increase in the antibody dose of H89 / L118 was observed.

Without particular limitation, the reason for obtaining such a result can be discussed as follows. When the antibody was administered at 2 mg / kg and 8 mg / kg, H533 / L118 showed the same human IL-8 elimination rate. The proportion of free IL-8 shown in the endosome can reach a level close to 100%, because the IL-8 binding at the acidic pH of H553 / L118 is weak enough even under the condition of 8mg / kg administration. In other words, this suggests that although the human IL-8 elimination effect of H89 / L118 can reach the maximum at a dose of 2 mg / kg, its effect can be weakened at a high dose of about 8 mg / kg. On the other hand, even at high doses of 8mg / kg, H553 / L118 can still achieve 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 to maintain this antibody's inhibitory effect on human IL-8 in vivo for a long period of time, it is maintained stably (neutralized by IL-8 of this antibody) while the antibody being administered is present in vivo (for example, in plasma). Stability of activity) IL-8 neutralizing activity is also important. Therefore, the stability of these antibodies in mouse plasma was evaluated by the following methods.

According to a method known in the art, 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 reach 1 mL. Also, add sodium azide (sodium azide) to a final concentration of 0.1% as an antibacterial agent. Each antibody (Hr9, H89 / L118, and H553 / L118) was then added to the mouse plasma to a final concentration of 0.2 mg / mL. At this point, a portion of the sample is collected as a starting sample. Store the remaining samples at 40 ° C. After 1 and 2 weeks of storage, a part of each sample was collected and used as a sample for 1 week and a sample for 2 weeks. All samples were frozen at -80 ° C and stored until each analysis was performed.

Anti-IL-8 antibodies contained in mouse plasma were then used to evaluate their human IL-8 neutralizing activity, 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, which is an artificial cell line that transmits signals in human IL-8- to emit chemical electricity Electroluminescence. Although not particularly limited, this cell can be used to evaluate human IL-8 neutralizing activity possessed by an anti-human IL-8 antibody. When human IL-8 is added to the cell culture fluid, a certain amount of chemiluminescence appears in a manner that depends on the concentration of human IL-8 added. 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, it can block human IL-8 signal transmission. Therefore, chemiluminescence caused by the added human IL-8 can be inhibited by the anti-human IL-8 antibody. The chemiluminescence will be weaker than the absence of the antibody, or there will be no chemiluminescence at all. Therefore, with the increase of the neutralizing activity of human IL-8 with the antibody, the degree of chemiluminescence becomes weaker; and with the decrease of the neutralizing activity of the human IL-8 with the antibody, the degree of chemiluminescence increases.

This is also the same for antibodies that have been added to mouse plasma and stored for a certain period of time. If the neutralizing activity of this antibody has not changed due to storage in mouse plasma, the extent of the above-mentioned chemical electroluminescence before and after storage will not change. Should change. On the other hand, if the neutralizing activity of the antibody is reduced because it is stored in mouse plasma, use The degree of chemical electroluminescence of the stored antibody will be increased compared to before storage.

Next, the above-mentioned cell lines were used to check whether the antibodies stored in mouse plasma maintained neutralizing activity. The cell line was first suspended in an AssayComplete (tm) Cell Plating 0 Reagent, and then seeded at 5,000 cells / well in a 384-well plate. One day after starting cell culture, the following experiment was performed to determine the concentration of human IL-8 to be added. A series of diluted human IL-8 solutions with a final human IL-8 concentration ranging from 45 nM (400 ng / mL) to 0.098 nM (0.1 ng / mL) was added to the cell culture solution. Then, according to the experimental steps of the product, a detection reagent is added, and the relative chemiluminescence level is detected using a chemiluminescence detector. From the results, the reactivity of the cells to human IL-8 was confirmed, and the 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 antibodies contained therein was then evaluated using the above-mentioned mouse plasma to which the anti-human IL-8 antibody was added. The human plasma IL-8 and the aforementioned anti-human IL-8 antibody-containing mouse plasma were added to the cell culture. The amount of mouse plasma to be added was determined to contain a gradient concentration of anti-human IL-8 antibodies ranging from 2 μg / mL (13.3 nM) to 0.016 μg / mL (0.1 nM). Then add detection reagents according to the product experiment steps, and use the chemiluminescence detector to detect the relative chemiluminescence level.

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 is not added and the antibody is in the well is 0%, but only when human IL-8 is added but The average relative chemiluminescence level when no antibody was in the well was 100%.

The results of the human IL-8 inhibition assay using human CXCR2-expressing cells are shown in Figure 25, and Figure 25 A shows the results from the starting sample (mouse blood The pulp is not treated with a preservative). Figure 25B shows the results of the samples stored at 40 ° C for 1 week, and Figure 25C shows the results of the samples stored at 40 ° C for 2 weeks.

Results For Hr9 and H89 / L118, no difference in human IL-8 neutralizing activity was observed before and after storage in mouse plasma. On the other hand, H553 / L118 showed a reduction in human IL-8 neutralizing activity after 2 weeks of storage. Therefore, the human IL-8 neutralizing activity of H553 / L118 in mouse plasma is 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]

Using a computer simulation (in silico) system to make antibodies with reduced predicted immunogenicity scores

(13-1) Predicted immunogenicity scores of various IL-8 binding antibodies

The production of anti-drug antibodies (ADA) will affect the efficacy and pharmacokinetics of therapeutic antibodies, and sometimes cause serious side effects; therefore, the clinical availability and drug efficacy of therapeutic antibodies may be limited by the production of ADA. The immunogenicity of therapeutic antibodies is known to be affected by many factors, and many reports have described the importance of effector T cell epitopes in therapeutic antibodies.

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

Here EpiMatrix is used to calculate the immunogenicity score of each anti-IL-8 antibody. EpiMatrix is a system for predicting the immunogenicity of a protein of interest. It cuts the amino acid sequence of a protein to be predicted by 9 amino acids from Design peptide sequences and calculate their 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) (De Groot et al., Clin. Immunol. 131 (2): 189-201 (2009)).

The immunogenicity scores of the heavy and light chains of each anti-IL-8 antibody calculated in the above manner are shown in the "EpiMatrix score" column of Table 19. 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 mainly in natural antibody sequences, and is thought to be a sequence that suppresses immunogenicity by activating regulatory T cells (Tregs).

Regarding the scores, the total scores for the heavy and light chains are shown in the "total" column.

Based on 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, which is known as a humanized anti-human IL-8 antibody For reduction.

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

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

(13-2) Manufacturing modified antibodies with lower predicted immunogenicity scores

As mentioned above, compared to hWS-4, the immunogenicity scores of H89 / L118, H496 / L118, and H553 / L118 are lower; however, it can be seen from Table 19 that the immunogenicity score of the heavy chain is higher than that of the light chain, suggesting that There is still room for improvement of heavy chain amino acid sequences, especially from the viewpoint of immunogenicity. A search was then performed on the heavy chain variable region of H496 to find amino acid modifications that could reduce the immunogenicity fraction. As a result of searching, 3 variants were found, namely H496v1, in which the alanine at position 52c of Kabat numbering was replaced with aspartic acid, and H496v2, where the glutamic acid at position 81 according to Kabat numbering was replaced by Threonine, H496v3, in which serine at position 82b of Kabat numbering was replaced with aspartic acid. In addition, H1004 containing all of these three modifications was produced.

The results of the immunogenicity scores calculated in a similar manner to that of 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 score than H496. H1004, which contains three modifications in combination, achieves a marked improvement in 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, which combine heavy and light chains, also show very low immunogenicity scores.

The frequency of ADA generated by these combinations is then predicted in a similar manner as in 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 have significantly lower immunogenicity scores, also showed an improvement in the predicted value of ADA production frequency, with a predicted value of 0%.

(13-3) Measurement of IL-8 binding affinity of H1004 / L395

Manufactured H1004 / L395, which is an antibody that includes H1004-IgG1m (SEQ ID NO: 91) as the heavy chain and L395-k0MT (SEQ ID NO: 82) as the light chain. The binding affinity of H1004 / L395 for human IL-8 was measured using BIACORE T200 (GE Healthcare) as described below.

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

An appropriate amount of Protein A / G (PIERCE) was fixed on Sensor chip CM4 (GE Healthcare) by amine coupling method, and the antibody of interest was captured. Human IL-8 is then interacted with the antibody captured on the sensor chip by injecting a diluted human IL-8 solution or running buffer (as a reference solution). For the running buffer, one of the above buffers was used, and human IL-8 was diluted with each buffer. To regenerate the sensor chip, 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) were used. The KD (M) of human IL-8 of each antibody was calculated based on the kinetic parameters calculated from the measured induction map, combining the 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. It has the same KD for human IL-8 at neutral pH, but has a larger KD and koff at acidic pH. The dissociative nature of IL-8.

[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

By the evaluation of Example 13, H1004 / L395 was obtained, which had a pH-dependent IL-8 binding ability and a lower immunogenicity score. A detailed investigation was then carried out to produce variants with these advantageous properties and stability in mouse plasma.

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

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

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

Then, the stability of IL-8 binding when this antibody was stored in PBS was evaluated as shown below.

Each antibody was dialyzed against DPBS (Sigma-Aldrich) overnight, and then the antibody concentration was adjusted to 0.1 mg / mL. At this point, some of this antibody sample 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, using the starting sample and the sample of the heating acceleration test, a BIACORE measurement of IL-8 binding affinity was performed as follows.

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

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

The amount of human IL-8 binding per 1000 RU of the captured antibody was calculated for the starting sample and the sample for the accelerated heating test. In addition, the ratio of the human IL-8 binding level of the starting sample to the heating accelerated test sample was calculated.

Human IL-8 junction of the starting sample relative to the sample of the accelerated heating test The results of the combined level ratio are also shown in Table 22.

Using the above test, H1009 / L395 was obtained, which is an antibody comprising H1009-IgG1m (SEQ ID NO: 92) as a heavy chain and L395-k0MT as a light chain.

As shown in Table 22, compared to H89 / L118, H1009 / L395 has slightly improved human IL-8 binding affinity at neutral pH, but has reduced binding affinity at acidic pH, that is, pH dependence has been Further strengthening. When exposed to severe conditions, such as 50 ° C in PBS, H1009 / L395 has a slightly improved stability in terms of IL-8 binding compared to H89 / L118.

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

(14-2) Stability Evaluation of H1009 / L395

Then, in a manner similar to Example 12-3, whether the IL-8 neutralizing activity of H1009 / L395 was stably maintained in mouse plasma was evaluated. Here, H1009 / L395-F1886s which will be described in detail in Example 19 will be used. This antibody has the same variable region as H1009 / L395, and its constant region has a modification that enhances FcRn binding under acidic pH conditions and a modification that reduces FcγR binding compared to natural human IgG1. The variable region of H1009 / L395, especially the region surrounding HVR, is responsible for IL-8 binding and the IL-8 neutralizing activity of antibodies, and the introduction of modifications into the constant region is not believed to affect these properties.

The stability evaluation 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 to a final concentration of 0.1%. Each antibody (Hr9, H89 / L118 or H1009 / L395-F1886s) was added to the above mouse plasma at a final concentration of 0.4 mg / mL. At this point, a portion of the sample is collected as a starting sample. Store the remaining samples at 40 ° C. After 1 and 2 weeks of storage, a part of each sample was collected and used as a sample for 1 week and a sample for 2 weeks. All samples were frozen at -80 ° C and stored until each analysis was performed.

Human CXCR2-expressing cells were used to measure human IL-8 neutralizing activity in a similar manner as in Example 12-3. 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 human IL-8 inhibition analysis using human CXCR2-expressing cells and the above-mentioned antibodies are shown in Figure 28A, Figure 28A shows the starting sample (unpreserved in mouse plasma), and Figure 28B shows storage at 40 ° C The results of the samples for 1 week, Fig. 28C shows the results for the samples stored at 40 ° C for 2 weeks.

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

(14-3) Mouse PK analysis using H1009 / L395

The rate of elimination of human IL-8 in mice by H1009 / H395 was evaluated as follows. H1009 / L395, H553 / L118 and H998 / L63 were used as antibodies. Administration of mice, collection of blood, and measurement of human IL-8 concentration in mouse plasma were performed 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 rate (CL) values from mouse plasma are shown in Table 23.

Results When the dose of H1009 / L395 was 2mg / kg, the human IL-8 elimination rate in mice was equivalent to H553 / L118, showing that H1009 / L395 was in the endosome. Almost 100% free IL-8 was reached. The clearance rate (CL) value quantitatively represents the rate of elimination of human IL-8 from mouse plasma, which is shown to be approximately 30 times higher than H998 / L63.

It is not particularly limited, and the effect of increasing the human IL-8 elimination rate can be understood as follows. Generally, in a living body, the antigen is maintained at a substantially constant concentration, and the rate of production and elimination of the antigen is also maintained at a substantially constant value. When the antibody is administered under such conditions, even if the rate of antigen production is not affected, the rate of antigen elimination may change because the antigen and antibody form a complex. Generally, since the rate of antigen elimination is greater than the rate of antibody elimination, in this case, the rate of elimination of the antigen that has formed a complex with the antibody decreases. When the rate of antigen elimination decreases, the concentration of antigen in plasma increases, but the degree of increase in this case can also be defined by the ratio of the rate of elimination when the antigen is present alone to the rate of elimination when the antigen forms a complex. That is, compared with the elimination rate when the antigen is present alone, if the elimination rate is reduced by one tenth when the complex is formed, the concentration of the antigen in the plasma of the organism to which the antibody is administered may be increased 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 the antibody is administered to the organism can be defined as the antigen CL before and after the antibody administration under each condition.

Here, when H998 / L63 and H1009 / L395 are administered, there is a 30-fold difference in the CL of human IL-8, suggesting that when these antibodies are administered to humans, the increased levels of human IL-8 in plasma will There are about 30 times difference. In addition, a 30-fold difference in human IL-8 concentration in plasma indicates that the amount of antibodies to completely block the biological activity of human IL-8 also differed 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 this antibody amount is very small. When H1009 / L395 and H998 / L63 is administered to humans individually at the same dose, and H1009 / L395 can block the biological activity of IL-8 for a longer time with greater strength. 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, has also been shown to be an antibody with superior effects from the viewpoint of neutralizing IL-8 in vivo.

[Example 15]

Evaluation of extracellular matrix binding using pH-dependent IL-8 binding antibody H1009 / L395

The excellent effect of H1009 / L395 shown in Example 14 to eliminate human IL-8 by 30 times is surprising. It is known that when a pH-dependent antigen-binding antibody is administered, the rate of antigen elimination depends on the rate of uptake of the antibody-antigen complex into the cell. That is, if the rate of uptake of pH-dependent antigen-binding antibodies into cells is increased, the antigen-eliminating effect of pH-dependent antibodies will increase when an antigen-antibody complex is formed compared to when no complex is formed. Methods known to increase the rate of uptake of antibodies into cells include: conferring FcRn binding capacity of the antibody at neutral pH conditions (WO 2011/122011), and methods of increasing the binding capacity of antibodies to FcγR (WO 2013/047752) And a method for promoting the formation of a complex comprising a multivalent antibody and a multivalent antigen (WO 2013/081143).

But the above technique is not used in the constant region of H1009 / L395. Also, although it is known that IL-8 will form homodimers, human IL-8 bound by H1009 / L395 was found to exist in the form of a unit body, because H1009 / L395 recognizes homologs of human IL-8. The body forms a surface. Therefore, antibodies do not form multivalent complexes.

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

Then, the inventors discussed the following as possible factors that may bring about the above-mentioned properties of a pH-dependent IL-8 binding antibody represented by H1009 / L395. However, the following are only possible inferences by the inventor based on the technical background, and the content of the disclosure C is not limited to the following discussion.

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

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

As mentioned above, affinity for extracellular matrix is also a factor that may affect uptake into cells. It was then checked whether the binding of the extracellular matrix to the antibody alone and the human IL-8-antibody were different.

ECL (electroluminescence) method to assess the amount of antibody bound to extracellular matrix

The extracellular matrix (BD Matrigel basement matrix / manufactured by BD) was diluted to 2 mg / mL using TBS (Takara, T903). 5 μL of the diluted extracellular matrix was dispensed into MULTI-ARRAY 96 well plates, high binding, bare plates (manufactured by Meso Scale Discovery: MSD), and immobilized overnight at 4 ° C. It was then blocked with 20 mM ACES buffer (pH 7.4) containing 150 mM NaCl, 0.05% Tween20, 0.5% BSA, 0.01% NaN ₃ .

Antibodies to be evaluated were prepared as follows. 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), and then buffer 2 (20 mM ACES buffer , Containing 150 mM NaCl, 0.05% TWeen20, 0.1% BSA, and 0.01% NaN ₃ , pH 7.4) and prepared 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 the antibody to the antibody sample, and then diluting each antibody with buffer-1 So that the antibody concentration is 9 μg / mL, 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 at room temperature for 1 hour for complex formation.

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

The results are shown in Figure 30. Interestingly, all anti-IL-8 antibodies, such as H1009 / L395, show little binding to the extracellular matrix when the antibody alone (-IL8), but when forming a complex with human IL-8 ((+ hIL8 )), Will bind to the extracellular matrix.

As described above, the properties of anti-IL-8 antibodies that have affinity for extracellular matrix by binding to human IL-8 have not been elucidated. Also, without limitation, combining such properties with a pH-dependent IL-8 binding antibody can effectively increase the rate of IL-8 elimination.

[Example 16]

Mouse PK assay using non-FcRn-binding antibodies

The following method was used to confirm whether a complex of human IL-8 and pH-dependent IL-8 binding antibody was formed, and whether the uptake of the complex into cells was increased in mice.

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

Because antibodies with this Fc region lack FcRn binding affinity under acidic pH conditions, they will not transfer from endosomes to plasma. Therefore, such antibodies will be eliminated from plasma faster in vivo than antibodies that include natural Fc regions. In this case, when the antibody including the natural Fc region is taken up into the cell, only a portion that is not salvaged by FcRn will be degraded after being transferred to the lysosomal body. Zone, all antibodies taken into the cell will be degraded in the lysosome. More specifically, the rate at which an antibody comprising such a modified Fc region is eliminated from plasma may be equivalent to the rate at which it enters a cell. That is, the intracellular uptake rate of an antibody with deleted FcRn binding affinity can also be confirmed by measuring the rate of elimination of this antibody from plasma.

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

For transgenic mice (B6.mFcRn-/-. HFcRn Tg line 32 + / + mice when anti-human IL-8 antibodies were administered alone; Jackson Laboratories; Methods Mol. Biol. 602: 93-104 (2010)), and the biodynamics of anti-IL-8 antibodies when human IL-8 and anti-human IL-8 antibodies were simultaneously administered to human FcRn transgenic mice. An anti-human IL-8 antibody solution (200 μg / mL) and a mixed solution of human IL-8 (10 μg / mL) and an anti-human IL-8 antibody (200 μg / mL) were individually administered once to the tail vein at 10 mL / kg. . In this case, since the anti-human IL-8 antibody is present in sufficient excess 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. The separated plasma is frozen at -20 ° C or lower until measurement.

The concentration of anti-human IL-8 antibody in mouse plasma was measured by electrochemiluminescence method. First, the streptavidin Gold Multi-ARRAY plate (Meso Scale Discovery) that had been blocked overnight at room temperature with a PBS-Tween solution containing 5% BSA (w / v) was used to make anti-human Kappa light chain goat IgG Biotin ( IBL) was reacted at room temperature for 1 hour to produce an anti-human antibody immobilized plate. Prepare a sample for the calibration curve. The concentration of anti-human IL-8 antibody in the plasma is 3.20, 1.60, 0.800, 0.400, 0.200, 0.100, and 0.0500 μg / mL, and a sample for the measurement of mouse plasma is diluted 100. Times or higher. Each sample was mixed with human IL-8, and then distributed to an anti-human antibody immobilization plate at 50 μL per well, followed by stirring at room temperature for 1 hour. Human IL-8 was adjusted to a final concentration of 333ng / mL.

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

The results of the antibody concentration in mouse plasma are shown in FIG. 31, and the clearance rate of this antibody under each condition is shown in 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 an increase of at least 2.2 times. It is marked "at least 2.2 times" for the following reasons: One is the possibility that the actual value is 5 times, 10 times, or 30 times. Because the rate of elimination of human IL-8 is 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 decreases rapidly after administration. More specifically, even if human IL-8 is administered at the same time, not all of the H1009 / L395-F1942m in plasma are present as human IL-8-binding type. In fact, most of them have been in free form after about 7 hours of administration. presence. Since the uptake rate is evaluated under such conditions, the intracellular uptake rate of the complex of H1009 / L395-F1942m and human IL-8 actually increases by 5 times, 10 times, or 30 times compared to the uptake rate of H1009 / L395-F1942m. Times, but the results are only partially reflected in the experimental department; therefore, the effect may be 2.2 times. 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 a value of 2.2 times.

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 than the antibody alone, and it is more positively charged. At the same time, the affinity of the complex to the extracellular matrix is increased more than the affinity of the antibody alone. For example, increasing the isoelectric point and promoting extracellular matrix binding are considered to promote Factors that antibodies take up into living cells. From mouse experiments, the intracellular uptake of the complex of H1009 / L395 and human IL-8 increased 2.2 times or more than the uptake rate of H1009 / L395. From the above, the theoretical explanation and the in-tube nature and in vivo phenomena consistently support the following hypothesis: H1009 / L395 forms a complex with human IL-8 to promote uptake of the complex into the cell, and results in a significant increase in elimination of human IL-8.

To date, some antibodies against IL-8 have been reported, but there is no report about the increased binding affinity to the extracellular matrix and the increased uptake of the complex into the cells after forming a complex with IL-8.

Based on the observation that when the antibody and IL-8 form a complex, intracellular uptake of the anti-IL-8 antibody increases, it is believed that the anti-IL-8 antibody that has formed a complex with IL-8 in the plasma will enter the cell quickly. Free antibodies that do not form a complex with IL-8 tend to remain in 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 cell, and then it can bind to another IL- 8 molecules; therefore, increased intracellular uptake after 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 C.

[Example 17]

Prediction of the immunogenicity of pH-dependent IL-8 binding antibody H1009 / L395 using a computer simulation system

Then, similar to the method of Example 13-1, the immunogenicity score of H1009 / L395 and the frequency of ADA generation were predicted. The results are shown in Table 25 and Figure 32. In Figure 32, H1009 / L395 is labeled "H1009L395".

[TABLE 25]

The results in Table 25 show that H1009 / L395 and H1004 / L395 have the same degree of low immunogenicity score. Moreover, in Fig. 32, the result of ADA generation frequency predicted for H1009 / L395 is 0%, and the result for H1004 / L395 is similar.

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

[Example 18]

Malay monkey PK analysis using H89 / L118 variants with improved FcRn binding ability under acidic pH conditions

As described in the previous examples, among the antibodies with natural IgG1 as the constant region, the pH-dependent IL-8 binding antibody H1009 / L395 is an antibody with superior properties. Such an antibody can also be used as an antibody containing an amino acid substitution in the constant region, as shown in Example 5 which includes an Fc region having an increased FcRn binding at acidic pH. Therefore, the use of H89 / L118 to confirm that the Fc region that promotes FcRn binding at acidic pH can also be used as a pH-dependent IL-8 binding antibody.

(18-1) H89 / L118 Fc region modified antibody produced at an acidic pH with enhanced FcRn binding

Various modifications described in Example 5-1 to enhance FcRn binding were introduced into the Fc region of H89 / L118. Specifically, the modifications used in F1847m, F1848m, F1886m, F1889m, F1927m, and F1168m were introduced into H89-IgG1 This Fc region was used to make the following variants: (a) H89 / L118-IgG1, including H89-IgG1m (SEQ ID NO: 94) as the heavy chain and L118-K0MT as the light chain; (b) H89 / L118-F1168m including H89-F1168m (SEQ ID NO: 95) as heavy chain and L118-K0MT as light chain; (c) H89 / L118-F1847m, including H89-F1847m (SEQ ID NO: 96) as heavy chain and L118-K0MT as light chain (D) H89 / L118-F1848m, including H89-F1848m (SEQ ID NO: 97) as the heavy chain and L118-K0MT as the light chain; (e) H89 / L118-F1886m, including H89-F1886m (SEQ ID NO : 98) as the heavy chain and L118-K0MT as the light chain; (f) H89 / L118-F1889m, including H89-F1889m (SEQ ID NO: 99) as the heavy chain and L118-K0MT as the light chain; and (g) H89 / L118-F1927m, including H89-F1927m (SEQ ID NO: 100) as the heavy chain and L118-K0MT as the light chain. The Malay monkey PK analysis method using these antibodies was performed according to the following formula.

(18-2) Novel Malay Monkey PK Assay Containing Fc Region Variant Antibodies

After administration of the anti-human IL-8 antibody to the Malay monkey, the biokinetics of the anti-human IL-8 antibody was evaluated. The anti-human IL-8 antibody solution was administered intravenously at 2 mg / kg once. Blood was collected 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. The separated plasma was frozen at -60 ° C or lower until measurement.

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

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

The above results confirm that all Fc region variants show longer retention in plasma compared to antibodies with the natural IgG1 Fc region. In particular, H89 / L118-F1886m has optimal hemodynamics.

[Example 19]

Fc region with low binding ability to FcγRs

It is known that the Fc region of natural human IgG1 binds to the Fcγ receptor (hereinafter referred to as FcγR) in various cells of the immune system and displays effector functions on target cells, such as ADCC and ADCP.

On the other hand, IL-8 is a soluble interleukin, and the use of anti-IL-8 antibodies as medicine is mainly expected to show pharmacological effects by neutralizing the function of IL-8 in the presence of excessive IL-8. The location where IL-8 is excessive is not particularly limited, and may be, for example, an inflammation site. It is known that various immune cells generally aggregate and activate at such an inflammation site. Unexpected activation signals transmitted via the Fc receptor to these cells and causing activities such as ADCC and ADCP in unexpected cells are not always advantageous. Therefore, it is not particularly limited, and 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 antibody with lower binding to FcγR

In order to reduce the binding ability to various human and Malay monkey FcγR, amino acid modification was further introduced into the Fc region of H1009 / L395-F1886m. Specifically, H1009-F1886m heavy chain was replaced by the following substitutions to make H1009-F1886s (SEQ ID NO: 81): According to EU numbering, L at position 235 was replaced by R, and G at position 236 was replaced by R, The 239-bit S is replaced by K. Similarly, the following substitutions are made for H1009-F1886m to make H1009-F1974m (SEQ ID NO: 80): According to the EU numbering method, replace L at 235 with R and G with 236 at R. In accordance with EU numbering, The region from positions 327 to 331 was replaced with the natural human IgG4 sequence. H1009 / L395-F1886s and H1009 / L395-F1974m are manufactured as antibodies with these heavy chains and L395-k0MT as the light chain.

(19-2) Confirmation of affinity for various human FcγR

Then, the affinity of H1009 / L395-F1886s or H1009 / L395-F1974m to soluble FcyRIa or FcyRIIIa in humans or Malay monkeys was confirmed by the following method.

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

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

(19-3) Fc variant mouse IL-8 elimination assay

Then for H1009 / L395-F1886s and H1009 / L395-F1974m, the following experiments were performed to confirm the rate of human IL-8 elimination and the retention of antibodies in mouse plasma. 3 doses of H1009 / L395-F1886s are used here, that is, 2mg / kg, 5 mg / kg and 10 mg / kg are being evaluated, so the effect of increasing antibody dose on H1009 / L395-F1886s can also be evaluated.

For human FcRn gene transgenic mice (B6.mFcRn-/-. HFcRn Tg line 32 + / + mice; Jackson Laboratories; Methods Mol. Biol. 602: 93-104 (2010)), simultaneous administration of human IL-8 After anti-human IL-8 antibody, the biodynamics of human IL-8 was evaluated. A single 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 via the tail vein in a single administration. In this case, since the anti-human IL-8 antibody is present in sufficient excess compared to human IL-8, almost all human IL-8 is considered to have bound to this antibody. Blood was collected 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. The separated plasma is frozen at -20 ° C or lower until measurement.

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

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

Then, when the dose of H1009 / L395-F1886s antibody was changed, although the IL-8 concentration in plasma was slightly different after 1 day of administration, there was no significant difference in the clearance rate of human IL-8 at the dose of 2mg / kg and 10mg / kg. This strongly suggests that even if the antibody is administered at a high dose, the antibody including the variable region of H1009 / L395 shows sufficient IL-8 elimination effect.

(19-4) Malay monkey PK analysis of Fc variant

Then use H1009 / L395-F1886s or H1009 / L395-F1974m to verify the plasma retention of antibodies in Malay monkeys as follows.

The biological kinetics of the anti-human IL-8 antibody was evaluated in the case where the anti-human IL-8 antibody was administered alone to the Malay monkey and when the human IL-8 and the anti-human IL-8 antibody were administered simultaneously. An anti-human IL-8 antibody solution (2 mg / mL) or a mixed solution of human IL-8 (100 μg / kg) and an anti-human IL-8 antibody (2 mg / kg) was administered intravenously in a single 1 mL / kg. Blood was collected 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. The separated plasma was frozen at -60 ° C or lower until measurement.

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

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

When H1009 / L395-F1886s and human IL-8 are administered at the same time, the change in plasma concentration is equivalent to that in the case where this antibody is administered alone. Not particularly limited The following discussion can be drawn from the above findings. As described above, compared with the case of H1009 / L395 alone, the intracellular uptake of the complex of H1009 / L395 and human IL-8 showed an increase. Generally, it is believed that high molecular weight proteins enter cells in a non-specific or receptor-dependent manner and then transfer to lysosomes, which are degraded by various degrading enzymes present in the lysosomes. Therefore, if the rate of protein uptake into cells increases, plasma retention of the protein may also worsen. However, if the antibody has the property of endosome to return to plasma through FcRn; as long as there is sufficient FcRn rescue function, even if the intracellular uptake rate is accelerated, plasma retention will not be affected. Here, even the simultaneous administration of H1009 / L395-F1886s and human IL-8 to Malay monkeys does not affect plasma retention. This shows the possibility that even if the rate of antibody uptake into H1009 / L395-F1886s into cells is increased, FcRn can still be enough to remedy it, allowing it to return to plasma.

Yet another Fc variant H1009 / L395-F1974m also shows equivalent plasma retention relative to H1009 / L395-F1886s. These Fc variants have been introduced with different modifications as described above for reduced binding capacity of various FcyRs, but have not been shown to have an effect on the plasma retention of the antibodies themselves. From the above, H1009 / L395-F1886s and H1009 / L395-F1974m show significant prolonged plasma retention in Malay monkeys, and compared to antibodies with the natural IgG1 Fc region, they are extremely satisfactory.

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

As described above, H1009 / L395 having a natural Fc region of IgG is sufficiently useful; however, a variant of H1009 / L395 including a functionally improved Fc region can also be suitably used as an antibody for enhanced use. Specifically, it can increase FcRn binding under acidic pH conditions to prolong plasma retention and maintain long-term effects. It is also possible to use a variant that includes a modification to the Fc region to reduce the binding ability to FcγR as a highly safe therapeutic antibody to avoid unwanted activation of immune cells and cytotoxic activity when administered to an organism. As such an Fc variant, the use of F1886s or F1974m is particularly advantageous, but is not limited to these Fc variants; as long as the Fc variant has a similar function, therapeutic properties including other modified Fc regions can be used Antibodies serve as an embodiment of disclosure C.

Therefore, the antibody capabilities of Rev. C, including H1009 / L395-F1886s and H1009 / L395-F1974m, produced by the fine research of the present invention Conditions are maintained that the biological activity of human IL-8 is safely and strongly inhibited for a long period of time. Here, the known levels of anti-IL-8 antibodies cannot be reached, and it has been 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 antibodies

The humanized anti-factor IXa / factor X bispecific antibody disclosed in WO2012 / 067176 will bind to human factor IXa and factor X, and induce the common aggregation activity of blood. The 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 : 331) / common L chain (SEQ ID NO: 332)) In this embodiment, F8M includes two different H chains and two common L chains. F8M is manufactured according to the method described in the example 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 based on the method of Reference Example 2: (a) F8M-F1847mv, a conventional antibody, including F8M-F1847mv1 (SEQ ID NO: 323) and F8M-F1847mv2 (SEQ ID NO: 324) as the heavy chain and F8ML (SEQ ID NO: 325) as the light chain; (b) F8M-F1868mv, a conventional antibody, including F8M-F1868mv1 (SEQ ID NO : 326) and F8M-F1868mv2 (SEQ ID NO: 327) as the heavy chain and F8ML (SEQ ID NO: 325) as the light chain; and (c) F8M-F1927mv, a conventional antibody, including F8M-F1927mv1 (SEQ ID NO: 328) and 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 and enhances FcRn as mentioned in Example 5. The same Fc variant sequence that binds and reduces rheumatoid factor binding is as follows:

(20-2) Pharmacokinetics of monoclonal antibodies, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv in Malay monkeys

The pharmacokinetics of male Malay monkeys after intravenous administration of a single antibody, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv at a dose of 0.6 mg / kg to a single bolus were evaluated. The plasma concentrations of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were determined by sandwich ELISA. Pharmacokinetic parameters were calculated using WinNonlin ver 6.4 software. As shown in Table 30, the half lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv are 29.3 days, 54.5 days, and 35.0 days, respectively. The PK study using F8M of the Malay monkey was performed at a dose of 6 mg / kg on different days, and the results showed that the half-life was 19.4 days. It is known that the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv are longer than F8M. This suggests that the half-life of anti-Factor IXa / X bispecific antibodies may be extended by the same modification of the Fc region sequence as described in Example 5.

[Example 21]

Assessment of IgE clearance from plasma using increased pi variants of Fab

In order to increase the clearance of human IgE, a pH-dependent antigen-binding antibody was used in this example to evaluate the substitution of increased pI in the Fab portion of the antibody. The method of adding amino acid substitution to the variable region of this antibody to increase pI is not particularly limited, but can be implemented by the method disclosed in WO2007 / 114319 or WO2009 / 041643. The substitution of amino acids introduced into the variable region should preferably reduce the number of negatively charged amino acids (such as aspartic acid and glutamic acid) and increase the number of positively charged amino acids (such as arginine and lysine). By. In addition, amino acid substitutions can be introduced at any position in the variable region of this antibody. Without particular limitation, the site for substitution by the introduction of amino acid is preferably a position where the amino acid side chain may be exposed on the surface of the antibody molecule.

(21-1) An antibody having an increased isoelectric point value by modifying an amino acid of a variable region

Test antibodies are arranged in Tables 32 and 33.

The heavy chain Ab1H003 (also referred to as H003, SEQ ID NO: 144) was obtained by introducing a substituted H32R with increased pI to Ab1H (SEQ ID NO: 38). Other heavy chain variants were also prepared according to the method shown in Reference Example 1 by introducing each substitution shown in Table 32 to Ab1H. All heavy chain variants are represented by Ab1L (SEQ ID NO: 39) as the light chain. The pH-dependent binding status of this antibody is summarized in Table 5 (Ab1).

Similarly, we also evaluated the substitution of increased pI in the light chain.

The light chain Ab1L001T (also referred to as L001, SEQ ID NO: 164) was prepared using the substitution G16K introduced into Ab1L with increased pI. Other light chain variants were also introduced according to the method shown in Reference Example 1 and substituted into Ab1L as shown in Table 33. To implement. All light chain variants behaved as Ab1H as the heavy chain.

(21-2) Human FcγRIIb binding assay using BIACORE using variants with increased pI

For the produced Fc region-containing antibody, the binding between soluble human FcγRIIb and the antigen-antibody complex was performed using a binding assay using BIACORE T200 (GE Healthcare). Soluble human FcγRIIb (NCBI accession NM_004001.3) is prepared in the form of a His-tagged molecule by methods known in the art. An appropriate amount of anti-His antibody was immobilized on a sensor chip CM5 (GE Healthcare) by an amine coupling method using a His capture kit (GE Healthcare) to capture human FcγRIIb. The antibody-antigen complex is then injected with running buffer (as a reference solution) and interacts with human FcyRIIb that has been captured on the sensor chip. 20 mM N- (2-acetamido) -2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl ₂ and 0.05% (w / v) Tween 20, pH 7.4 were used as running buffers, and each buffer was also used. Solution diluted soluble human FcyRIIb. To regenerate the sensor chip, 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 induction map obtained from the measurement and when the binding amount of Ab1H / Ab1L (original Ab1) was defined as 1.00. To calculate the parameters, BIACORE T100 Evaluation Software (GE Healthcare) was used.

The SPR analysis results are summarized in Tables 32 and 33. Some variants have shown enhanced binding to human FcyRIIb immobilized on a BIACORE sensor chip.

An antibody prepared by introducing a modification with increased pI into the variable region of the antibody has a variable region with more positive charge than before the modification. Therefore, it can be considered that the Coulomb interaction between the variable region (positive charge) and the surface of the induction wafer (negative charge) is enhanced by the modification of increasing pI. In addition, such an effect can be expected to also occur on the surface of a negatively charged cell membrane; therefore, an effect of accelerating the uptake into the living body of a cell can also be expected.

Here, it is about 1.2 compared to binding the original Ab1 to hFcγRIIb. This variant, which binds to hFcγRIIb twice or more, is believed to have a strong charge effect by binding antibody to hFcγRIIb on the sensor wafer.

Among the heavy chain variants with increased pI, Q13K, G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G or Q105R substituted (according to Kabat numbering) antibodies alone or in combination showed higher binding to hFcγRIIb. A single amino acid substitution or combination of these substitutions in the heavy chain is presumed to have a strong charge effect on hFcγRIIb bound to the sensor wafer. Therefore, one or more positions that are expected to increase the rate or rate of uptake into the living body of a cell by introducing an increase in pI into the variable region of the antibody heavy chain may include, for example, positions 13, 15, 64, according to Kabat numbering 77, 82a, 82b, 85 and 105. The amino acid substitution introduced at such a position may be aspartic acid, glycine, serine, arginine, or lysine, and preferably arginine or lysine.

A light chain variant added to pI-, 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 FcyRIIb. A single amino acid substitution or combination of these substitutions in the light chain is presumed to have a strong charge effect on human FcγRIIb bound to the sensor chip. Therefore, one or more positions that are expected to increase the rate or rate of uptake into the cell's living body by introducing a modified pI into the variable region of the antibody light chain may include, for example, positions 16, 24, 25, according to Kabat numbering, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77, and 79. The amino acid substitution introduced to such a position may be arginine or lysine.

(21-3) pI-increased cellular uptake of Fab region-containing antibody

To assess intracellular uptake using manufactured antibodies containing Fab region variants The rate of hFcγRIIb-expressing cell lines was similar to that described in (4-5) above, except that the amount of antigen taken up was represented by a relative value relative to the value of Ab1H / Ab1L (original Ab1) (let's say 1.00).

The quantitative results of cell uptake are summarized in Tables 32 and 33. Strong fluorescence from this antigen in cells was observed in various Fc variants. Here, when the fluorescence intensity of the antigen that enters the cells of this variant is taken up about 1.5 times or more than the fluorescence intensity of the original Ab1, it is considered that the antigen has a strong charge effect into the cells.

Among the heavy chain variants with increased pI, antibodies bearing P41R, G44R, T77R, D82aN, D82aG, D82aS, S82bR, or E85G substitution (according to Kabat numbering) alone or in combination showed stronger antigen uptake into cells. The substitution of a single amino acid or a combination of heavy chains is presumed to have a strong charge effect on the uptake of antigen-antibody complexes into cells. It is therefore expected that the introduction of an increase in pI into the variable region of the antibody heavy chain results in faster or more uptake of the antigen-antibody complex into the cell at one or more positions, which may include, for example, positions 41, 44 according to Kabat numbering , 77, 82a, 82b, or 85. The amino acid substitution introduced at such a position may be aspartic acid, glycine, serine, arginine, or lysine, and preferably arginine or lysine.

Light chain variants that increase in pI, alone or in combination G16K, Q24R, A25R, A25K, S26R, S26K, E27R, E27Q, E27K, Q37R, G41R, Q42K, S52K, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substituted (according to Kabat numbering) antibodies showed strong 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 the uptake of antigen-antibody complexes into cells. Variants with 4 or more amino acid substitutions tend to be less variants than with amino acid substitutions Show strong charge effect. It is therefore expected that the introduction of an increase in pI into the variable region of the antibody light chain will result in the uptake of the antigen-antibody complex into the cell at one or more positions, which may include, for example, positions 16, 24 according to Kabat numbering , 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77, or 79. The amino acid substitution introduced at such a position may be glutamic acid, arginine, or lysine, and preferably arginine or lysine.

Although not limited to a specific theory, this result can be explained as follows: The antigen and antibody added to the cell culture medium form an antigen-antibody complex in the culture medium. This antigen-antibody complex binds to human FcyRIIb expressed on the cell membrane via the antibody Fc region, and is taken up into the cells in a receptor-dependent manner. The antibody used in this experiment binds the antigen in a pH-dependent manner; therefore, the antibody can dissociate from the antigen in the endosome (acid pH conditions) inside the cell. Because the dissociated antigen will be transported to the lysosome and accumulated, it will fluoresce in the cell. Therefore, the strong fluorescence intensity in the cell is thought to indicate that the uptake of the antigen-antibody complex into the cell occurs faster or more.

(21-4) Assessment of clearance of human IgE in mouse co-injection model

Anti-IgE antibodies (raw Ab1, Ab1H / Ab1L013, Ab1H / Ab1L014, Ab1H / Ab1L007) with pH-dependent antigen binding were tested in a mouse co-injection model to assess their ability to accelerate the clearance of IgE from plasma. In a co-injection model, C57BL6J mice (Jackson Laboratories) were administered a single intravenous injection of IgE that had been premixed with anti-IgE antibodies. All groups received 0.2 mg / kg IgE and 1.0 mg / kg anti-IgE antibodies. The total IgE plasma concentration was determined by an anti-IgE ELISA. First, anti-human IgE (Selection 107, MABTECH) was assigned to a microwell plate (Nalge nunc International) and allowed to stand at room temperature. It was left for 2 hours or left overnight at 4 ° C to prepare an anti-human IgE antibody-immobilized plate. Samples and samples against the standard curve were mixed with excess anti-IgE antibody (made in-house) to form a homogeneous structure of the immune complex. These samples were added to an anti-human IgE antibody-immobilized plate and allowed to stand overnight at 4 ° C. These samples were then sequentially reacted with human GPC3 nuclear protein (made in-house), biotinylated anti-GPC3 antibody (made in-house), streptavidin Poly HRP80 conjugate (Stereospecific Detection Technologies) for 1 hour. SuperSignal (registered trademark) ELISA Pico chemiluminescent substrate (Thermo Fisher Scientific) was then added. Chemiluminescence was read with SpectraMax M2 (Molecular Devices). Human IgE concentration was calculated using SOFTmax PRO (Molecular Devices). Figure 37 depicts the time course of IgE plasma concentration in C57BL6J mice.

After administration of a Fab variant with increased pH-bound pI, the total plasma IgE concentration was lower than that of the original Ab1. These results show that this antigen-antibody immune complex with a high pI variant with pH-dependent antigen binding will bind more strongly to plasma membrane receptors such as FcγR, increasing the cellular uptake of the antigen-antibody immune complex. This antigen, which is taken up into the cells, is effectively released from the antibody in the endosome to accelerate the elimination of IgE. In-tube experiments showed weak potency in IgE concentrations of Ab1H / Ab1L007-treated mice, which were higher than those of other antibodies containing increased pI Fab variants. These results also imply that the sensitivity of the in-tube system using the fluorescence intensity used by the above-mentioned InCell Analyzer 6000 will be higher than that of the BIACORE system using the above-mentioned tube in order to estimate the antigen clearance of plasma in vivo.

[Example 22]

Assessment of C5 clearance from plasma using pI-increased Fab variants

In order to increase the clearance of human IgE, a pH-dependent antigen-binding antibody was used in this example to evaluate the substitution of increased pI in the Fab portion of the antibody.

(22-1) Preparation of C5 [Expression and purification of recombinant human C5]

Recombinant human C5 (NCBI GenBank accession number: NP_001726.2, SEQ ID NO: 207) was temporarily expressed using 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 then eluted with a NaCl gradient. The 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 obtained 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 combined and applied to a CHT ceramic hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). Human C5 eluate was applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). The fractions containing human C5 were combined and stored at -150 ° C. Recombinant human C5 prepared from the factory or plasma derived from human C5 (CALBIOCHEM, Cat # 204888) was used for the study here.

Performance and purification of recombinant Malay C5 (NCBI GenBank accession number: XP_005580972, SEQ ID NO: 208) was performed in exactly the same manner as the human part.

(22-2) Preparation of synthetic calcium library (calcium libary)

A gene library of antibody heavy chain variable regions was used as a synthetic human heavy chain library consisting of 10 heavy chain libraries. Selection of reproductive series frameworks VH1-2, VH1-69, VH1-2, VH1-69, VH1-2 VH3-23, VH3-66, VH3-72, VH4-59, VH4-61, VH4-b, VH5-51, and VH6-1 are libraries. The synthetic human heavy chain library has diversity in the antibody binding site of the human B cell antibody library.

A gene library of antibody light chain variable regions is designed, which has a calcium-binding motif and has diversity at positions that may contribute to antigen recognition, referring to the human B cell antibody library. The design of a gene library of antibody light chain variable regions that exhibits properties against calcium-dependent binding antigens is described in WO 2012/073992.

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

(22-3) Calcium-dependent anti-C5 antibody

The phage presentation library was diluted with TBS supplemented with BSA and CaCl ₂ to a final concentration of 4% and 1.2 mM, respectively. As a panning method, referring to the general experimental procedure, the conventional magnetic bead selection method (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 are coated with NeutrAvidin (Sera-Mag SpeedBeads NeutrAvidin) or streptavidin-coated beads (Dynabeads M-280 streptavidin). Human C5 (CALBIOCHEM (Cat # 204888) is labeled with EZ-Link NHS-PEG4-Biotin (PIERCE, Cat No. 21329).

For the initial round of phage selection, the phage presentation library was incubated with biotinylated human C5 (312.5nM) for 60 minutes at room temperature. Magnetic beads were then used to capture phages that exhibited Fab variant binding.

After incubating with magnetic beads at room temperature for 15 minutes, the magnetic beads were washed three times with 1 mL of TBS containing 1.2 mM CaCl ₂ and 0.1% Tween20, and the magnetic beads were washed twice with 1 mL of TBS containing 1.2 mM CaCl ₂ . Magnetic beads were suspended in TBS containing 1 mg / mL trypsin for 15 minutes to elute phage. The eluted phages were infected with ER2738 and rescued with helper phages. The rescued phages were precipitated with polyethylene glycol to resuspend the TSA supplemented with BSA and CaCl _{2 at a} final concentration of 4% and 1.2 mM, respectively, and used for panning in the next round.

After the first round of panning, the phage was selected in a calcium-dependent manner, where this antibody binds more strongly to C5 in the presence of calcium ions. Panning in rounds 2 and 3 was performed in the same way as round 1, except for the following differences: 50nM (round 2) or 12.5nM (round 3) biotinylated antigen, 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 its calcium dependence. After selection, the selected phage colonies were converted to the IgG format.

The binding capacity of the transformed IgG antibody to human C5 was evaluated under two different conditions: binding and dissociation at 1.2 mM CaCl ₂ -pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl ₂ ), and binding to 1.2 mM CaCl _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. The induction diagrams of these antibodies are shown in Figure 38.

(22-4) Identification of anti-C5 bispecific antibodies

From the isolates isolated from Example B-3, nine pH or calcium-dependent anti-C5 antibody clones were selected for further analysis (CFP0008 ,, 0011, 0015, 0016, 0017, 0018, 0019, 0020, 0021 ). Some amino acid substitutions use methods known to those skilled in the art to introduce the CFP0016 heavy chain variable region to improve the properties of the antibody, such as physicochemical properties. The CFP0016 variant, CFP0016H019, is used for further analysis, not CFP0016. The amino acid sequences of the VH and VL regions of these 9 antibodies are described in Table 34. In this table, names enclosed in parentheses represent abbreviated names.

Full-length genes encoding the nucleotide sequences of the heavy and light chains of antibodies are synthesized and prepared according to the general methods known to those skilled in the art. The heavy and light chain expression vectors are prepared by inserting the obtained plastid fragments into the vector for expression in mammalian cells. The obtained expression vectors are sequenced according to general methods known to those skilled in the art. For antibody expression, the prepared plastids were temporarily transfected into a FreeStyle293-F cell line (Thermo Fisher Scientific). Purification of the expressing antibodies from the conditioned medium is performed by the general method known to those skilled in the art using rProtein A Sepharose Fast Flow (GE Healthcare).

(22-5) Production and characterization of pH-dependent anti-C5 bispecific antibodies

A bispecific antibody that recognizes two different epitopes of C5 and is manufactured using a combination of CFP0020 and CFP0018. The bispecific antibody system is prepared in an IgG format with two different clones at the Fab of each binding site of the antibody using general methods known to those skilled in the art. In this bispecific IgG antibody, the two heavy chains include mutually different heavy chain constant regions (GldP1, SEQ ID NO: 227 and G1dN1, SEQ ID NO: 228) in order to efficiently form two heavy chain heterodimers. Element body. An anti-C5 bispecific antibody including the binding site of anti-C5 MAb "X" and anti-C5 MAb "Y" is represented by "X // Y".

By introducing some amino acids into the heavy and light chain CDRs by a general method known to those skilled in the art, we have obtained a common light chain 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).

Optimal 20 // 18 kinetic parameters against recombinant human C5 were evaluated using a BIACORE T200 instrument (GE Healthcare) at 37 ° C under two different conditions (e.g. (A) binding and dissociation at pH 7.4, (B) binding at pH 7.4 , Dissociates at pH 5.8). Protein A / G (Pierce, Cat No. # 21186) or anti-human IgG (Fc) antibody (in the human antibody capture kit; GE Healthcare, Cat No. BR-1008-39) was immobilized on the amine coupling method Series S CM4 (GE Healthcare, Cat No. BR-1005-34). The anti-C5 antibody is captured on the immobilized molecule and then injected with human C5. The running buffers used were ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20). The dynamic parameters for both pH conditions were determined by using BIACORE T200 evaluation software, version 2.0 (GE Healthcare), by fitting the sensor map with a 1: 1 combination -RI (without a large number of effect adjustments) model. The kinetic parameters, that is, the binding rate (ka), dissociation rate (kd), and binding affinity (KD) of pH 7.4, and the dissociation rate (kd) determined only by calculating the dissociation phase at each pH condition are described in Table 35 . Optimizing 20 // 18, the dissociation of human C5 is faster at pH 5.8 than the dissociation rate at pH 7.4.

(22-6) Modification of amino acids in variable regions to produce antibodies with increased isoelectric point

Test antibodies are collated in Tables 36 and 37.

The heavy chain, CFP0020H0261-001-G1dP1 (also referred to as 20H001, SEQ ID NO: 232), was prepared by introducing P41R / G44R with increased pI to CFP0020H0261-G1dP1 (SEQ ID NO: 229). Similarly, the heavy chain, CFP0018H0012-002-G1dN1 (also referred to as 18H002, SEQ ID NO: 251), was prepared by introducing T77R / E85R with increased pI into CFP0018H0012-G1dN1 (SEQ ID NO: 230). Other heavy chain variants were also prepared by introducing 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 and two heavy chain variants of the CFPO018H0012-G1dN1 variant and CFP0020L233-k0 (SEQ ID NO: 231) as a light chain were performed together to obtain a bispecific antibody.

Similarly, we also evaluated the substitution of increased pI in the light chain. The light chain, CFP0020L233-001-k0 (also referred to as 20L233-001, SEQ ID NO: 271) was prepared by introducing a substituted G16K with an increase in pI to CFP0020L233-k0. Other light chain variants were also prepared according to the method shown in Reference Example 1 by introducing substitutions to CFP0020L233-k0 as shown in Table 37. All light chain variants and CFP0020H0261-G1dP1 as heavy chains were shown together with CFP0018H0012-G1dN1 to obtain bispecific antibodies.

(22-7) Human FcγRIIb binding analysis using BIACORE using variants with increased pI

For the antibody containing the Fc region variant, the binding analysis of soluble hFcγRIIb and the antigen-antibody complex was performed using BIACORE T200 (GE Healthcare). Soluble hFcγRIIb is formed as a His-labeled molecule according to methods known in the art. An appropriate amount of an anti-His antibody was immobilized on an induction wafer CM5 (GE Healthcare) using a His capture kit (GE Healthcare) using an amine coupling method to capture hFcγRIIb. The antibody-antigen complex is then injected with a running buffer (as a reference solution) to interact with hFcγRIIb captured on the sensor wafer. 20 mM N- (2-acetamido) -2-aminomethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl ₂ and 0.05% (w / v) Tween 20, pH 7.4 were used as running buffers, and each buffer was used To dilute soluble hFcγRIIb. To regenerate the sensor chip, 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 based on the sensorgrams obtained from the measurements and showed that when the CFP0020H0261-G1dP1 / CFP0018H0012-G1dN1 / CFP0020L233-k0 (original Ab2) binding quantity was defined as 1.00 relative value. To calculate the parameters, BIACORE T100 evaluation software (GE Healthcare) was used.

The results of the SPR analysis are summarized in Tables 36 and 37. There are some variants showing enhanced binding to hFcγRIIb immobilized on a BIACORE sensor chip. When this variant binds to hFcγRIIb at about 1.2 times or more compared to the original Ab2 binding to hFcγRIIb, it is believed that the antibody has a strong charge effect on the binding of hFcγRIIb on the sensor wafer.

Among the heavy chain variants with increased pI, antibodies with L63R, F63R, L82K or S82bR substitutions (according to Kabat numbering) showed higher binding to hFcγRIIb. A single amino acid substitution or combination of these substitutions in the heavy chain is presumed to have a strong charge effect on the binding of hFcγRIIb in the sensor wafer. Therefore, it is expected that the in vivo uptake into cells will be faster or more rapid by introducing modifications that increase pI into the variable region of the antibody heavy chain. The one or more positions may include, for example, positions 63, 82, or 82b according to Kabat numbering. The amino acid substitution introduced at such a position may be arginine or lysine.

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) show higher binding to hFcγRIIb. A single amino acid substitution in the light chain or a combination of these substitutions is believed to have a strong charge effect on the binding of human FcyRIIb in a sensor chip. It is therefore expected that the introduction of an increase in pI into the variable region of the antibody light chain results in faster or more in vivo uptake into the cell at one or more positions, which may include, for example, positions 16, 27, 41, according to Kabat numbering, 52, 56, 65, 69, 74, 76, 77 or 79. The amino acid substitution introduced at such a position may be arginine or lysine. Variants with 4 or more amino acid substitutions tend to show a stronger charge effect than variants with less amino acid substitutions.

(22-8) pI-increased cellular uptake of Fab region-containing antibody

In order to evaluate the rate of intracellular uptake into hFcγRIIb-expressing cell lines using antibodies made with Fab region variants, the following analysis was performed.

A MDCK (Madin-Darby canine kidney) cell line that continuously expresses hFcγRIIb was produced according to a known method. These cells were used to assess the intracellular uptake of the antigen-antibody complex. Specifically, human C5 was labeled with Alexa555 (Life Technlogies) according to established experimental procedures, and an antigen-antibody complex was formed in a culture solution having 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 a culture plate of the above-mentioned MDCK cells that continued to express hFcγRIIb, incubated for 1 hour, and then the fluorescence intensity of the antigen that entered the cells was quantified using the InCell Analyzer 6000 (GE healthcare). Uptake antigen The amount is expressed as a value relative to the original Ab2 value, which is taken as 1.00.

The quantitative results of cell uptake are shown in Tables 36 and 37. Strong fluorescence from this antigen was observed in cells in some heavy and light chain variants. Here, compared with the fluorescence intensity of the original Ab2, when the fluorescence intensity of the cells taken up by the antigen into the variant is about 1.5 times or more, it is considered that the antigen has a strong charge effect when taken up into the cells.

pI increased heavy chain variants with G8R, L18R, Q39K, P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN, S82bR, T83R, A85R or E85G substitution (according to Kabat numbering) Antibodies showed stronger antigen uptake into cells. A single amino acid substitution or combination of these substitutions in the heavy chain is presumed to have a strong charge effect on the uptake of antigen-antibody complexes into cells. It is therefore expected that faster or more uptake of the antigen-antibody complex into the cell by introduction of a modified increase in pI into the variable region of the antibody heavy chain may include, for example, positions 8, 18 according to Kabat numbering , 39, 41, 44, 63, 64, 77, 82, 82a, 82b, 83, or 85. The amino acid substitution introduced to such a position may be aspartic acid, glycine, serine, arginine or lysine and preferably arginine or lysine.

Among pI-increased light chain variants, antibodies substituted with G16K, Q27R, S27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R, or Q79K (according to Kabat numbering) show strong uptake of antigen Into the cell. A single amino acid substitution or combination of these substitutions in the light chain is presumed to have a strong charge effect on the uptake of antigen-antibody complexes into cells. Variants with 4 or more amino acid substitutions tend to have a stronger charge effect than variants with less amino acid substitutions. As in Example 21 and (21-3), the combination of 42K and 76R substitution in the IgE anti-system is effective. In the case of C5 antibodies, the amino acid at position 42 of the Kabat numbering It is lysine, so we can observe the charge effect of 42K / 76R by single substitution of 76R. The fact that the variant with the 76R substitution has a strong charge effect shows that regardless of the antigen, the 42K / 76R combination also shows a strong charge effect in the C5 antibody. It is therefore expected that faster or more uptake of the antigen-antibody complex into the cell by introduction of a modified increase in pI into the variable region of the antibody light chain may include, for example, positions 16, 27 according to Kabat numbering , 41, 52, 56, 65, 69, 74, 76, 77, or 79. The amino acid substitution introduced at such a position may be arginine or lysine.

(22-9) Evaluate clearance rate of C5 co-injection model in mice

Co-injection models of mice with some anti-C5 bispecific antibodies (raw Ab2, 20L233-005, 20L233-006, and 20L233-009) were tested to assess the ability to expedite the clearance of C5 from plasma. In a co-injection model, C57BL6J mice (Jackson Laboratories) were administered a single intravenous injection of C5 that had been premixed with anti-C5 bispecific antibodies. All groups received 0.1 mg / kg C5 and 1.0 mg / kg anti-C5 bispecific antibodies. The total C5 plasma concentration was determined by an anti-C5 ELISA. First, the anti-human C5 mouse IgG was distributed to an ECL plate, and an anti-human C5 mouse IgG immobilized plate was prepared overnight at 5 ° C. The samples and samples for the 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 the plate was incubated at room temperature for 1 hour, a sulfur-conjugated anti-HRP antibody was added. ECL signals were read with Sector Imager 2400 (Meso Scale discovery). Human C5 concentration was calculated from ECL signals in the standard curve using SOFTmax PRO (Molecular Devices). Figure 39 depicts the C5 plasma concentration time curve in C57BL6J mice.

Compared to the original Ab2, all bispecific antibodies tested with pI-increase in this study demonstrated rapid C5 clearance from plasma. Therefore, it is suggested that T74K / S77R, S76R / Q79K and Q37R amino acid substitutions in the light chain will also accelerate the elimination of C5-antibody immune complexes in vivo. In addition, the C5 elimination of 20L233-005 and 20L233-006 is faster than that of 20L233-009, which is consistent with in-tube angiography and BIACORE analysis. These results suggest that even in vivo, in vivo test tube systems using the InCell Analyzer 6000 to measure fluorescence intensity or locations in the BIACORE system described above that appear unlikely to contribute to plasma antigen clearance can still be used in a more sensitive in vivo The system finds contributors. These results also suggest that in order to estimate the clearance rate of antigens from plasma in vivo, the sensitivity of the in-tube system using the fluorescence intensity of the above-mentioned InCell Analyzer 6000 may be higher than that of the above-mentioned BIACORE system.

[Example 23]

Assessment of IgE clearance in plasma using pI-increased Fc variants

In order to increase clearance of human IgE or human C5, pH-dependent antibodies were used to evaluate the pI-increased substitutions in the Fc portion of the antibodies. The method of substituting an amino acid to the constant region of an antibody to increase pI is not particularly limited, and for example, it can be performed 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

Tested antibodies are collated in Table 38. The heavy chain, Ab1H-P1394m (SEQ ID NO: 307), was prepared by introducing a substitution of Q311K with increased pI to 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. Expresses all the heavy chain variants, and Ab1L as the light chain.

(23-2) Human FcγRIIb binding analysis using 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 and (21-2). The analysis results are shown in Table 38. Here, when the binding of the variant to hFcγRIIb is about 1.2 times or more compared to the original Ab1 binding to hFcγRIIb, it can be considered that the antibody binds to hFcγRIIb on the sensor wafer with a strong charge effect.

Among the variants with increased pI with a single amino acid substitution relative to the original Ab1, some variants such as P1398m, P1466m, P1482m, P1512m, P1513m and P1514m formed the highest antigen-antibody complexes for hFcγRIIb binding. D413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K or G402K single amino acid substitutions are speculated for The binding of hFcγRIIb on the sensor chip has a strong charge effect. It is therefore expected that the introduction of an increase in pI into the antibody constant region or Fc region results in accelerated uptake into one or more positions in the cell, which may include, for example, positions 311, 343, 384, 401, 402, or 413 according to Kabat numbering, According to EU numbering. The amino acid substitution introduced to such a position may be arginine or lysine.

(23-3) pI-increased cellular uptake of antibodies containing Fc region variants

In order to evaluate the intracellular uptake of the antigen-antibody complex formed by the antibodies described in Table 38, the cytographic analysis method similar to that described in Examples 21 and (21-3) was performed. The analysis results are shown in Table 38. Here, compared with the fluorescence intensity of the original Ab1, the variants of the antigen entering the cell have about 1.5 times or more fluorescence intensity, which is considered to have a strong charge effect on the antigen entering the cell.

Among the variants with increased pI relative to the original Ab1 with a single amino acid substitution, 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 when the antigen-antibody complex is taken up into cells. It is therefore expected that the introduction of increased pI modifications into the constant or Fc region of the antibody to cause uptake of the antigen-antibody complex into the cell faster or more positions may include, for example, positions 311, 315, 342, according to EU numbering, 343, 384, 401, or 413. The amino acid substitution introduced to such a position may be arginine or lysine.

(23-4) Evaluate clearance of human IgE in mouse co-injection model

Test mouse co-injection models of some pH-dependent antigen-binding anti-IgE antibodies (original Ab1, P1466m, P1469m, P1470m, P1480m, P1482m, P1512m, P1653m) to evaluate their ability to accelerate the clearance of IgE from plasma. The analysis method is implemented in a manner similar to that of Example 21, (21-4). Figure 40 depicts the plasma concentration time curve in C57BL6J mice.

After administration of a high pI variant with pH-dependent antigen binding (only a single amino acid substitution), the total plasma IgE concentration was lower than the original concentration except P1480m. P1480m, which showed weak efficacy in both test tubes and failed to accelerate the elimination of IgE. In mice treated with high pI variants without pH-dependent antigen binding, the total plasma IgE concentration was significantly higher than that of pH-dependent antigens. Combined high pI variant (data not shown). These results show that the cellular uptake of the antigen-antibody immune complex is increased by the introduction of modifications that increase pI. Antigens that are taken up into cells and complexed with pH-dependent antigen-binding antibodies can be efficiently released from the antibodies in the endosomes, thereby speeding up the elimination of IgE. These results suggest that even though the substitution position tested by the BIACORE system in the above-mentioned test tube does not seem to contribute significantly to the clearance of antigens from plasma in vivo, contributors can still be found using more sensitive in vivo systems. These results also suggest that in order to estimate the clearance of antigens from plasma in vivo, the sensitivity of measuring the fluorescence intensity in the test tube system using the above-mentioned InCell Analyzer 6000 may be higher than that of the BIACORE system in the test tube.

Reference Example 1

Expression vector for constructing amino-substituted IgG antibodies

Mutants were prepared using the method described in the attached instruction manual using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). A plastid fragment containing this mutant was inserted into an animal cell expression vector to construct a desired H chain and L chain expression vector. The nucleotide sequence of the obtained expression vector is determined by methods known in the art.

Reference Example 2

Performance and purification of IgG antibodies

Antibodies were expressed using the following methods. The HEK293H cell line (Invitrogen) from human embryonic kidney cancer cells was suspended in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Cells were seeded 10mL per dish for adherent cells (diameter 10cm; CORNING), a cell density of 5 to 6 x 10 ⁵ cells / mL, and cultured in a CO ₂ incubator _{(37 ℃, 5% CO 2} ) 1 days. It was then drawn to remove the medium and 6.9 mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plastids were introduced into cells by a lipofection method. 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 a supernatant. The antibody was purified from the obtained culture supernatant using a method known in the art using rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences). To determine the concentration of the purified antibody, the absorbance at 280 nm was measured using a spectrophotometer. According to the method described in Pace et al., Protein Science 4: 2411-2423 (1995), the antibody concentration was calculated from the measured value using the absorbance coefficient.

Reference Example 3

Preparation of soluble human IL-6 receptor

A recombinant soluble human IL-6 receptor as an antigen was prepared by the following method. A method known in the technical field is used to construct a CHO cell line that continuously expresses the soluble human IL-6 receptor. The soluble human IL-6 receptor is composed of amino acid sequences 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 by culturing this CHO cell lines appear. Soluble human IL-6 receptor was purified from the obtained culture supernatant of CHO cell line in two steps: Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. The fraction eluted as the main peak in the final step was used as the final purified product.

<110> Sino-Foreign Pharmaceutical Co., Ltd.

<120> IL-8 binding antibody and use thereof

<130> C1-A1502Y1P2

<150> JP 2015-185254

<151> 2015-09-18

<160> 332

<170> PatentIn version 3.5

<210> 1

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Vk1

<400> 1

<210> 2

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Vk2

<400> 2

<210> 3

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Vk3

<400> 3

<210> 4

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Vk4

<400> 4

<210> 5

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 6RL # 9-IgG1

<400> 5

<210> 6

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> 6KC4-1 # 85-IgG1

<400> 6

<210> 7

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Calcium binding domain

<400> 7

<210> 8

<211> 115

<212> PRT

<213> Homo sapiens

<400> 8

<210> 9

<211> 112

<212> PRT

<213> Homo sapiens

<400> 9

<210> 10

<211> 449

<212> PRT

<213> Homo sapiens

<400> 10

<210> 11

<211> 214

<212> PRT

<213> Homo sapiens

<400> 11

<210> 12

<211> 433

<212> PRT

<213> Homo sapiens

<400> 12

<210> 13

<211> 214

<212> PRT

<213> Homo sapiens

<400> 13

<210> 14

<211> 445

<212> PRT

<213> Homo sapiens

<400> 14

<210> 15

<211> 219

<212> PRT

<213> Homo sapiens

<400> 15

<210> 16

<211> 447

<212> PRT

<213> Homo sapiens

<400> 16

<210> 17

<211> 214

<212> PRT

<213> Homo sapiens

<400> 17

<210> 18

<211> 330

<212> PRT

<213> Homo sapiens

<400> 18

<210> 19

<211> 326

<212> PRT

<213> Homo sapiens

<400> 19

<210> 20

<211> 377

<212> PRT

<213> Homo sapiens

<400> 20

<210> 21

<211> 327

<212> PRT

<213> Homo sapiens

<400> 21

<210> 22

<211> 107

<212> PRT

<213> Homo sapiens

<400> 22

<210> 23

<211> 105

<212> PRT

<213> Homo sapiens

<400> 23

<210> 24

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-IgG1

<400> 24

<210> 25

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3 (High_pI) -IgG1

<400> 25

<210> 26

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-IgG1-F939

<400> 26

<210> 27

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3 (High_pI) -F939

<400> 27

<210> 28

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-IgG1-F1180

<400> 28

<210> 29

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3 (High_pI) -F1180

<400> 29

<210> 30

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-IgG1-F11

<400> 30

<210> 31

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3 (High_pI) -F11

<400> 31

<210> 32

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> VL3-CK

<400> 32

<210> 33

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> VL3 (High_pI) -CK

<400> 33

<210> 34

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H54

<400> 34

<210> 35

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> L28

<400> 35

<210> 36

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H (WT)

<400> 36

<210> 37

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> L (WT)

<400> 37

<210> 38

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H

<400> 38

<210> 39

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1L

<400> 39

<210> 40

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<223> Ab2H

<400> 40

<210> 41

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> Ab2L

<400> 41

<210> 42

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab3H

<400> 42

<210> 43

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> Ab3L

<400> 43

<210> 44

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> hGPC3-derived peptide

<400> 44

<210> 45

<400> 45

000

<210> 46

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-IgG1m

<400> 46

<210> 47

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-YTE

<400> 47

<210> 48

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-LS

<400> 48

<210> 49

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-N434H

<400> 49

<210> 50

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-F1847m

<400> 50

<210> 51

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-F1848m

<400> 51

<210> 52

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-F1886m

<400> 52

<210> 53

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-F1889m

<400> 53

<210> 54

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-F1927m

<400> 54

<210> 55

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-F1168m

<400> 55

<210> 56

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-IgG1

<400> 56

<210> 57

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> OHBL-CK

<400> 57

<210> 58

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-LS

<400> 58

<210> 59

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-N434A

<400> 59

<210> 60

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-F1847m

<400> 60

<210> 61

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-F1848m

<400> 61

<210> 62

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-F1886m

<400> 62

<210> 63

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-F1889m

<400> 63

<210> 64

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> OHBH-F1927m

<400> 64

<210> 65

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> VH3-F1718

<400> 65

<210> 66

<211> 77

<212> PRT

<213> Artificial sequence

<220>

<223> IL8 molecule

<400> 66

<210> 67

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1 for WS4

<400> 67

<210> 68

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2 for WS4

<400> 68

<210> 69

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3 for WS4

<400> 69

<210> 70

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1 for WS4

<400> 70

<210> 71

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2 for WS4

<400> 71

<210> 72

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3 for WS4

<400> 72

<210> 73

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2 for H1009

<400> 73

<210> 74

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3 for H1009

<400> 74

<210> 75

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2 for L395

<400> 75

<210> 76

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3 for L395

<400> 76

<210> 77

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of Hr9

<400> 77

<210> 78

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of H1009

<400> 78

<210> 79

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> L395 light chain variable domain

<400> 79

<210> 80

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H1009-F1974m

<400> 80

<210> 81

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H1009-F1886s

<400> 81

<210> 82

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> L395-k0MT

<400> 82

<210> 83

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> hWS4H-IgG1

<400> 83

<210> 84

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> hWS4L-k0MT

<400> 84

<210> 85

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Hr9-IgG1

<400> 85

<210> 86

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-IgG1

<400> 86

<210> 87

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> L63-k0MT

<400> 87

<210> 88

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> L118-k0MT

<400> 88

<210> 89

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H998-IgG1

<400> 89

<210> 90

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H553-IgG1

<400> 90

<210> 91

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H1004-IgG1m

<400> 91

<210> 92

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H1009-IgG1m

<400> 92

<210> 93

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> 1009-F1942m

<400> 93

<210> 94

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-IgG1m

<400> 94

<210> 95

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-F1168m

<400> 95

<210> 96

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-F1847m

<400> 96

<210> 97

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-F1848m

<400> 97

<210> 98

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-F1886m

<400> 98

<210> 99

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-F1889m

<400> 99

<210> 100

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H89-F1927m

<400> 100

<210> 101

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> H496-IgG1

<400> 101

<210> 102

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 102

<210> 103

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 103

<210> 104

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 104

<210> 105

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 105

<210> 106

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 106

<210> 107

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 107

<210> 108

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 108

<210> 109

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 109

<210> 110

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 110

<210> 111

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 111

<210> 112

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 112

<210> 113

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 113

<210> 114

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 114

<210> 115

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 115

<210> 116

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 116

<210> 117

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 117

<210> 118

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 118

<210> 119

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 119

<210> 120

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 120

<210> 121

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 121

<210> 122

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 122

<210> 123

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 123

<210> 124

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 124

<210> 125

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 125

<210> 126

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 126

<210> 127

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 127

<210> 128

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 128

<210> 129

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 129

<210> 130

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 130

<210> 131

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 131

<210> 132

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 132

<210> 133

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 133

<210> 134

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 134

<210> 135

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 135

<210> 136

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 136

<210> 137

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 137

<210> 138

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 138

<210> 139

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 139

<210> 140

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 140

<210> 141

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 141

<210> 142

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 142

<210> 143

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 143

<210> 144

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H003

<400> 144

<210> 145

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H005

<400> 145

<210> 146

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H010

<400> 146

<210> 147

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H012

<400> 147

<210> 148

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H013

<400> 148

<210> 149

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H014

<400> 149

<210> 150

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H016

<400> 150

<210> 151

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H018

<400> 151

<210> 152

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H026

<400> 152

<210> 153

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H027

<400> 153

<210> 154

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H028

<400> 154

<210> 155

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H029

<400> 155

<210> 156

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H030

<400> 156

<210> 157

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H031

<400> 157

<210> 158

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H032

<400> 158

<210> 159

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H034

<400> 159

<210> 160

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H035

<400> 160

<210> 161

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H039

<400> 161

<210> 162

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H041

<400> 162

<210> 163

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> H045

<400> 163

<210> 164

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L001

<400> 164

<210> 165

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L002

<400> 165

<210> 166

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L003

<400> 166

<210> 167

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L004

<400> 167

<210> 168

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L005

<400> 168

<210> 169

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L006

<400> 169

<210> 170

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L007

<400> 170

<210> 171

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L008

<400> 171

<210> 172

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L009

<400> 172

<210> 173

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L010

<400> 173

<210> 174

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L011

<400> 174

<210> 175

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L012

<400> 175

<210> 176

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L013

<400> 176

<210> 177

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L014

<400> 177

<210> 178

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L015

<400> 178

<210> 179

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L016

<400> 179

<210> 180

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L017

<400> 180

<210> 181

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L018

<400> 181

<210> 182

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L019

<400> 182

<210> 183

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L020

<400> 183

<210> 184

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L021

<400> 184

<210> 185

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L022

<400> 185

<210> 186

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L023

<400> 186

<210> 187

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L024

<400> 187

<210> 188

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L025

<400> 188

<210> 189

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L026

<400> 189

<210> 190

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L028

<400> 190

<210> 191

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L029

<400> 191

<210> 192

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L030

<400> 192

<210> 193

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L032

<400> 193

<210> 194

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L033

<400> 194

<210> 195

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L034

<400> 195

<210> 196

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L035

<400> 196

<210> 197

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L036

<400> 197

<210> 198

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L037

<400> 198

<210> 199

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L038

<400> 199

<210> 200

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L039

<400> 200

<210> 201

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L040

<400> 201

<210> 202

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L041

<400> 202

<210> 203

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L061

<400> 203

<210> 204

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> L062

<400> 204

<210> 205

<400> 205

000

<210> 206

<400> 206

000

<210> 207

<211> 1676

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant human C5

<400> 207

<210> 208

<211> 1676

<212> PRT

<213> Artificial sequence

<220>

<223> Malay C5

<400> 208

<210> 209

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0008H

<400> 209

<210> 210

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0008L

<400> 210

<210> 211

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0011H

<400> 211

<210> 212

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0011L

<400> 212

<210> 213

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0015H

<400> 213

<210> 214

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0015L

<400> 214

<210> 215

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0016H019

<400> 215

<210> 216

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0016L

<400> 216

<210> 217

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0017H

<400> 217

<210> 218

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0017L

<400> 218

<210> 219

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H

<400> 219

<210> 220

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018L

<400> 220

<210> 221

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0019H

<400> 221

<210> 222

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0019L

<400> 222

<210> 223

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H

<400> 223

<210> 224

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L

<400> 224

<210> 225

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0021H

<400> 225

<210> 226

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0021L

<400> 226

<210> 227

<211> 328

<212> PRT

<213> Artificial sequence

<220>

<223> G1dP1

<400> 227

<210> 228

<211> 328

<212> PRT

<213> Artificial sequence

<220>

<223> G1dN1

<400> 228

<210> 229

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-G1dP1_Optimization of 20 heavy chains

<400> 229

<210> 230

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-G1dN1_Optimization of 18 heavy chains

<400> 230

<210> 231

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-k0_Optimized 20 // 18 light chain

<400> 231

<210> 232

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-001-G1dP1

<400> 232

<210> 233

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-002-G1dP1

<400> 233

<210> 234

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-003-G1dP1

<400> 234

<210> 235

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-005-G1dP1

<400> 235

<210> 236

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-008-G1dP1

<400> 236

<210> 237

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-009-G1dP1

<400> 237

<210> 238

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-013-G1dP1

<400> 238

<210> 239

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-018-G1dP1

<400> 239

<210> 240

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-019-G1dP1

<400> 240

<210> 241

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-020-G1dP1

<400> 241

<210> 242

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-021-G1dP1

<400> 242

<210> 243

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-022-G1dP1

<400> 243

<210> 244

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-023-G1dP1

<400> 244

<210> 245

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-025-G1dP1

<400> 245

<210> 246

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-026-G1dP1

<400> 246

<210> 247

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-032-GldP1

<400> 247

<210> 248

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-035-GldP1

<400> 248

<210> 249

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-036-GldP1

<400> 249

<210> 250

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020H0261-037-GldP1

<400> 250

<210> 251

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-002-GldN1

<400> 251

<210> 252

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-003-GldN1

<400> 252

<210> 253

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-005-GldN1

<400> 253

<210> 254

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-008-GldN1

<400> 254

<210> 255

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-009-GldN1

<400> 255

<210> 256

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-013-GldN1

<400> 256

<210> 257

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-014-GldN1

<400> 257

<210> 258

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-016-GldN1

<400> 258

<210> 259

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-018-GldN1

<400> 259

<210> 260

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-019-GldN1

<400> 260

<210> 261

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-020-GldN1

<400> 261

<210> 262

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-021-GldN1

<400> 262

<210> 263

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-022-GldN1

<400> 263

<210> 264

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-023-GldN1

<400> 264

<210> 265

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-024-GldN1

<400> 265

<210> 266

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-025-GldN1

<400> 266

<210> 267

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-026-GldN1

<400> 267

<210> 268

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-027-GldN1

<400> 268

<210> 269

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-032-GldN1

<400> 269

<210> 270

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0018H0012-037-GldN1

<400> 270

<210> 271

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-001-k0

<400> 271

<210> 272

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-002-k0

<400> 272

<210> 273

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-003-k0

<400> 273

<210> 274

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-004-k0

<400> 274

<210> 275

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-005-k0

<400> 275

<210> 276

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-006-k0

<400> 276

<210> 277

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-007-k0

<400> 277

<210> 278

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-008-k0

<400> 278

<210> 279

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-009-k0

<400> 279

<210> 280

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-010-k0

<400> 280

<210> 281

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-011-k0

<400> 281

<210> 282

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-012-k0

<400> 282

<210> 283

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-013-k0

<400> 283

<210> 284

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-016-k0

<400> 284

<210> 285

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-017-k0

<400> 285

<210> 286

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-018-k0

<400> 286

<210> 287

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-019-k0

<400> 287

<210> 288

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-021-k0

<400> 288

<210> 289

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-022-k0

<400> 289

<210> 290

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-023-k0

<400> 290

<210> 291

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-024-k0

<400> 291

<210> 292

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-025-k0

<400> 292

<210> 293

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-027-k0

<400> 293

<210> 294

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-028-k0

<400> 294

<210> 295

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-029-k0

<400> 295

<210> 296

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-031-k0

<400> 296

<210> 297

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-032-k0

<400> 297

<210> 298

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-034-k0

<400> 298

<210> 299

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-036-k0

<400> 299

<210> 300

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-037-k0

<400> 300

<210> 301

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-038-k0

<400> 301

<210> 302

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-040-k0

<400> 302

<210> 303

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-041-k0

<400> 303

<210> 304

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-043-k0

<400> 304

<210> 305

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-044-k0

<400> 305

<210> 306

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> CFP0020L233-045-k0

<400> 306

<210> 307

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1394m

<400> 307

<210> 308

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1398m

<400> 308

<210> 309

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1466m

<400> 309

<210> 310

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1468m

<400> 310

<210> 311

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1469m

<400> 311

<210> 312

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1470m

<400> 312

<210> 313

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1471m

<400> 313

<210> 314

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1480m

<400> 314

<210> 315

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1481m

<400> 315

<210> 316

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1482m

<400> 316

<210> 317

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1483m

<400> 317

<210> 318

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1512m

<400> 318

<210> 319

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1513m

<400> 319

<210> 320

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1514m

<400> 320

<210> 321

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1515m

<400> 321

<210> 322

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ab1H-P1653m

<400> 322

<210> 323

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> F8M-F1847mv1

<400> 323

<210> 324

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<223> F8M-F1847mv2

<400> 324

<210> 325

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> F8ML

<400> 325

<210> 326

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> F8M-F1868mv1

<400> 326

<210> 327

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<223> F8M-F1868mv1

<400> 327

<210> 328

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> F8M-F1927mv1

<400> 328

<210> 329

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<223> F8M-F1927mv2

<400> 329

<210> 330

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> Q499-z121

<400> 330

<210> 331

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<223> J327-z119

<400> 331

<210> 332

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> L404-k

<400> 332

Claims (14)

An isolated anti-IL-8 antibody that binds to human IL-8. The anti-IL-8 antibody includes (a) HVR-H1, including the amino acid sequence of SEQ ID NO: 67; (b) HVR-H2 , Including the amino acid sequence of SEQ ID NO: 73; (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 ; (E) HVR-L2, including the amino acid sequence of SEQ ID NO: 75; and (f) HVR-L3, including the amino acid sequence of SEQ ID NO: 76, wherein the anti-IL-8 antibody is at pH 5. The dissociation constant ratio for human IL-8 between 8 and pH7.4, that is, [KD (pH5.8) / KD (pH7.4)], is 30 or more. The anti-IL-8 antibody according to item 1 of the patent application scope, wherein the anti-IL-8 antibody is a humanized anti-IL-8 antibody. The anti-IL-8 antibody according to item 1 of the patent application scope, wherein the anti-IL-8 antibody is a humanized anti-IL-8 antibody derived from a mouse antibody. An anti-IL-8 antibody comprising the heavy chain variable domain of SEQ ID NO: 78 and the light chain variable domain of SEQ ID NO: 79, and is a humanized IgG antibody. An anti-IL-8 antibody includes a heavy chain including the amino acid sequence of SEQ ID NO: 80 or SEQ ID NO: 81, and a light chain including the amino acid sequence of SEQ ID NO: 82. An isolated nucleic acid encoding an anti-IL-8 antibody as described in any one of claims 1 to 5 of the patent application scope. A vector comprising a nucleic acid as described in item 6 of the patent application. A host cell comprising a vector as described in item 7 of the patent application. A method for manufacturing an anti-IL-8 antibody, comprising culturing a host cell as described in item 8 of the patent application scope. The method according to item 9 of the patent application scope further comprises isolating the antibody from the host cell culture fluid. A pharmaceutical composition includes the anti-IL-8 antibody as described in any one of claims 1 to 5 and a pharmaceutically acceptable carrier. The use of the anti-IL-8 antibody according to any one of claims 1 to 5 of the scope of patent application, which is used for manufacturing a pharmaceutical composition for treating a condition in which an excessive amount of IL-8 exists. The use of the anti-IL-8 antibody according to any one of claims 1 to 5 of the scope of patent application, which is used for manufacturing a pharmaceutical composition for inhibiting angiogenesis. The use of the anti-IL-8 antibody according to any one of claims 1 to 5 of the scope of the patent application, which is used for manufacturing a pharmaceutical composition for inhibiting neutrophil migration promotion.