RU2799423C1 - Method of changing plasma retention and immunogenicity of antigen-binding molecules - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: different variants of a method for improving the pharmacokinetics of an antigen-binding molecule are proposed, where the method includes modification of the Fc region of the antigen-binding molecule. The antigen-binding molecule is a human IgG antibody containing an antigen-binding domain whose antigen-binding activity varies with pH and an Fc region having an FcRn binding activity at pH 7.4.

EFFECT: invention has demonstrated that modification of the Fc region of an antigen-binding molecule to an Fc region that does not form in the neutral pH range a heterotetrameric complex containing two FcRn molecules and an active Fcγ-receptor, improved the pharmacokinetics of the antigen-binding molecule and reduced the immune response to the antigen-binding molecule.

5 cl, 65 dwg, 84 tbl, 14 ex

Description

Technical field

The present invention relates to methods for improving the pharmacokinetics of an antigen-binding molecule in animals administered with the molecule, and to methods for reducing the immune response to an antigen-binding molecule by modifying the Fc region of the antigen-binding molecule, which has an antigen-binding domain whose antigen-binding activity varies with ion concentration, and Fc -region that has FcRn binding activity in the neutral pH range. The present invention also relates to antigen-binding molecules that exhibit improved pharmacokinetics or reduced immune response in animals administered with these molecules. Moreover, the present invention relates to methods for producing antigen-binding molecules and to pharmaceutical compositions containing such an antigen-binding molecule as an active ingredient.

State of the art

Antibodies are attracting attention as drugs because they are highly stable in plasma and have few side effects. At present, a number of such IgG-type pharmaceuticals are commercially available, and many antibody-based drugs are being developed today (Non-Patent Documents 1 and 2). Meanwhile, various methods applicable to the second generation of antibody-based drugs have been described, including methods that enhance effector function, antigen-binding capacity, pharmacokinetics, and stability, and methods that reduce the risk of developing immunogenicity (Non-Patent Document 3). As a rule, the required dose of drug based antibodies is very high. This, in turn, has led to problems such as high production costs as well as difficulties in obtaining subcutaneous formulations. In theory, the dose of an antibody-based pharmaceutical can be reduced by improving the pharmacokinetics of the antibody or by increasing the affinity between antibodies and antigens.

The literature describes methods for improving the pharmacokinetics of antibodies using artificial amino acid substitutions in the constant regions (non-patent documents 4 and 5). Similarly, affinity maturation is described as a method for improving antigen-binding ability or antigen neutralization activity (non-patent document b). This method makes it possible to increase the antigen-binding capacity by introducing an amino acid mutation in the CDR region of the variable region, and the like. Increasing the antigen-binding capacity provides an improvement in in vitro biological activity or allows a dose reduction, and further provides an improved in vivo efficacy (Non-Patent Document 7).

The ability of a single antibody molecule to neutralize an antigen depends on its affinity. By increasing the affinity, the antigen can be neutralized with less antibody. Various methods can be used to increase the affinity of an antibody (Non-Patent Document 6). Further, if affinity could be made infinite by covalently binding an antibody to an antigen, one antibody molecule could neutralize one antigen molecule (a bivalent antibody could neutralize two antigen molecules). However, the neutralization stoichiometry of one antibody against one antigen (one bivalent antibody against two antigens) is a limiting factor for the prior methods, and thus it is not possible to completely neutralize an antigen with less antibody than antigen. In other words, the affinity enhancing effect has a limiting value (Non-Patent Document 9). To increase the period of action of the neutralizing effect of a neutralizing antibody over a certain period, the antibody must be administered at a dose higher than the amount of antigen produced in the body during the same period. Thus, in the case of improving only the pharmacokinetics of antibodies or affinity maturation technology, as described above, there is a limitation in terms of reducing the required dose of the antibody. Thus, in order to maintain the effect of antibodies in neutralizing an antigen for a given period of time with antibodies in an amount less than the amount of antigen, a single antibody must neutralize a plurality of antigens.

Recently, an antibody that binds to an antigen in a pH-dependent manner has been described as a novel method for achieving the above-described object (Patent Document 1). Antibodies with pH dependent antigen binding, which bind strongly to antigen under neutral plasma conditions and dissociate from antigen under acidic conditions in the endosome, can dissociate from antigen in the endosome. When an antibody with pH-dependent antigen binding dissociates from the antigen and is recirculated to plasma via the FcRn, it can re-bind to another antibody. Thus, an antibody with pH dependent antigen binding can repeatedly bind to multiple antigens.

In addition, the retention time of antigen in plasma is very short compared to the retention time of antibodies recirculating to plasma by binding to FcRn. If an antibody with such a long plasma retention time binds an antigen, the plasma retention time of the antigen-antibody complex is extended to the plasma retention time of the antibody. Thus, due to binding to the antibody, the retention time of the antigen increases, and thus the plasma concentration of the antigen increases.

The IgG antibody has a longer plasma retention time as a result of FcRn binding. Binding between IgG and FcRn is observed only under acidic conditions (pH 6.0). In contrast, binding is practically not detected under neutral conditions (pH 7.4). The IgG antibody is taken up by cells nonspecifically. The antibody returns to the cell surface by binding to endosomal FcRn under the acidic conditions of endosomes and then dissociates from the FcRn under neutral plasma conditions. When FcRn binding under acidic conditions is lost due to the introduction of mutations in the Fc region of IgG, the absence of antibody recycled to plasma from the endosome significantly reduces the plasma retention time of the antibody. The described method for increasing the plasma retention time of an IgG antibody is to enhance FcRn binding under acidic conditions. The Fc region of the IgG antibody introduces amino acid mutations that increase FcRn binding under acidic conditions. This increases the efficiency of recycling to plasma from the endosome, resulting in increased plasma retention time. An important requirement for amino acid substitution is that there is no enhancement of FcRn binding under neutral conditions. If an IgG antibody binds to FcRn under neutral conditions, the antibody returns to the cell surface by binding to FcRn under acidic endosome conditions and does not dissociate from FcRn under neutral plasma conditions. In this case, plasma retention, on the other hand, is lost because the IgG antibody is not recycled into the plasma. For example, an IgG1 antibody modified by making amino acid substitutions so that the resulting antibody is capable of binding to mouse FcRn under neutral conditions (pH 7.4) has been described to exhibit very low plasma retention when administered to mice (Non-Patent Document 10) . Moreover, the IgG1 antibody was modified by introducing amino acid substitutions so that the resulting antibody showed increased binding to human FcRn under acidic conditions (pH 6.0) and at the same time became able to bind to human FcRn under neutral conditions (pH 7.4) (non-patent document 10, 11 and 12). The resulting antibody has been described as showing neither improvement nor change in plasma retention when administered to cynomolgus monkeys. Thus, antibody engineering technology to improve antibody function has focused only on increasing the plasma retention time of the antibody by enhancing binding to human FcRn under acidic conditions without enhancing it under neutral conditions (pH 7.4). To date, no benefit has been described for increasing binding to human FcRn under neutral conditions (pH 7.4) by introducing amino acid substitutions in the Fc region of an IgG antibody. Even if the affinity of the antibody for the antigen is increased, the elimination of the antigen from the plasma cannot be enhanced. The pH-dependent antigen-binding antibodies described above have been reported to be more effective as a method of enhancing antigen elimination from plasma compared to typical antibodies (Patent Document 1).

Thus, a single antibody with pH-dependent antigen binding binds to multiple antigens and is able to facilitate elimination of the antigen from plasma compared to typical antibodies. Thus, antibodies with pH-dependent antigen binding have effects not achieved by typical antibodies. However, to date, there has been no description of antibody engineering methods to further improve the ability of antibodies with pH dependent antigen binding to repeatedly bind antigens and provide increased antigen clearance from plasma.

Meanwhile, the immunogenicity of antibody-based pharmaceuticals is very important in terms of plasma retention, efficacy and safety when administered to a human.

It has been described that if antibodies are produced against administered antibody-based pharmaceuticals in the human body, they cause undesirable effects such as accelerating the elimination of antibody-based pharmaceuticals from plasma, reducing efficacy and inducing a hypersensitivity reaction, and affecting safety (Non-Patent Document 13 ).

First of all, when considering the immunogenicity of pharmaceuticals based on antibodies, it is necessary to understand the functions of natural antibodies in vivo. First, most antibody-based pharmaceuticals are antibodies that belong to the IgG class, and Fcγ receptors (hereinafter also referred to as FcγR) are known to exist as Fc receptors that function by binding to the Fc region of IgG antibodies. FcγRs are expressed on the cell membranes of dendritic cells, NK cells, macrophages, neutrophils, adipocytes, and others; and they are known to transmit activating or inhibitory intracellular signals to immune cells upon binding to the IgG Fc region. For the human FcγR protein family, the FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms are known, and their allotypes are also described (Non-Patent Document 14). Two allotypes have been described for human FcγRIIa: Arg (hFcγRIIa(R)) and His (hFcγRIIa(H)) at position 131. Moreover, two allotypes have been described for human FcγRIIIa: Val (hFcγRIIIa (V)) and Phe (hFcγRIIIa(F )) at position 158. Meanwhile, for the mouse FcγR protein family, FcγRI, FcγRIIb, FcγRIII, and FcγRIV are described (Non-Patent Document 15).

Human FcγRs include the activating receptors FcγRIa, FcγRIIa, FcγRIIIa and FcγRIIIb, and the inhibitory receptor FcγRIIb. Similarly, mouse FcγRs include the activating receptors FcγRI, FcγRIII and FcγRIV, and the inhibitory receptor FcγRIIb.

When an activating FcγR binds to the immune complex, it phosphorylates immunoreceptor tyrosine activating motifs (ITAMs) contained in the intracellular domain or common γ-chain FcR (interaction partner), activates the SYK signal transmitter, and triggers an inflammatory immune response by initiating activation of the signal transduction cascade (non-patent document 15).

It has been demonstrated that certain amino acid residues in the hinge region and the CH2 domain of an antibody, and a sugar chain attached to Asn at position 297 of the CH2 domain according to the EU numbering system (Non-Patent Documents 15- 17). Focusing on antibodies mutated at the regions described above, mutants with different FcγR binding properties were investigated, and Fc region mutants were obtained that have a higher affinity for activating FcγRs (Patent Documents 2-5).

Meanwhile, FcγRIIb, which is an inhibitory FcγR, is the only FcγR expressed on B cells (Non-Patent Document 18). Interaction of the Fc region of an antibody with FcγRIIb has been described to suppress the primary immune response of B cells (Non-Patent Document 19). Moreover, it is described that when FcγRIIb on B cells and B cell receptor (BCR) bind through the immune complex, B cell activation is suppressed in blood and antibody production by B cells is suppressed (Non-Patent Document 20). This immunosuppressive signaling mediated by BCR and FcγRIIb requires an immunoreceptor tyrosine inhibitory motif (ITIM) contained in the intracellular domain of FcγRIIb (Non-Patent Documents 21 and 22). This immunosuppressive action is caused by ITIM phosphorylation. As a result of phosphorylation, SH2-containing inositol polyphosphate 5-phosphatase (SHIP) is recruited, transmission of other activating FcγR signaling cascades is inhibited, and the inflammatory immune response is suppressed (Non-Patent Document 23).

Due to this property, FcγRIIb is promising as a means to directly reduce the immunogenicity of antibody-based pharmaceuticals. Exendin-4 (Ex4) is a foreign protein to mice, however, even when an IgG1 (Ex4/Fc) fusion molecule is administered to mice, no antibodies are produced. Meanwhile, anti-Ex4 antibodies are produced by introducing a molecule (Ex4/Fc mut) which is obtained by modifying Ex4/Fc so that it does not bind to FcγRIIb on B cells (Non-Patent Document 24). This result indicates that Ex4/Fc binds FcγRIIb on B cells and inhibits the production of mouse anti-Ex4 antibodies on B cells.

Moreover, FcγRIIb is also expressed on dendritic cells, macrophages, activated neutrophils, mast cells and basophils. FcγRIIb inhibits activating FcγR functions such as phagocytosis and release of inflammatory cytokines in these cells and suppresses inflammatory immune responses (Non-Patent Document 25).

The importance of the immunosuppressive functions of FcγRIIb has been established to date by studies using FcγRIIb knockout mice. There are reports that in FcγRIIb knockout mice, humoral immunity is not properly regulated (Non-Patent Document 26), increased susceptibility to collagen-induced arthritis (CIA) (Non-Patent Document 27), has lupus-like symptoms, and has Goodpasture's syndrome-like symptoms (Non-Patent Document 28).

Moreover, it has been described that inadequate regulation of FcγRIIb is associated with human autoimmune diseases. For example, the relationship between genetic polymorphisms in the transmembrane region and promoter region of FcγRIIb and the incidence of systemic lupus erythematosus (SLE) has been described (Non-Patent Documents 29, 30, 31, 32 and 33), and a decrease in FcγRIIb expression on the surface of B cells in patients with SLE (non-patent document 34 and 35).

Based on mouse models and clinical data itself, FcγRIIb is believed to play a role in the control of autoimmune diseases and inflammatory diseases, mainly through the involvement of B cells, and is a promising target molecule for the control of autoimmune diseases and inflammatory diseases.

It is known that IgG1, commonly used as a commercially available antibody-based pharmaceutical, not only binds to FcγRIIb, but also strongly binds to an activating FcγR (Non-Patent Document 36). It is possible to develop pharmaceutical preparations based on antibodies having better immunosuppressive properties compared to the immunosuppressive properties of IgG1, using an Fc region with increased FcγRIIb binding, or increased selectivity of FcγRIIb binding compared to activating FcγR. For example, it has been suggested that the use of an antibody having a variable region that binds to BCR and an Fc with enhanced FcγRIIb binding can inhibit B cell activation (Non-Patent Document 37).

However, FcγRIIb shares 93% sequence identity in the extracellular region with the extracellular region of FcγRIIa, which is one of the activating FcγRs, and they are structurally very similar. There are FcγRIIa, H-type and R-type allotypes in which the amino acid at position 131 is His (H type) or Arg (R type), and each even reacts differently with antibodies (Non-Patent Document 38). Thus, in order to obtain an Fc region that specifically binds to FcγRIIb, the most difficult problem may be to communicate to the Fc region of an antibody the property of selectively increased FcγRIIb binding activity, which involves reducing or not increasing binding activity to each FcγRIIa allotype while increasing FcγRIIb binding activity. .

A case of enhancing the binding specificity of FcγRIIb by introducing amino acid mutations in the Fc region has been described (Non-Patent Document 39). According to this document, the mutants were designed so that, compared to IgG1, they retained their binding to FcγRIIb to a greater extent than to FcγRIIa, which has two polymorphic forms. However, compared to native IgG1, all of the mutants described in this document as having increased specificity for FcγRIIb were found to have reduced FcγRIIb binding. Thus, it is considered difficult for mutants to induce an FcγRIIb-mediated immunosuppressive response more than IgG1.

There is also a report on enhancing FcγRIIb binding (Non-Patent Document 37). In this document, FcγRIIb binding was increased by introducing mutations such as S267E/L328F, G236D/S267E and S239D/S267E to the Fc region of the antibody. Among them, the antibody introduced with the S267E/L328F mutation bound more strongly to FcγRIIb. This mutant has been shown to retain binding to FcγRIa and type H FcγRIIa at levels comparable to native IgG1. Even if FcγRIIb binding was enhanced relative to IgG1, only increased FcγRIIa binding but not increased FcγRIIb binding is expected to have an effect on cells such as platelets that express FcγRIIa but not FcγRIIb (Non-Patent Document 25). For example, it has been described that platelets are activated through an FcγRIIa-dependent mechanism in systemic erythematosis, and platelet activation correlates with severity (Non-Patent Document 40). According to another report, the mutation described above increased binding to FcγRIIa type R by several hundred times, to the same extent as binding to FcγRIIb, and did not increase binding specificity to FcγRIIb compared to FcγRIIa type R (patent document 17). Moreover, in cell types that express both FcγRIIa and FcγRIIb, such as dendritic cells and macrophages, binding selectivity for FcγRIIb relative to FcγRIIa is required for inhibitory signaling; however, such selectivity could not be achieved for type R.

FcγRIIa type H and type R occur with almost equal frequency among Caucasians and African Americans (non-patent documents 41 and 42). Thus, there are certain limitations on the use of antibodies with increased binding to FcγRIIa type R for the treatment of autoimmune diseases. Even if FcγRIIb binding was enhanced relative to activating FcγRs, the fact that binding to any of the FcγRIIa polymorphs is enhanced cannot be overlooked from the point of view of their use as a drug for autoimmune diseases.

When antibody-based pharmaceutical preparations targeting FcγRIIb are prepared for the treatment of autoimmune diseases, it is important that Fc-mediated binding activity to any of the FcγRIIa polymorphs is not increased or preferably decreased, and that FcγRIIb binding activity is enhanced relative to native IgG. However, there have been no reports of mutants having the properties described above, and thus there is a need to develop such mutants.

The prior art documents of the present invention are presented below.

Documents of the prior art

[Patent Documents]

[Patent document 1] WO 2009/125825

[Patent document 2] WO 2000/042072

[Patent document 3] WO 2006/019447

[Patent Document 4] WO 2004/0992 49

[Patent document 5] WO 2004/029207

[Non-Patent Documents]

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[Non-Patent Document 2] Pavlou AK, Belsey MJ., The therapeutic antibosies market to 2008., Eur J Pharm Biopharm. (2005) 59(3), 389-396

[Non-Patent Document 3] Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. (2005) 20(1), 17-29

[Non-Patent Document 4] Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J. Immunol. (2006) 176(1), 346-356

[Non-Patent Document 5] Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat. Biotechnol. (1997) 15(7), 637-640

[Non-Patent Document 6] Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for greatly improving the affinity antibody by using combinatorial libraries., Proc. Natl. Acad. sci. U.S.A. (2005) 102 (24), 8466-8471

[Non-Patent Document 7] Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA., Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory Syncytial Virus Infection in the Upper and Lower Respiratory Tract., J. Mol. Biol. (2007) 368, 652-665

[Non-Patent Document 8] Hanson CV, Nishiyama Y, Paul S., Catalytic antibodies and their applications., Curr Opin Biotechnol. (2005) 16(6), 631-636

[Non-Patent Document 9] Rathanaswami P, Roalstad S, Roskos L, Su QJ, Lackie S, Babcook J., Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukine-8., Biochem. Biophys. Res. commun. (2005) 334(4), 1004-1013

[Non-Patent Document 10] Dall'Acqua WF, Woods RM, Ward ES, Palaszynski SR, Patel NK, Brewah YA, Wu H, Kiener PA, Langermann S., Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences ., J. Immunol. (2002) 169(9), 5171-5180

[Non-Patent Document 11] Yeung YA, Leabman MK, Marvin JS, Qiu J, Adams CW, Lien S, Starovasnik MA, Lowman HB., Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates., J. Immunol. (2009) 182 (12), 7663-7671

[Non-Patent Document 12] Datta-Mannan A, Witcher DR, Tang Y, Watkins J, Wroblewski VJ., Monoclonal antibody clearance.

Impact of modulating the interaction of IgG with the neonatal Fc receptor., J. Biol. Chem. (2007) 282(3), 1709-1717

[Non-Patent Document 13] Niebecker R, Kloft C., Safety of therapeutic monoclonal antibodies., Curr. drug safe. (2010) 5(4), 275-286

[Non-Patent Document 14] Jefferis R, Lund J., Interaction sites on human IgG-Fc for FcgammaR: current models., Immunol. Lett. (2002) 82, 57-65

[Non-Patent Document 15] Nimmerjahn F, Ravetch JV., Fcgamma receptors as regulators of immune responses., Nat. Rev. Immunol. (2008) 8(1), 34-47

[Non-Patent Document 16] M. Clark, Antibody Engineering IgG Effector Mechanisms., Chemical Immunology (1997), 65, 88-110

[Non-Patent Document 17] Greenwood J, Clark M, Waldmann H., Structural motifs involved in human IgG antibody effector functions., Eur. J. Immunol. (1993) 23, 1098-1104

[Non-Patent Document 18] Amigorena S, Bonnerot C, Choquet D, Fridman WH, Teillaud JL., Fc gamma RII expression in resting and activated in lymphocytes., Eur. J. Immunol. (1989) 19, 1379-1385

[Non-Patent Document 19] Nicholas R, Sinclair SC, Regulation of the immune response. I. Reduction in ability of specific antibody to inhibit long-lasting IgG immunological priming after removal of the Fc fragment., J. Exp. Med. (1969) 129, 1183-1201

[Non-Patent Document 20] Heyman B., Feedback regulation by IgG antibodies., Immunol. Lett. (2003) 88, 157-161

[Non-Patent Document 21] S Amigorena, C Bonnerot, Drake, JR, D Choquet, W Hunziker, JG Guillet, P Webster, C Sautes, I Mellman, and WH Fridman, Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. , Science (1992) 256, 1808-1812

[Non-Patent Document 22] Muta, T., Kurosaki, T., Misulovin, Z., Sanchez, M., Nussenzweig, M.C., and Ravetch, J.V., A 13-amino-acid motif in the cytoplasmic domain of FcγRIIB modulates B- cell receptor signaling., Nature (1994) 368, 70-73

[Non-Patent Document 23] Ravetch JV, Lanier LL., Immune inhibitory receptors., Science (2000) 290, 84-89

[Non-Patent Document 24] Liang Y, Qiu H, Glinka Y, Lazarus AH, Ni H, Prud'homme GJ, Wang Q., Immunity against a therapeutic xenoprotein/Fc construct delivered by gene transfer is reduced through binding to the inhibitory receptor FcγRIIb ., J. Gene Med. (2011) doi:10.1002/jgm.1598

[Non-Patent Document 25] Smith KG, Clatworthy MR., FcgammaRIIB in autoimmunity and infection: evolutionary and therapeutic implications., Nat. Rev. Immunol. (2010) 10, 328-343

J, Mattsson R, Verbeek JS, Heyman В., IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice., J. Immunol. (1999) 163, 618-622[Non-Patent Document 26] Wernersson S, Karlsson MC,

J, Mattsson R, Verbeek JS, Heyman B., IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice., J. Immunol. (1999) 163, 618-622

[Non-Patent Document 27] Joachim L. Schultze, Sabine Michalak, Joel Lowne, Adam Wong, Maria H. Gilleece, John G. Gribben, and Lee M. Nadler, Human Non-Germinal Center In Cell Interleukin (IL)-12 Production Is Primarily Regulated by T Cell Signals CD40 Ligand, Interferon γ, and IL-10: Role of B Cells in the Maintenance of T Cell Responses., J. Exp. Med. (1999) 189, 187-194

[Non-Patent Document 28] Nakamura, A., Yuasa, T., Ujike, A., Ono, M., Nukiwa, T., Ravetch, J.V., Takai, T., Fey receptor IIB-deficient mice develop Goodpasture's syndrome upon immunization with type IV collagen: A novel murine model for autoimmune glomerular basement membrane disease., J. Exp. Med. (2000) 191, 899-906

[Non-Patent Document 29] Blank MC, Stefanescu RN, Masuda E, Marti F, King PD, Redecha PB, Wurzburger RJ, Peterson MG, Tanaka S, Pricop L., Decreased transcription of the human FCGR2B gene mediated by the -343 G/ C promoter polymorphism and association with systemic lupus erythematosus., Hum. Genet. (2005) 117, 220-227

[Non-Patent Document 30] Olferiev M, Masuda E, Tanaka S, Blank MC, Pricop L., The Role of Activating Protein 1 in the Transcriptional Regulation of the Human FCGR2B Promoter Mediated by the -343 G ->C Polymorphism Associated with Systemic Lupus Erythematosus., J. Biol. Chem. (2007) 282, 1738-1746

[Non-Patent Document 31] Lv J, Yang Y, Zhou X, Yu L, Li R, Hou P, Zhang H., FCGR3B copy number variation is not associated with lupus nephritis in a Chinese population., Arthritis Rheum. (2006) 54, 3908-3917

[Non-Patent Document 32] Floto RA, Clatworthy MR, Heilbronn KR, Rosner DR, MacAry PA, Rankin A, Lehner PJ, Ouwehand WH, Allen JM, Watkins NA, Smith KG., Loss of function of a lupus-associated FcgammaRIIb polymorphism through exclusion from lipid rafts., Nat. Med. (2005) 11, 1056-1058

[Non-Patent Document 33] Li DH, Tung JW, Tarner IH, Snow AL, Yukinari T, Ngernmaneepothong R, Martinez OM, Parnes JR., CD72 Down-Models BCR-Induced Signal Transduction and Diminishes Survival in Primary Mature B Lymphocytes., J Immunol. (2006) 176, 5321-5328

[Non-Patent Document 34] Mackay M, Stanevsky A, Wang T, Aranow C, Li M, Koenig S, Ravetch JV, Diamond B., Selective dysregulation of the FcgammallB receptor on memory B cells in SLE., J. Exp. Med. (2006) 203, 2157-2164

[Non-Patent Document 35] Su K, Yang H, Li X, Li X, Gibson AW, Cafardi JM, Zhou T, Edberg JC, Kimberly RP., Expression profile of FcgammaRIIb on leukocytes and its dysregulation in systemic lupus erythematosus., J. Immunol. (2007) 178, 3272-3280

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M., Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses., Blood (2009) 113, 3716-

[Non-Patent Document 37] Chu SY, Vostiar I, Karki S, Moore GL, Lazar GA, Pong E, Joyce PF, Szymkowski DE, Desjarlais JR., Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies., Mol. Immunol. (2008) 45, 3926-3933

[Non-Patent Document 38] Warmerdam PA, van de Winkel JG, Gosselin EJ, Capel PJ., Molecular basis for a polymorphism of human Fc gamma receptor II (CD32), J. Exp. Med. (1990) 172, 19-25

[Non-Patent Document 39] Armour, KL, van de Winkel, JG, Williamson, LM, Clark, MR., Differential binding to human FcgammaRIIa and FcgammaRIIb receptors by human IgG wildtype and mutant antibodies., Mol. Immunol. (2003) 40, 585-593

[Non-Patent Document 40] Science Translational Medicine (2010) Vol. 2, Issue 47, p. 47ra63

[Non-Patent Document 41] Salmon JE, Millard S, Schachter LA, Arnett FC, Glnzler EM, Gourley MF, Ramsey-Goldman R, Peterson MG, Kimberly RP., Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans. , J. Clin. Invest. (1996) 97, 1348-1354

[Non-Patent Document 42] Manger K, Repp R, Spriewald BM, Rascu A, Geiger A, Wassmuth R, Westerdaal NA, Wentz B, Manger B, Kalden JR, van de Winkel JG., Fcgamma receptor IIa polymorphism in Caucasian patients with systemic lupus erythematosus: association with clinical symptoms., Arthritis Rheum. (1998) 41, 1181-1189

[Non-Patent Document 43] Qiao SW, Kobayashi K, Johansen FE, Sollid LM, Andersen JT, Milford E, Roopenian DC, Lencer WI, Blumberg RS., Dependence of antibody-mediated presentation of antigen on FcRn., Proc. Natl. Acad. sci. (2008) 105 (27) 9337-9342

[Non-Patent Document 44] Mi W, Wanjie S, Lo ST, Gan Z, Pickl-Herk B, Ober RJ, Ward ES., Targeting the neonatal fc receptor for antigen delivery using engineered fc fragments., J. Immunol. (2008) 181(11), 7550-7561.

The essence of the invention

[Problems solved by the invention]

In addition to the involvement of the activating FcγR described above, the so-called antigen presentation mechanism is very important as a factor in inducing an immune response to administered antibody-based pharmaceuticals. Antigen presentation refers to an immunological mechanism in which, after intracellular internalization and degradation of foreign antigens such as bacteria and endogenous antigens, antigen presenting cells such as macrophages and dendritic cells present parts of the antigens on the cell surface. The presented antigens are recognized by T cells and other cells and activate both cellular and humoral immunity.

The antigen presentation cascade by dendritic cells involves the internalization of the antigen as an immune complex (the complex formed between a polyvalent antibody and antigen) into cells, degradation in the lysosome, and presentation of the resulting peptides derived from the antigen by class II MHC molecules. FcRn plays an important role in this cascade; and it has been described that when FcRn-deficient dendritic cells or immune complexes that are unable to bind FcRn are used, antigen presentation and eventual T cell activation do not occur (Non-Patent Document 43).

When animals are injected with an antigenic protein as a foreign substance, they often produce antibodies against the injected antigenic protein. For example, when mice are injected with the soluble human IL-6 receptor as a foreign protein, they produce mouse antibodies against the soluble human IL-6 receptor. On the other hand, even when mice are injected with a human IgG1 antibody as a foreign protein, they hardly produce mouse anti-human IgG1 antibodies. This difference indicates that the rate of elimination of the introduced foreign protein from plasma may have an effect.

As described in Reference Example 4, a human IgG1 antibody is able to bind mouse FcRn under acidic conditions, and thus, like mouse antibodies, a human IgG1 antibody is recycled through mouse FcRn when incorporated into endosomes. For this reason, when a human IgG1 antibody is administered to normal mice, the elimination of the antibody from plasma is very slow. Meanwhile, the soluble human IL-6 receptor is not recycled through mouse FcRn and thus is rapidly eliminated after administration. On the other hand, as described in Reference Example 4, the production of mouse antibodies against soluble anti-human IL-6R antibody was observed in normal mice injected with soluble human IL-6 receptor, while the production of mouse antibodies against human IgG1 antibody was not observed. in normal mice injected with a human IgG1 antibody. In other words, the soluble human IL-6 receptor, which is rapidly eliminated, is more immunogenic in mice than the human IgG1 antibody, which is slowly eliminated.

Part of the elimination cascade of these foreign proteins (human soluble IL-6 receptor and human IgG1 antibody) from plasma is thought to occur by uptake by antigen-presenting cells. Foreign proteins incorporated into antigen-presenting cells associate with class II MHC molecules after intracellular processing and are transported to the cell membrane. Presentation of the antigen to antigen-specific T cells (eg, T cells that specifically respond to the human soluble IL-6 receptor or human IgG1 antibody) then induces activation of the antigen-specific T cells. In this context, presumably for a foreign protein that is slowly eliminated from plasma, processing in antigen-presenting cells is difficult, and as a result, antigen presentation to antigen-presenting T cells is unlikely to occur.

Binding to FcRn under neutral conditions is known to adversely affect plasma retention of the antibody. After IgG antibody binds to FcRn under neutral conditions, even if it returns to the cell surface under acidic conditions of endosomes as a result of binding to FcRn, the IgG antibody cannot be recycled to plasma without dissociating from FcRn under neutral conditions in plasma; and this adversely affects plasma retention. For example, according to the report (Non-Patent Document 10), when an antibody that becomes capable of binding to mouse FcRn under neutral conditions (pH 7.4) as a result of amino acid substitutions made in IgG1 was administered to mice, plasma retention of the antibody was impaired. Meanwhile, it has been described that when an antibody confirmed to bind to human FcRn under neutral conditions (pH 7.4) was administered to cynomolgus monkeys, the plasma retention of the antibody was not prolonged, but rather remained unchanged (Non-Patent Documents 10 -12). When the plasma retention time of an antigen-binding molecule is reduced due to increased binding to FcRn under neutral conditions (pH 7.4), immunogenicity may become higher due to accelerated elimination of the antigen-binding molecule.

Moreover, FcRn has been described to be expressed in antigen-presenting cells and involved in antigen presentation. According to a report published regarding the assessment of the immunogenicity of a protein obtained by fusion of myelin basic protein (MBP), although not an antigen-binding molecule, with the Fc region of mouse IgG1 (hereinafter abbreviated as MBP-Fc), T cells, which respond in a MBP-Fc-specific manner, are activated and proliferate when cultured in the presence of MBP-Fc. In this aspect, T cell activation is known to be enhanced in vitro when a modification is added to the MBP-Fc Fc region that enhances FcRn binding to increase incorporation into antigen presenting cells via FcRn expressed on antigen presenting cells. It has been described that, regardless of the accelerated elimination from plasma as a result of the introduction of a modification that enhances FcRn binding, in vivo T cell activation was rather worsened (Non-Patent Document 44). Thus, immunogenicity is not necessarily enhanced when elimination is accelerated by enhancing FcRn binding.

As described above, there is not enough research to understand how increased FcRn binding to an antigen-binding molecule that has an FcRn-binding domain under neutral conditions (pH 7.4) affects the plasma retention and immunogenicity of the antigen-binding molecule. Thus, there is no disclosed method for increasing the plasma retention and immunogenicity of antigen-binding molecules having FcRn binding activity under neutral conditions (pH 7.4).

It has been found that the elimination of antigen from plasma can be accelerated by using an antigen-binding molecule that contains an antigen-binding domain of an antigen-binding molecule whose antigen-binding activity varies with ion concentration and an Fc region that has FcRn binding activity in the neutral pH range. However, not enough research has been done to understand how increased FcRn binding activity by the Fc region in the neutral pH range affects plasma retention of antigen-binding molecules and immunogenicity. During testing, the inventors of the present invention found the problem that, as a result of enhancing the activity of the Fc region for FcRn binding in the neutral pH range, the retention time of the antigen-binding molecule in plasma decreases (pharmacokinetics deteriorates) and the immunogenicity of the antigen-binding molecule increases (immune response on the antigen-binding molecule is enhanced).

The present invention has been made in view of the circumstances described above. It is an object of the present invention to provide methods for improving the pharmacokinetics in animals administered with an antigen-binding molecule by modifying the Fc region of the antigen-binding molecule, which contains an antigen-binding domain of the antigen-binding molecule, the antigen-binding activity of which varies depending on the concentration of ions, and an Fc region, which has binding activity FcRn in the neutral pH range. It is another object of the present invention to provide methods for reducing an immune response to an antigen binding molecule by modifying an Fc region of an antigen binding molecule that contains an antigen binding domain of an antigen binding molecule whose antigen binding activity varies with ion concentration, and an Fc region that has FcRn binding activity in range of neutral pH values. Another object of the present invention is to provide antigen-binding molecules that exhibit improved pharmacokinetics or reduced immune response in vivo when administered to animals. Another object of the present invention is to provide methods for the preparation of such antigen-binding molecules, as well as pharmaceutical compositions containing antigen-binding molecules as an active ingredient.

[Troubleshooting Tools]

The inventors of the present invention have carried out purposeful studies to achieve the objectives described above. As a result, the present inventors found that an antigen-binding molecule that contains an antigen-binding domain of an antigen-binding molecule whose antigen-binding activity varies depending on the concentration of ions, and an Fc region that has an FcRn binding activity in the neutral pH range, formed a heterocomplex consisting of four molecules: antigen-binding molecule/two FcRn molecules/activating Fcγ receptor (Fig. 48). The present inventors also demonstrated that tetramer formation adversely affected pharmacokinetics and immune response. The present inventors have demonstrated that the pharmacokinetics of an antigen binding molecule is improved by modifying the Fc region of such an antigen binding molecule into an Fc region that does not form a heterotetrameric complex containing two FcRn molecules and activating the Fcγ receptor in the neutral pH range. The present inventors have also demonstrated that the immune response in animals administered with an antigen-binding molecule can be altered by modifying the Fc region of such an antigen-binding molecule into an Fc region that, in the neutral pH range, does not form a tetrameric complex containing two FcRn molecules and activating Fcγ receptor. The present inventors also demonstrated that the immune response to the antigen-binding molecule was reduced by modification to an Fc region that, in the neutral pH range, does not form a heterotetrameric complex containing two FcRn molecules and activating the Fcγ receptor. Moreover, the inventors of the present invention have discovered antigen-binding molecules and methods for producing them, and furthermore, they have discovered that, when administered, pharmaceutical compositions containing such an antigen-binding molecule or an antigen-binding molecule obtained by the production method of the present invention have improved properties when administered. , such as improved pharmacokinetics and reduced immune response when administered to a living body, compared to conventional antigen-binding molecules; and thereby accomplished the present invention.

More specifically, the present invention relates to the following.

[1] The method according to any of (a) or (b) below, wherein the method comprises modifying an Fc region of an antigen-binding molecule containing an antigen-binding domain, the antigen-binding activity varies depending on the ion concentration, and an Fc region that has an FcRn binding activity in neutral pH range, into an Fc region that does not form a heterocomplex containing two FcRn molecules and one activating Fcγ receptor molecule in the neutral pH range:

(a) a method for improving the pharmacokinetics of an antigen-binding molecule; And

(b) a method for reducing the immunogenicity of an antigen-binding molecule.

[2] The method according to [1], wherein modification to an Fc region that does not form said heterocomplex comprises modifying an Fc region to an Fc region whose binding activity to an activating Fcγ receptor is lower than the binding activity of the Fc region native human IgG with an activating Fcγ receptor.

[3] The method according to [1] or [2], wherein the activating Fcγ receptor is human FcγRIa, human FcYRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F).

[4] The method according to any one of [1]-[3], which comprises substituting an amino acid of said Fc region at one or more of amino acid positions 235, 237, 238, 239, 270, 298, 325, and 329, as specified according to EU numbering.

[5] The method of [4], which comprises replacing the amino acid of said Fc region, as indicated by EU numbering, in any one or more of:

amino acid at position 234 to any of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr and Trp;

amino acid at position 235 to any of Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val and Arg;

amino acid at position 236 to any of Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro and Tyr;

amino acid at position 237 to any of Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr and Arg;

amino acid at position 238 to any of Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp and Arg;

amino acid at position 239 to any of Gln, His, Lys, Phe, Pro, Trp, Tyr and Arg;

amino acid at position 265 to any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val;

amino acid at position 266 to any of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp and Tyr;

amino acid at position 267 to any of Arg, His, Lys, Phe, Pro, Trp and Tyr;

amino acid at position 269 to any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val;

amino acid at position 270 to any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val;

amino acid at position 271 to any of Arg, His, Phe, Ser, Thr, Trp and Tyr;

amino acid at position 295 to any of Arg, Asn, Asp, Gly, His, Phe, Ser, Trp and Tyr;

amino acid at position 296 to any of Arg, Gly, Lys and Pro;

amino acid at position 297 on Ala;

amino acid at position 298 to any of Arg, Gly, Lys, Pro, Trp and Tyr;

amino acid at position 300 to any of Arg, Lys and Pro;

amino acid at position 324 on Lys or Pro;

the amino acid at position 325 to any of Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr and Val;

the amino acid at position 327 to any of Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val;

amino acid at position 328 to any of Arg, Asn, Gly, His, Lys, and Pro;

the amino acid at position 329 to any of Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, and Arg;

amino acid at position 330 on Pro or Ser;

amino acid at position 331 to any of Arg, Gly and Lys; or

amino acid at position 332 to any of Arg, Lys and Pro.

[6] The method according to [1], wherein modification to an Fc region that does not form said heterocomplex comprises modifying an Fc region to an Fc region that has a higher binding activity to an inhibitory Fcγ receptor than an activating Fcγ receptor .

[7] The method according to [6], wherein the inhibitory Fcγ receptor is human FcγRIIb.

[8] The method according to [6] or [7], wherein the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F).

[9] The method according to any one of [6]-[8], which comprises changing the amino acid at position 238 or 328 as indicated by EU numbering.

[10] The method according to [9], which includes replacing the amino acid at position 238 with Asp or the amino acid at position 328 with Glu, as indicated by EU numbering.

[11] The method according to [9] or [10], which includes replacing any one or more amino acids from:

amino acid at position 233 on Asp;

amino acid at position 234 on Trp or Tyr;

amino acid at position 237 to any of Ala, Asp, Glu, Leu, Met, Phe, Trp and Tyr;

amino acid at position 239 on Asp;

amino acid at position 267 to any of Ala, Gln and Val;

amino acid at position 268 to any of Asn, Asp and Glu;

amino acid at position 271 on Gly;

amino acid at position 326 to any of Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, and Thr;

amino acid at position 330 to any of Arg, Lys and Met;

amino acid at position 323 to any of Ile, Leu and Met; And

amino acid at position 296 on Asp; where amino acids are indicated according to EU numbering.

[12] The method according to any of [1]-[11], wherein the Fc region contains one or more amino acids that differ from the amino acids of the native Fc region at amino acid positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 3 85, 386, 387, 389, 424, 428, 433, 434 and 436 of the indicated Fc region, as indicated by EU numbering.

[13] The method according to [12], wherein the amino acids of said Fc region, indicated according to EU numbering, are a combination of one or more of:

Met at amino acid position 237;

Ile at amino acid position 248;

any of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp and Tyr at amino acid position 250;

any of Phe, Trp and Tyr at amino acid position 252;

Thr at amino acid position 254;

Glu at amino acid position 255;

any of Asp, Asn, Glu and Gln at amino acid position 256;

any of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr and Val at amino acid position 257;

His at amino acid position 258;

Ala at amino acid position 265;

Ala or Glu at amino acid position 286;

His at amino acid position 289;

Ala at amino acid position 297;

Gly at amino acid position 298;

Ala at amino acid position 303;

Ala at amino acid position 305;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr at amino acid position 307;

any of Ala, Phe, Ile, Leu, Met, Pro, Gln and Thr at amino acid position 308;

any of Ala, Asp, Glu, Pro and Arg at amino acid position 309;

any of Ala, His and Ile at amino acid position 311;

Ala or His at amino acid position 312;

Lys or Arg at amino acid position 314;

any of Ala, Asp and His at amino acid position 315;

Ala at amino acid position 317;

Val at amino acid position 332;

Leu at amino acid position 334;

His at amino acid position 360;

Ala at amino acid position 376;

Ala at amino acid position 380;

Ala at amino acid position 382;

Ala at amino acid position 384;

Asp or His at amino acid position 385;

Pro at amino acid position 386;

Glu at amino acid position 387;

Ala or Ser at amino acid position 389;

Ala at amino acid position 424;

any of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at amino acid position 428;

Lys at amino acid position 433;

any of Ala, Phe, His, Ser, Trp and Tyr at amino acid position 434; And

any of His, Ile, Leu, Phe, Thr and Val at amino acid position 436.

[14] The method according to any one of [1] to [13], wherein said antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies depending on the concentration of calcium ions.

[15] The method according to [14], wherein said antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies such that the antigen-binding activity at a low calcium ion concentration is lower than the antigen-binding activity at a high calcium ion concentration.

[16] The method according to any one of [1] to [13], wherein said antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies with pH.

[17] The method of [16], wherein said antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies such that the antigen-binding activity in the acidic pH range is lower than the antigen-binding activity in the neutral pH range.

[18] The method according to any one of [1]-[17], wherein the antigen-binding domain is the variable region of an antibody.

[19] The method according to any one of [1]-[18], wherein the antigen-binding molecule is an antibody.

[20] The method according to [1], wherein a modification to an Fc region that does not form said heterocomplex comprises a modification to an Fc region in which one of the two polypeptides constituting the Fc region has FcRn binding activity in the neutral pH range and the other has no FcRn binding activity in the neutral pH range.

[21] The method according to [20], which comprises replacing an amino acid at any one or more of positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 4 36, as indicated by EU numbering, in the amino acid sequence of one of the two polypeptides that make up the specified Fc region.

[22] The method according to [21], which comprises replacing an amino acid of said Fc region in any or more of:

amino acid at position 237 on Met;

amino acid at position 248 on Ile;

amino acid at position 250 on Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr;

amino acid at position 252 on Phe, Trp or Tyr;

amino acid at position 254 on Thr;

amino acid at position 255 on Glu;

amino acid at position 256 on Asp, Asn, Glu or Gln;

amino acid at position 257 on Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;

amino acid at position 258 on His; amino acid at position 265 on Ala;

amino acid at position 286 on Ala or Glu;

amino acid at position 289 on His;

amino acid at position 297 on Ala;

amino acid at position 298 on Gly;

amino acid at position 303 on Ala;

amino acid at position 305 on Ala;

amino acid at position 307 on Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;

amino acid at position 308 on Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;

amino acid at position 309 on Ala, Asp, Glu, Pro, or Arg;

amino acid at position 311 on Ala, His or Ile;

amino acid at position 312 on Ala or His;

amino acid at position 314 on Lys or Arg;

amino acid at position 315 on Ala, Asp or His;

amino acid at position 317 on Ala;

amino acid at position 332 on Val;

amino acid at position 334 on Leu;

amino acid at position 360 on His;

amino acid at position 376 on Ala;

amino acid at position 380 on Ala;

amino acid at position 382 on Ala;

amino acid at position 384 on Ala;

amino acid at position 385 on Asp or His;

amino acid at position 386 on Pro;

amino acid at position 387 on Glu;

amino acid at position 389 on Ala or Ser;

amino acid at position 424 on Ala;

amino acid at position 428 on Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;

amino acid at position 433 on Lys;

amino acid at position 434 on Ala, Phe, His, Ser, Trp or Tyr; And

amino acid at position 436 on His, Ile, Leu, Phe, Thr or Val; where amino acids are indicated according to EU numbering.

[23] The method according to any one of [20] to [22], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies depending on the calcium concentration.

[24] The method according to [23], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies such that the antigen-binding activity at a low calcium concentration is lower than the antigen-binding activity at a high calcium concentration.

[25] The method according to any one of [20]-[22], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies with pH.

[26] The method according to [25], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies such that the antigen-binding activity in the acidic pH range is lower than the antigen-binding activity in the neutral pH range.

[27] The method according to any one of [20]-[26], wherein the antigen-binding domain is the variable region of an antibody.

[28] The method according to any one of [20]-[27], wherein the antigen-binding molecule is an antibody.

[29] An antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of ions, and an Fc region that has FcRn binding activity in the neutral pH range, where the Fc region contains one or more amino acids selected from:

Ala at amino acid position 234;

Ala, Lys or Arg at amino acid position 235;

Arg at amino acid position 236;

Arg at amino acid position 238;

Lys at amino acid position 239;

Phe at amino acid position 270;

Ala at amino acid position 297;

Gly at amino acid position 298;

Gly at amino acid position 325;

Arg at amino acid position 328; And

Lys or Arg at amino acid position 329; where amino acids are indicated according to EU numbering.

[30] An antigen-binding molecule according to [29], which contains one or more amino acids selected from:

Lys or Arg at amino acid position 237;

Lys at amino acid position 238;

Arg at amino acid position 239; And

Lys or Arg at amino acid position 329; where amino acids are indicated according to EU numbering.

[31] An antigen-binding molecule comprising an antigen-binding domain whose antigen-binding activity varies depending on the concentration of ions, and an Fc region in which one of the two polypeptides constituting the Fc region has FcRn binding activity in the neutral pH range, and the other does not has FcRn binding activity in the neutral pH range.

[32] An antigen-binding molecule according to any one of [29]-[31], wherein the Fc region contains one or more amino acids that differ from the amino acids of the native Fc region, at any of amino acid positions 237, 248, 250, 252, 254, 255 256 257 258 265 286 289 297 303 305 307 308 309 311 312 314 315 317 332 334 360 376 380 382 3 84, 385, 386, 387, 389, 424, 428, 433, 434 and 436, indicated according to the EU numbering in the amino acid sequence of one of the two polypeptides that make up the Fc region.

[33] An antigen-binding molecule according to [32], which contains a combination of one or more amino acids of the specified Fc region from:

Met at amino acid position 237;

Ile at amino acid position 248;

Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at amino acid position 250;

Phe, Trp or Tyr at amino acid position 252;

Thr at amino acid position 254;

Glu at amino acid position 255;

Asp, Asn, Glu or Gln at amino acid position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257;

His at amino acid position 258;

Ala at amino acid position 265;

Ala or Glu at amino acid position 286;

His at amino acid position 289;

Ala at amino acid position 297;

Ala at amino acid position 303;

Ala at amino acid position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid position 308;

Ala, Asp, Glu, Pro, or Arg at amino acid position 309;

Ala, His or Ile at amino acid position 311;

Ala or His at amino acid position 312;

Lys or Arg at amino acid position 314;

Ala, Asp or His at amino acid position 315;

Ala at amino acid position 317;

Val at amino acid position 332;

Leu at amino acid position 334;

His at amino acid position 360;

Ala at amino acid position 376;

Ala at amino acid position 380;

Ala at amino acid position 382;

Ala at amino acid position 384;

Asp or His at amino acid position 385;

Pro at amino acid position 386;

Glu at amino acid position 387;

Ala or Ser at amino acid position 389;

Ala at amino acid position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid position 428;

Lys at amino acid position 433;

Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434; And

His, Ile, Leu, Phe, Thr, or Val at amino acid position 436; where amino acids are indicated according to EU numbering.

[34] The antigen-binding molecule according to any one of [29]-[33], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies depending on the concentration of calcium ions.

[35] An antigen-binding molecule according to [34], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies such that the antigen-binding activity at a low calcium concentration is lower than the antigen-binding activity at a high calcium concentration.

[36] An antigen-binding molecule according to any one of [29]-[33], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies with pH.

[37] The antigen-binding molecule according to [36], wherein the antigen-binding domain is an antigen-binding domain whose antigen-binding activity varies such that the antigen-binding activity in the acidic pH range is lower than the antigen-binding activity in the neutral pH range.

[38] An antigen-binding molecule according to any one of [29]-[37], wherein the antigen-binding domain is the variable region of an antibody.

[39] An antigen-binding molecule according to any one of [29]-[38], wherein the antigen-binding molecule is an antibody.

[40] A polynucleotide encoding an antigen-binding molecule according to any one of [29]-[39].

[41] A vector that is operably linked to a polynucleotide according to [40].

[42] The cell into which the vector is entered according to [41].

[43] The method for producing an antigen-binding molecule according to any one of [29] to [39], which includes the step of collecting an antigen-binding molecule from a cell culture according to [42].

[44] A pharmaceutical composition that contains, as an active ingredient, an antigen-binding molecule according to any one of [29] to [39] or an antigen-binding molecule obtained by the production method according to [43].

Moreover, the present invention relates to kits for use in the methods of the present invention, which contain an antigen-binding molecule of the present invention or an antigen-binding molecule obtained by the preparation method of the present invention. The present invention also relates to agents for improving the pharmacokinetics of an antigen-binding molecule and agents for reducing the immunogenicity of an antigen-binding molecule, which contain, as an active ingredient, an antigen-binding molecule of the present invention or an antigen-binding molecule obtained by the production method of the present invention. The present invention also relates to methods for the treatment of immune/inflammatory diseases, which include the step of administering to the individual an antigen-binding molecule of the present invention or an antigen-binding molecule obtained by the production method of the present invention. In addition, the present invention relates to the use of antigen-binding molecules of the present invention or antigen-binding molecules obtained by the production method of the present invention for obtaining agents for improving the pharmacokinetics of antigen-binding molecules and agents for reducing the immunogenicity of antigen-binding molecules. The present invention also relates to antigen-binding molecules of the present invention or antigen-binding molecules obtained by the production method of the present invention for use in the methods of the present invention.

[Effects of invention]

The present invention relates to methods for improving the pharmacokinetics of antigen-binding molecules and methods for reducing the immunogenicity of antigen-binding molecules. The present invention provides antibody therapy without adverse in vivo effects compared to conventional antibodies.

Brief description of the drawings

In FIG. 1 is a graph showing the effects on soluble antigen of an existing neutralizing antibody and an antibody that binds to the antigen in a pH dependent manner and exhibits enhanced FcRn binding under neutral conditions.

In FIG. 2 is a graph showing plasma concentration over time following intravenous or subcutaneous administration of Fv4-IgG1 or Fv4-IgG1-F1 to normal mice.

In FIG. 3 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to human FcγRIa.

In FIG. 4 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to human FcγRIIa (R).

In FIG. 5 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to human FcγRIIa (H).

In FIG. 6 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to human FcγRIIb.

In FIG. 7 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to human FcγRIIIa (F).

In FIG. 8 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to mouse FcγRI.

In FIG. 9 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to mouse FcγRIIb.

In FIG. 10 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to mouse FcγRIII.

In FIG. 11 is a graph demonstrating that in the human FcRn bound state, Fv4-IgG1-F157 binds to mouse FcγRIV.

In FIG. 12 is a graph demonstrating that, when bound to mouse FcRn, Fv4-IgG1-F20 binds to mouse FcγRI, mouse FcγRIIb, mouse FcγRIII, and mouse FcγRIV.

In FIG. 13 is a graph showing that, when bound to mouse FcRn, mPM1-mIgG1-mF3 binds to mouse FcγRIIb and mouse FcγRIII.

In FIG. 14 is a graph showing the plasma concentration over time of Fv4-IgG1-F21, Fv4-IgG1-F140, Fv4-IgG1-F157, and Fv4-IgG1-F424 in human FcRn transgenic mice.

In FIG. 15 is a graph showing the plasma concentration over time of Fv4-IgG1 and Fv4-IgG1-F760 in human FcRn transgenic mice.

In FIG. 16 is a graph showing the plasma concentration over time of Fv4-IgG1-F11, Fv4-IgG1-F890, Fv4-IgG1-F947, Fv4-IgG1-F821, Fv4-IgG1-F939 and Fv4-IgG1-F1009 in transgenic mice with human FcRn.

In FIG. 17 is a graph showing the plasma concentration over time of mPM1-mIgG1-mF14, mPM1-mIgG1-mF38, mPM1-mIgG1-mF39 and mPM1-mIgG1-mF40 in normal mice.

In FIG. 18 is a graph showing the result of immunogenicity evaluation using Fv4-IgG1-F21 and Fv4-IgG1-F140.

In FIG. 19 is a graph showing the result of immunogenicity evaluation using hA33-IgG1-F21 and hA33-IgG1-F140.

In FIG. 20 is a graph showing the result of immunogenicity evaluation using hA33-IgG1-F698 and hA33-IgG1-F699.

In FIG. 21 is a graph showing the result of immunogenicity evaluation using hA33-IgG1-F698 and hA33-IgG1-F763.

In FIG. 22 is a graph showing the titers of mouse antibody produced against Fv4-IgG1-F11 at 3, 7, 14, 21 and 28 days after administration to human FcRn transgenic mice.

In FIG. 23 is a graph showing the titers of mouse antibody produced against Fv4-IgG1-F821 at 3, 7, 14, 21 and 28 days after administration to human FcRn transgenic mice.

In FIG. 24 is a graph showing the titers of mouse antibody produced against Fv4-IgG1-F890 at 3, 7, 14, 21 and 28 days after administration to human FcRn transgenic mice. B is an increase in A.

In FIG. 25 is a graph showing the titers of mouse antibody produced against Fv4-IgG1-F939 at 3, 7, 14, 21 and 28 days after administration to human FcRn transgenic mice.

In FIG. 26 is a graph showing the titers of mouse antibody produced against Fv4-IgG1-F947 at 3, 7, 14, 21 and 28 days after administration to human FcRn transgenic mice.

In FIG. 27 is a graph showing the titers of mouse antibody produced against Fv4-IgG1-F1009 at 3, 7, 14, 21 and 28 days after administration to human FcRn transgenic mice.

In FIG. 28 is a graph showing the titers of mouse antibody produced against mPM1-IgG1-mF14 at 14, 21 and 28 days after administration to normal mice.

In FIG. 29 is a graph showing the titers of mouse antibody produced against mPM1-IgG1-mF39 at 14, 21 and 28 days after administration to normal mice.

In FIG. 30 is a graph showing the titers of mouse antibody produced against mPM1-IgG1-mF38 at 14, 21 and 28 days after administration to normal mice.

In FIG. 31 is a graph showing the titers of mouse antibody produced against mPM1-IgG1-mF40 at 14, 21 and 28 days after administration to normal mice.

In FIG. 32 is a graph showing plasma antibody concentrations for Fv4-IgG1-F947 and Fv4-IgG1-FA6a/FB4a at 15 minutes, seven hours, one, two, three, four, and seven days after administration to human FcRn transgenic mice.

In FIG. 33 is a graph showing the change in binding of each of the B3 mutants to FcγRIIb and FcγRIa.

In FIG. 34 is a graph showing the change in binding of each of the B3 mutants to FcγRIIb and FcγRIIa (H).

In FIG. 35 is a graph showing the change in binding of each of the B3 mutants to FcγRIIb and FcγRIIa (R).

In FIG. 36 is a graph showing the change in binding of each of the B3 mutants to FcγRIIb and FcγRIIIa.

In FIG. 37 is a graph showing the plasma kinetics of soluble human IL-6 receptor in normal mice and mouse plasma titer against soluble human IL-6 receptor.

In FIG. 38 is a graph showing plasma kinetics of soluble human IL-6 receptor in normal mice injected with mouse anti-CD4 antibody and mouse plasma titer of anti-soluble human IL-6 receptor.

In FIG. 39 is a graph showing the plasma kinetics of an anti-IL-6 receptor antibody in normal mice.

In FIG. 40 is a graph showing the concentration of soluble human IL-6 receptor over time after co-administration of soluble human IL-6 receptor and anti-IL-6 receptor antibody to human FcRn transgenic mice.

In FIG. 41 is a diagram showing the structure of the CDR3 Fab fragment of the heavy chain of the 6RL#9 antibody determined by X-ray crystallography.

In FIG. 42 is a graph showing plasma antibody concentration over time for H54/L28-IgG1, 6RL#9-IgG1 and FH4-IgG1 in normal mice.

In FIG. 43 is a graph showing the plasma concentration of soluble human IL-6 receptor over time in normal mice treated with H54/L28-IgG1, 6RL#9-IgG1, or FH4-IgG1.

In FIG. 44 is a graph showing plasma antibody concentrations over time for H54/L28-N434W, 6RL#9-N434W and FH4-N434W in normal mice.

In FIG. 45 is a graph showing the plasma concentration of soluble human IL-6 receptor over time in normal mice treated with H54/L28-N434W, 6RL#9-N434W, or FH4-N434W.

In FIG. 46 is an ion exchange chromatogram for an antibody containing the human Vk5-2 sequence and an antibody containing the hVk5-2_L65 sequence that has a modified glycosylation sequence from the human Vk5-2 sequence. The solid line corresponds to the chromatogram for an antibody containing the human Vk5-2 sequence (heavy chain: CIM_H, SEQ ID NO: 108; and light chain: hVk5-2, SEQ ID NO: 4). The dotted line corresponds to the chromatogram for an antibody containing the sequence hVk5-2_L65 (heavy chain: CIM_H (SEQ ID NO: 108); and light chain: hVk5-2_L65 (SEQ ID NO: 107)).

In FIG. 47 is a diagram showing the alignment of IgG1, IgG2, IgG3, and IgG4 constant region sequences, which are numbered according to the EU numbering system.

In FIG. 48 is a schematic diagram showing the formation of a tetrameric complex consisting of one Fc region molecule that has FcRn binding activity in the neutral pH range, two FcRn molecules, and one FcγR molecule.

In FIG. 49 is a schematic diagram showing the interaction of two FcRn molecules and one FcγR molecule with an Fc region that has FcRn binding activity in the neutral pH range and lower activating FcγR binding activity than the native Fc region.

In FIG. 50 is a schematic diagram showing the interaction of two FcRn molecules and one FcγR molecule with an Fc region that has FcRn binding activity in the neutral pH range and selective binding activity for inhibitory FcγR.

In FIG. 51 is a schematic diagram showing the interaction of two FcRn molecules and one FcγR molecule with an Fc region in which only one of the two FcRn-binding domain polypeptides has FcRn binding activity in the neutral pH range and the other has no FcRn binding activity at range of neutral pH values.

In FIG. 52 is a graph showing the relationship between the intended amino acid distribution (denoted as model) and amino acid distribution (denoted as library) for sequence information on 290 clones isolated from E. coli that were introduced with an antibody gene library that binds with antigens in a Ca-dependent manner. The horizontal axis indicates the positions of amino acids in the Kabat numbering system. The vertical axis indicates the % distribution of amino acids.

In FIG. 53 is a graph showing the relationship between the intended amino acid distribution (denoted as model) and amino acid distribution (denoted as library) for sequence information for 132 clones isolated from E. coli that were introduced with an antibody gene library that binds to antigens in a pH-dependent manner. The horizontal axis indicates the positions of amino acids in the Kabat numbering system. The vertical axis indicates the % distribution of amino acids.

In FIG. 54 is a graph showing the plasma concentration over time of Fv4-IgG1-F947 and Fv4-IgG1-F1326 in human FcRn transgenic mice administered Fv4-IgG1-F947 or Fv4-IgG1-F1326.

In FIG. 55 is a graph showing the relative magnitude of FcγRIIb binding activity for each PD variant on the horizontal axis and the relative magnitude of type R FcγRIIa binding activity for each PD variant on the vertical axis. The binding value of each PD variant to each FcγR was divided by the binding value of IL6R-F652, which is the pre-change control antibody (changed Fc by replacing Pro at position 238 (indicated by EU numbering) with Asp) in each FcγR; and then the resulting value was multiplied by 100 and used as the relative value of the binding activity for each PD variant with each FcγR. Plot F652 in the figure shows the value for IL6R-F652.

In FIG. 56 is a graph in which the vertical axis shows the relative magnitude of FcγRIIb binding activity for variants obtained by making each change in GpH7-B3 that does not have the P238D change, and the horizontal axis shows the relative magnitude of FcγRIIb binding activity for variants obtained by making each change in IL6R-F652 that has change P238D. The FcγRIIb binding value of each variant was divided by the FcγRIIb binding value of the pre-modified antibody; and then the obtained value was multiplied by 100 and used as the value of the relative binding activity. In this case, region A contains changes that exhibit the effect of enhancing FcγRIIb binding in both cases when the change is made to GpH7-B3 that does not have P238D and when the change is made to IL6R-F652 that has P238D. Region B contains changes that show the effect of enhancing FcγRIIb binding when introduced into GpH7-B3, which does not have P238D, but does not show the effect of enhancing FcγRIIb binding when introduced into IL6R-F652, which has P238D.

In FIG. 57 shows the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 58 is an overlay image of the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the model structure of the Fc(WT) complex/FcγRIIb extracellular region, with respect to the FcγRIIb extracellular region and Fc CH2 domain A by least-squares fitting based on the distances between the pairs Ca atoms.

In FIG. 59 shows a comparison of the detailed structure in the P238D region after superposition of the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the model structure of the Fc(WT) complex/FcγRIIb extracellular region, in relation to Fc CH2 domain A only or Fc CH2 domain B only by approximations by the least squares method based on the distances between pairs of Сα atoms.

In FIG. 60 shows that a hydrogen bond can be found between the Gly backbone at position 237 (listed according to EU numbering) in Fc's A CH2 domain, and Tyr at position 160 in FcγRIIb in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 61 shows that an electrostatic interaction can be detected between Asp at position 270 (listed according to EU numbering) in the CH2 domain B of Fc, and Arg at position 131 of FcγRIIb in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 62 is a graph showing the relative magnitude of FcγRIIb binding activity for each variant 2B on the horizontal axis and the relative magnitude of type R FcγRIIa binding activity for each variant 2B on the vertical axis. The binding value of each variant 2B to each FcγR was divided by the binding value of the control antibody before change (changed Fc with Pro at position 238 (referenced by EU numbering) replaced by Asp) for each FcγR; and then the resulting value was multiplied by 100, and used as the value of the relative binding activity of each option 2B against each FcγR.

In FIG. 63 shows Glu at position 233 (indicated according to EU numbering) in the Fc chain A and surrounding residues in the FcγRIIb extracellular region in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 64 shows Ala at position 330 (indicated according to EU numbering) in the Fc chain A and surrounding residues in the FcγRIIb extracellular region in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex.

In FIG. 65 shows structures of Pro at position 271 (indicated according to EU numbering) of the B chain of Fc after superposition of the crystal structures of the Fc(P238D) complex/FcγRIIb extracellular region and the Fc(WT) complex/FcγRIIIa extracellular region by least-squares fitting based on distances between atom pairs Cα, in relation to the B-chain Fc.

Method for carrying out the invention

The definitions and detailed description below are provided to help understand the present invention illustrated in the present description.

Amino acids

Within the scope of the present invention, amino acids are described by one or three letter codes or both, e.g. Ala/A, Leu/L, Arg/R, Lys/K, Asn/N, Met/M, Asp/D, Phe/F , Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T, Gly/G, Trp/W, His/H, Tyr/Y, Ile/I or Val/V.

Antigens

Within the scope of the present invention, "antigens" are not particularly limited in their structure, provided that they contain epitopes to which antigen-binding domains bind.

Other antigens include, for example, the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, active, active A, active AB, active B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9 , ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1 antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av/b3 integrin, Ax1, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS ), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BACAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b -NGF, BOK, bombesin, bone neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA ), cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a , CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG -2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15 , CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (decay accelerating factor), des-(1-3) -IGF-I (IGF-1 brain), Dhh, digoxin, DNCM-1, DNase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB- 1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activating factor (FAP), Fas, FcRI, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3 , Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF- 8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1 GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb/IIIa ( GPIIb/IIIa), GM-CSF, gp 130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, gB HCMV envelope glycoprotein, gH HCMV envelope glycoprotein, UL HCMV, Hematopoietic Growth Factor (HGF), HebB gp120, Heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), Herpes Simplex Virus (HSV) gB Glycoprotein, gD Glycoprotein HSV, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), gp120 HIV, loop V3 gp 120 IIIB HIV, HLA, HLA-DR, HM1,24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV ), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R , IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL -6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain, Insulin-like growth factor 1, Alpha-2 integrin, Alpha-3 integrin, Alpha-4 integrin, Alpha4/beta1 integrin, Alpha4/beta7 integrin, Alpha5 integrin (alpha V), alpha5/beta1 integrin, alpha5/beta3 integrin, alpha6 integrin, beta1 integrin, beta2 integrin, interferon-gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1 , latent TGF-1 bpl, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y associated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L -selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface protein, luteinizing hormone, lymphotoxin-beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP , M-CSF, MDC, Mer, metalloproteases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP -11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP , Mucin (Muc1), MUC18, Müllerian Inhibitory Substance, Mug, MuSK, NAIP, NAP, NCAD, Adherin N-C, NCA90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4 or -6, Neuroturin, Nerve Growth Factor (NGF ), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGGI, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP , PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A-chain, B-chain relaxina, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T- cellular receptor (eg, T cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, PLAP-like testis alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, pan-specific TGF- beta, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymus Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha-beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD ), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2) , TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD 120a, p55-60), TNFRSF1B (TNF RII CD 120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (0X40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3 , LARD, TR-3, TRAMP, WSL-1), TNFSF10 (Apo-2 TRAIL ligand, TL2), TNFSF11 (ODF TRANCE/RANK ligand, OPG ligand), TNFSF12 (Apo-3 TWEAK ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (IVEM LIGHT I ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a connectin , DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (gp34 OX40 ligand, TXGP1), TNFSF5 (CD154 CD40 ligand, gp39, HIGM1, IMD3, TRAP), TNFSF6 ( Apo-1 Fas Ligand, APT1 Ligand), TNFSF7 (CD70 CD27 Ligand), TNFSF8 (CD153 CD30 Ligand), TNFSF9 (CD137 Ligand 4-1BB), TP-1, t-PA, Thro, TRAIL, TRAIL R , TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated CA125, tumor-associated antigen expressing Lewis-Y-associated carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A , WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90 , IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R , Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a , C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor Xlla, factor XIII, factor XIIIa , TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA and SIP; and receptors for hormones and growth factors.

"Epitope" means an antigenic determinant in an antigen and refers to the antigenic site to which the antigen-binding domain of the antigen-binding molecule described herein binds. Thus, for example, an epitope can be defined according to its structure. Alternatively, an epitope may be defined according to the antigen-binding activity of an antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope may be indicated by the amino acid residues that form the epitope. Alternatively, when the epitope is a sugar chain, the epitope may be indicated by its defined sugar chain structure.

A linear epitope is an epitope that contains an epitope in which its primary amino acid sequence is recognized. Such a linear epitope typically contains at least three and most often at least five, for example, about 8-10 or 6-20 amino acids in its particular sequence.

In contrast to a linear epitope, a "conformational epitope" is an epitope in which the primary amino acid sequence containing the epitope is not the sole determinant of the recognized epitope (e.g., the primary amino acid sequence of the conformational epitope is not necessarily recognized by the epitope-defining antibody). Conformational epitopes may contain more amino acids than linear epitopes. An antibody that recognizes a conformational epitope recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds and forms a three-dimensional structure, the amino acids and/or polypeptide backbones that form the conformational epitope are adjacent to each other, and the epitope becomes recognizable to an antibody. Methods for determining epitope conformations include, but are not limited to, X-ray crystallography, 2D nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance, for example. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

Binding Activity

Examples of a method for assessing epitope binding by a test antigen-binding molecule containing an antigen-binding domain for IL-6R are described below. According to the examples below, methods for evaluating epitope binding of an antigen-binding molecule of interest containing an antigen-binding domain to an antigen other than IL-6R can also be appropriately carried out.

For example, that an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R recognizes a linear epitope in an IL-6R molecule can be confirmed, for example, as described below. For the above purpose, a linear peptide is synthesized containing the amino acid sequence forming the extracellular domain of IL-6R. The peptide can be synthesized chemically or it can be obtained by genetic engineering methods using the region encoding the amino acid sequence corresponding to the extracellular domain in the cDNA of IL-6R. The test antigen-binding molecule containing the antigen-binding domain for IL-6R is then evaluated for its binding activity to a linear peptide containing an amino acid sequence forming an extracellular domain. For example, an immobilized linear peptide can be used as an antigen in an ELISA to evaluate the binding activity of an antigen-binding molecule to a peptide. Alternatively, the binding activity of a linear peptide can be assessed in terms of the level at which the linear peptide inhibits the binding of an antigen-binding molecule to cells expressing IL-6R. These assays can demonstrate the binding activity of an antigen-binding molecule to a linear peptide.

The presence of recognition of the investigated antigen-binding molecule containing the antigen-binding domain for IL-6R conformational epitope can be assessed as follows. For the above purpose, cells expressing IL-6R are obtained. The presence of recognition of an antigen-binding molecule of interest containing an IL-6R antigen-binding domain, a conformational epitope, can be determined when it binds strongly to IL-6R expressing cells upon contact, but does not substantially bind to an immobilized linear peptide containing an amino acid sequence forming an IL extracellular domain -6R. In the context of the present invention, "substantially does not bind" means that the binding activity is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the cell binding activity, expressing human IL-6R.

Methods for evaluating the binding activity of an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R to IL-6R expressing cells include, for example, the methods described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). In particular, the evaluation can be carried out based on the principle of ELISA or fluorescence-activated cell sorting (FACS) using IL-6R expressing cells as antigen.

In an ELISA format, the binding activity of an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R to IL-6R expressing cells can be quantified by comparing the signal levels generated by the enzymatic reaction. Specifically, the polypeptide complex of interest is added to an ELISA plate on which IL-6R expressing cells are immobilized. The antigen-binding molecule bound to the cells is then detected using an enzyme-labeled antibody that recognizes the antigen-binding molecule of interest. Alternatively, when FACS is used, a dilution series of the antigen binding molecule of interest is prepared and the binding titer of the antibody to IL-6R expressing cells can be determined to compare the binding activity of the antigen binding molecule of interest to IL-6R expressing cells.

The binding of an antigen-binding molecule of interest to an antigen expressed on the surface of cells suspended in buffer or similar can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices:

FACSCanto™ II

FACSAria™

FACSArray™

FACSVantage™ SE

FACSCalibur™ (all are trade names of BD Biosciences)

EPICS ALTRA HyPerSort

Cytomics FC 500

EPICS XL-MCL ADC EPICS XL ADC

Cell Lab Quanta/Cell Lab Quanta SC (all are Beckman Coulter trade names).

Preferred methods for assessing the binding activity of an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R to an antigen include, for example, the following method. First, IL-6R expressing cells are reacted with the antigen-binding molecule of interest, and then they are stained with a FITC-labeled secondary antibody that recognizes the antigen-binding molecule. The antigen-binding molecule to be tested is appropriately diluted with an appropriate buffer to obtain the molecule at the desired concentration. For example, the molecule can be used at a concentration in the range of 10 µg/mL to 10 ng/mL. Then determine the intensity of fluorescence and the number of cells using FACSCalibur (BD). Fluorescence intensity obtained by analysis using the CELL QUEST (BD) software, i.e. the geometric mean reflects the amount of antibody bound to the cells. Therefore, the binding activity of an antigen-binding molecule under investigation, which reflects the amount of bound antigen-binding molecule under investigation, can be determined by determining the geometric mean.

Whether an antigen-binding molecule of interest containing an antigen-binding domain for IL-6R shares a common epitope with another antigen-binding molecule can be assessed based on competition between the two molecules for the same epitope. Competition between antigen-binding molecules can be detected using cross-blocking assays and the like. For example, a preferred cross-blocking assay is a competitive ELISA.

In particular, in the cross-blocking assay, IL-6R protein immobilized on the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competitive antigen-binding molecule, and then the antigen-binding molecule is added thereto. The amount of an antigen-binding molecule of interest bound to the IL-6R protein in the wells is indirectly correlated with the binding capacity of a candidate competitive antigen-binding molecule that competes for binding to the same epitope. Therefore, the higher the affinity of a competitive antigen-binding molecule for the same epitope, the lower the binding activity of the antigen-binding molecule of interest to IL-6R protein-coated wells.

The amount of antigen-binding molecule of interest bound to the wells via IL-6R can be readily determined by prelabeling the antigen-binding molecule. For example, a biotin labeled antigen binding molecule is detected using an avidin/peroxidase conjugate and an appropriate substrate. In particular, a cross-blocking assay that uses enzyme labels such as peroxidase is referred to as a "competitive ELISA assay".

The antigen binding molecule can also be labeled with other labeling agents that provide detection or measurement. In particular, radioactive labels, fluorescent labels, and the like are known.

When a candidate competitive antigen-binding molecule can block binding of the antigen-binding molecule of interest containing an antigen-binding domain for IL-6R by at least 20%, preferably by at least 20-50%, and more preferably by at least 50% compared to binding activity in a control experiment conducted in the absence of a competitive antigen-binding molecule, it is determined that the antigen-binding molecule under test substantially binds to the same epitope to which the competitive antigen-binding molecule binds or competes for binding to the same epitope.

When the structure of the epitope to which the test antigen-binding molecule binds, containing the antigen-binding domain for IL-6R, has already been identified, then whether the test and control antigen-binding molecules share a common epitope can be assessed by comparing the binding activity of the two antigen-binding molecules to the peptide obtained by introducing amino acid mutations in the peptide that forms the epitope.

To measure the above binding activity, for example, the binding activity of test and control antigen-binding molecules against the linear peptide in which the mutation has been introduced is compared in the ELISA format described above. In addition to ELISA methods, binding activity to a mutant peptide bound to a column can be determined by passing test and control antigen-binding molecules through the column and then quantifying the antigen-binding molecule eluted in the elution solution. Methods for adsorbing a mutant peptide onto a column, for example in the form of a fusion peptide to GST, are known.

Alternatively, when the identified epitope is a conformational epitope, then whether test and control antigen-binding molecules share a common epitope can be assessed by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced in the epitope are obtained. Test and control antigen-binding molecules are added to a cell suspension obtained by suspending the cells in an appropriate buffer such as PBS. The cell suspensions are then properly washed with buffer and a FITC-labeled antibody that recognizes the test and control antigen-binding molecules is added to them. Fluorescence intensity and the number of cells stained with labeled antibody are determined using FACSCalibur (BD). Test and control antigen-binding molecules are appropriately diluted with an appropriate buffer and used at desired concentrations. For example, they can be used at a concentration in the range of 10 μg/ml to 10 ng/ml. Fluorescence intensity determined by analysis using CELL QUEST (BD) software, i.e. geometric mean, reflects the amount of labeled antibody bound to the cells. Therefore, the binding activity of test and control antigen-binding molecules, as reflected by the amount of bound labeled antibody, can be determined by measuring the geometric mean.

In the method described above, that the antigen-binding molecule "substantially does not bind to cells expressing the mutant IL-6R" can be assessed, for example, by the following method. First, test and control antigen-binding molecules bound to cells expressing the mutant IL-6R are stained with labeled antibody. The fluorescence intensity of the cells is then determined. When using the FACSCalibur to detect fluorescence by flow cytometry, the determined fluorescence intensity can be analyzed using the CELL QUEST software. From the geometric mean values in the presence and absence of the polypeptide complex, a comparative value (ΔGeo-Mean) can be calculated using the following formula to determine the ratio of the increase in fluorescence intensity due to binding by an antigen-binding molecule.

ΔGeo-Mean = Geo-Mean (in the presence of the polypeptide complex) / Geo-Mean (in the absence of the polypeptide complex)

The geometric mean comparative value (ΔGeo-Mean value for the mutant IL-6R molecule) determined by the assay described above, which reflects the amount of the antigen-binding molecule of interest bound to cells expressing the mutant IL-6R, is compared with the comparative ΔGeo-Mean value, which reflects the amount of the antigen-binding molecule of interest bound to IL-6R expressing cells. In this case, it is particularly preferred that the concentrations of the antigen-binding molecule of interest used to determine the comparative ΔGeo-Mean values for IL-6R expressing cells and cells expressing mutant IL-6R are adjusted to be equal or substantially equal. As a control antigen-binding molecule, an antigen-binding molecule that has been confirmed to recognize an epitope in IL-6R is used.

If the comparative ΔGeo-Mean of the test antigen-binding molecule for cells expressing the mutant IL-6R is less than the comparative ΔGeo-Mean of the test antigen-binding molecule for IL-6R-expressing cells by at least 80%, preferably by 50%, more preferably by 30 %, and particularly preferably by 15%, then the antigen-binding molecule under investigation "substantially does not bind to cells expressing the mutant IL-6R". The formula for determining the Geo-Mean value (geometric mean) is described in the CELL QUEST (BD biosciences) software user manual. If the comparison shows that the comparative values are essentially equivalent, then it can be determined that the epitope for the test and control antigen-binding molecules is the same.

Antigen binding domain

Within the scope of the present invention, an "antigen-binding domain" may be any structure, provided that it binds to the antigen of interest. Such domains preferably include, for example:

variable regions of heavy chains and light chains of antibodies;

a module of approximately 35 amino acids called the A domain, which is contained in the cell membrane protein avimer in vivo (WO 2004/044011, WO 2005/040229);

adnectin containing the 10Fn3 domain, which binds to the protein part of fibronectin, a glycoprotein expressed on the cell membrane (WO 2002/032925);

an affibody which consists of a three-stranded bundle of 58-amino acids based on the backbone of the IgG binding domain of protein A (WO 1995/001937);

Modeled ankyrin repeat proteins (DARPin), which is a region exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a subunit consisting of a turn containing 33 amino acid residues, two antiparallel helices, and loops repeatedly stacked (WO 2002/020565);

anticalins, and the like, which are four-loop domains that support one side of a barrel structure of eight circularly arranged anti-parallel strands that are highly conserved among lipocalin molecules such as gelatin-associated neutrophil lipocalin (NGAL ) (WO 2003/029462); And

a concave region formed by a parallel layer structure within a horseshoe-shaped structure formed by stacked repeats of the leucine-rich repeat (LRR) module of the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is used in the acquired immunity system in jawless vertebrates such as lamprey and hagfish (WO 2008/016854). Preferred antigen-binding domains of the present invention include, for example, antigen-binding domains having antibody heavy chain and light chain variable regions. Preferred examples of antigen binding domains include "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv 2 (scFv2)", "Fab" and "F(ab')2".

The antigen-binding domains of the antigen-binding molecules of the present invention can bind to an identical epitope. Such an epitope may be present, for example, in a protein containing the amino acid sequence of SEQ ID NO: 1. Alternatively, an epitope may be present in a protein containing amino acids at positions 20-365 of the amino acid sequence of SEQ ID NO: 1. Alternatively, each of the antigen-binding domains of the antigen-binding molecules of the present invention may bind to a different epitope. Within the scope of the present invention, a different epitope may be present, for example, in a protein containing the amino acid sequence of SEQ ID NO: 1. Alternatively, an epitope may be present in a protein containing amino acids at positions 20-365 of the amino acid sequence of SEQ ID NO: 1.

Specificity

"Specific" means that one of the molecules that specifically binds does not show any significant binding to molecules other than one or more binding partner molecules. Moreover, "specific" is also used when the antigen binding domain is specific for a particular epitope among a plurality of epitopes in the antigen. When an epitope bound by an antigen-binding domain is contained in several different antigens, antigen-binding molecules containing the antigen-binding domain can bind to different antigens that have that epitope.

Antibody

In the context of the present invention, "antibody" refers to a natural immunoglobulin or an immunoglobulin obtained by partial or complete synthesis. Antibodies can be isolated from natural sources such as natural plasma and serum, or from culture supernatants of antibody-producing hybridomas. Alternatively, antibodies can be partially or completely synthesized using methods such as genetic recombination. Preferred antibodies include, for example, antibodies of the immunoglobulin isotype or subclass thereof. Known human immunoglobulins include antibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. Of these isotypes, the antibodies of the present invention include IgG1, IgG2, IgG3 and IgG4.

Methods for producing an antibody with the desired binding activity are known to those skilled in the art. The following is an example that describes a method for producing an antibody that binds to IL-6R (anti-IL-6R antibody). Antibodies that bind to an antigen other than IL-6R can also be prepared according to the example described below.

Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Anti-IL-6R antibodies produced are preferably mammalian-derived monoclonal antibodies. Such mammalian-derived monoclonal antibodies include antibodies produced by hybridomas or host cells transformed with an expression vector carrying the antibody gene by genetic engineering techniques. Monoclonal antibodies of the present invention include "humanized antibodies" or "chimeric antibodies".

Monoclonal antibody-producing hybridomas can be obtained using known methods, for example, as described below. In particular, mammals are immunized by conventional immunization methods using the IL-6R protein as the sensitizing antigen. The resulting immune cells are fused with known parental cells by conventional cell fusion techniques. Anti-IL-6R antibody producing hybridomas can then be selected by screening monoclonal antibody producing cells using conventional screening methods.

In particular, monoclonal antibodies are prepared as described below. First, an IL-6R gene whose nucleotide sequence is shown in SEQ ID NO: 2 can be expressed to obtain an IL-6R protein shown in SEQ ID NO: 1, which will be used as a sensitizing antigen for producing an antibody. To do this, the sequence of the gene encoding IL-6R is inserted into a known expression vector, and suitable host cells are transformed with this vector. The desired human IL-6R protein is purified from host cells or their culture supernatants by known methods. To obtain soluble IL-6R from culture supernatants, for example, a protein consisting of amino acids at positions 1-357 in the IL-6R polypeptide sequence of SEQ ID NO: 1, such as described in Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968) express as soluble IL-6R instead of IL-6R protein SEQ ID NO: 1. Purified natural IL-6R protein can also be used as sensitizing antigen.

The purified IL-6R protein can be used as a sensitizing antigen for mammalian immunization. Also, a partial IL-6R peptide can be used as a sensitizing antigen. In this case, the partial peptide can be produced by chemical synthesis based on the amino acid sequence of human IL-6R, or by inserting the partial IL-6R gene into an expression vector. Alternatively, the incomplete peptide can be generated by degradation of the IL-6R protein by a protease. The length and region of the partial IL-6R peptide is not limited to specific embodiments. The preferred region can be arbitrarily selected from the amino acid sequence at amino acid positions 20-357 of the amino acid sequence of SEQ ID NO: 1. The number of amino acids forming the peptide to be used as the sensitizing antigen is preferably at least five or more, six or more, or seven or more. More specifically, a peptide of 8-50 residues, more preferably 10-30 residues, can be used as the sensitizing antigen.

Alternatively, a fusion protein obtained by fusing the desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used for the sensitizing antigen. For example, antibody Fc fragments and peptide tags are preferably used as sensitizing antigens. Vectors for the expression of such fusion proteins can be constructed by fusing in frame the genes encoding two or more desired polypeptide fragments and inserting the fusion gene into an expression vector as described above. Methods for making fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. Press). Methods for producing IL-6R for use as a sensitizing antigen and methods for immunization using IL-6R are specifically described in WO 2003/000883, WO 2004/022754, WO 2006/006693 and the like.

There is no particular limitation on the mammals to be immunized with the sensitizing antigen. However, it is preferred to select mammals based on their compatibility with the parental cells used for cell fusion. Generally, rodents such as mice, rats and hamsters, rabbits and monkeys are preferably used.

The above animals are immunized with the sensitizing antigen by known methods. As a rule, performed methods of immunization include, for example, intraperitoneal or subcutaneous injection of the sensitizing antigen in mammals. In particular, the sensitizing antigen is suitably diluted with PBS (phosphate buffered saline), physiological saline or the like. If desired, a conventional adjuvant such as Freund's complete adjuvant is mixed with the antigen and the mixture is emulsified. The sensitizing antigen is then administered to the mammal several times at intervals of 4-21 days. When immunized with a sensitizing antigen, suitable carriers may be used. In particular, when a low molecular weight partial peptide is used as the sensitizing antigen, it is sometimes desirable to couple the sensitizing antigenic peptide to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

Alternatively, hybridomas producing the desired antibody can be obtained using DNA immunization methods as described below. DNA immunization is an immunization method that stimulates the immune system by expressing a sensitizing antigen in an animal immunized by introducing a vector DNA designed to cause the gene encoding the antigenic protein to be expressed in the animal. Compared to conventional immunization methods in which a protein antigen is administered to the animals to be immunized, DNA immunization is superior in that:

- can be provided with stimulation of the immune system while maintaining the structure of the membrane protein, such as IL-6R; And

- there is no need to purify the antigen for immunization.

To obtain a monoclonal antibody of the present invention using DNA immunization, first, DNA expressing IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The resulting DNA is inserted into an appropriate expression vector, and then it is administered to the animal to be immunized. Preferably used expression vectors include, for example, commercially available expression vectors such as pcDNA3.1. Vectors can be introduced into the body using conventional methods. For example, DNA immunization is carried out using a gene gun to introduce expression vector-coated gold particles into the body cells of the animal to be immunized. Antibodies that recognize IL-6R. can also be obtained by the methods described in WO 2003/104453.

After immunizing the mammal as described above, an increase in serum IL-6R binding antibody titer was confirmed. The immune cells are then harvested from the mammal and then subjected to cell fusion. In particular, splenocytes are preferably used as immune cells.

As the cell to be fused with the above-mentioned immune cells, mammalian myeloma cells are used. The myeloma cells preferably contain a suitable selectable marker for screening. A selectable marker gives the cells characteristics for their survival (or death) under specific culture conditions. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) are known as selectable markers. HGPRT or TK deficient cells have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-responsive cells cannot synthesize DNA in HAT selective media and thus die. However, when cells are fused with normal cells, they can continue to synthesize DNA using the normal cell rescue pathway, and thus they can grow in HAT selective media.

Selection of cells deficient in HGPRT and TK can be carried out in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG) or 5'-bromodeoxyuridine, respectively. Normal cells die as they incorporate pyrimidine analogs into their DNA. Meanwhile, cells that are deficient in these enzymes can survive in a selective medium because they cannot incorporate these pyrimidine analogs. In addition, a selectable marker called G418 resistance conferred by the neomycin resistance gene confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogues). Various types of myeloma cells are known that are suitable for cell fusion.

For example, preferably myeloma cells can be used, including the following cells:

P3(P3x63Ag8, 653) (J. Immunol. (1979) 123 (4), 1548-1550);

P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978)81, 1-7);

NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519);

MPC-11 (Cell (1976) 8(3), 405-415);

SP2/0 (Nature (1978) 276 (5685), 269-270);

FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);

S194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);

R210 (Nature (1979) 277 (5692), 131-133), etc.

Cell fusion between immunocytes and myeloma cells is essentially carried out using known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73: 3-46).

More specifically, cell fusion can be carried out, for example, in a conventional culture medium in the presence of a cell fusion agent. Cell fusion agents include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ). If necessary, an auxiliary such as dimethyl sulfoxide can also be added to improve the efficiency of the fusion.

The ratio of immune cells to myeloma cells can be determined independently, preferably, for example, it is one myeloma cell for every one to ten immune cells. Culture media used for cell fusion include, for example, media that are suitable for growing cell lines, such as RPMI1640 media and MEM media, and other conventional culture media used for this type of cell culture. Additionally, whey supplements such as fetal calf serum (FCS) can preferably be added to the culture medium.

For cell fusion, predetermined amounts of the above immune cells and myeloma cells are mixed in the culture medium described above. The PEG solution (eg, average molecular weight is about 1000 to 6000) is then added, preheated to about 37° C., at a concentration typically of 30% to 60% (w/v). This mixture is then gently stirred to obtain the desired confluent cells (hybridoma). Then, the appropriate culture medium mentioned above is gradually added to the cells, and the mixture is repeatedly centrifuged to remove the supernatant. Thus, cell fusion agents and the like that are unfavorable for hybridoma growth can be removed.

Selection of hybridomas thus obtained can be carried out by culturing using a conventional selection medium, for example HAT medium (culture medium containing hypoxanthine, aminopterin and thymidine). Cells other than the desired hybridomas (non-fused cells) can be killed by continuing culturing in the HAT medium described above for a sufficient period of time. As a rule, the period is from several days to several weeks. Then, hybridomas producing the desired antibody are screened and cloned individually by conventional limiting dilution techniques.

Selection of hybridomas thus obtained can be carried out using a selection medium based on the selectable marker possessed by the myeloma used for cell fusion. For example, selection of cells deficient in HGPRT or TK can be carried out by culturing using HAT medium (culture medium containing hypoxanthine, aminopterin and thymidine). In particular, when HAT-responsive myeloma cells are used for cell fusion, cells successfully fused with normal cells can selectively proliferate in HAT media. Cells other than the desired hybridomas (non-fused cells) can be killed by continuing culturing in the HAT medium described above for a sufficient period of time. In particular, the selection of desired hybridomas can be carried out by culturing, typically for several days to several weeks. Hybridomas producing the desired antibody are then screened and individually cloned by conventional limiting dilution techniques.

The desired antibodies can preferably be selected and individually cloned by screening methods based on a known antigen/antibody reaction. For example, an IL-6R-binding monoclonal antibody can bind to IL-6R expressed on a cell surface. Such a monoclonal antibody can be screened using fluorescence activated cell sorting (FACS). FACS is a system that evaluates antibody cell surface binding by analyzing cells in contact with a fluorescent antibody using a laser beam and measuring the fluorescence emitted by individual cells.

To screen for hybridomas that produce the monoclonal antibodies of the present invention using FACS, IL-6R expressing cells are first obtained. Cells preferably used for screening are mammalian cells in which IL-6R is not naturally expressed. As a control, selective detection of antibody activity that binds to cell surface IL-6R can be performed using untransformed mammalian cells as host cells. In particular, hybridomas producing an anti-IL-6R monoclonal antibody can be isolated by selecting hybridomas that produce an antibody that binds to cells that express IL-6R in an unnatural manner, but not to host cells.

Alternatively, antibody binding activity to immobilized IL-6R expressing cells can be assessed based on the ELISA principle. For example, IL-6R expressing cells are immobilized on the wells of an ELISA plate. The hybridoma culture supernatants are contacted with the immobilized cells in the wells and detect antibodies that bind to the immobilized cells. When the monoclonal antibodies are mouse-derived, the cell-bound antibodies can be detected using an anti-mouse immunoglobulin antibody. Selection of hybridomas producing the desired antibody having antigen-binding ability is carried out by the screening described above, and they can be cloned by the limiting dilution method and the like.

Monoclonal antibody-producing hybridomas thus obtained can be passaged in conventional culture media and stored in liquid nitrogen for an extended period of time.

The hybridomas described above are cultured in a conventional manner and the desired monoclonal antibodies can be obtained from culture supernatants. Alternatively, hybridomas are administered to and grown in compatible mammals and monoclonal antibodies are obtained from ascitic fluid. The first of these methods is suitable for obtaining antibodies with high purity.

Preferably, antibodies encoded by antibody genes that are cloned from antibody-producing cells, such as the hybridomas described above, can also be used. The cloned antibody gene is inserted into a suitable vector and introduced into a host cell to express the antibody encoded by the gene. Methods for isolating antibody genes, inserting genes into vectors, and transforming host cells have already been developed, for example by Vandamme et al. (Eur. J. Biochem. (1990) 192(3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells expressing an anti-IL-6R antibody. For this purpose, total RNA is extracted from hybridomas first. Methods used to extract mRNA from cells include, for example:

- guanidine ultracentrifugation method (Biochemistry (1979) 18(24), 5294-5299), and

- AGPC method (Anal. Biochem. (1987) 162(1), 156-159)

The extracted mRNAs can be purified using an mRNA Purification Kit (GE Healthcare Bioscience) and the like. Alternatively, kits for extracting total mRNA directly from cells are also commercially available, such as the QuickPrep mRNA Purification Kit (GE Healthcare Bioscience). mRNA can be obtained from hybridomas using such kits. cDNAs encoding the V region of an antibody can be synthesized from the resulting mRNAs using reverse transcriptase. cDNA can be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Co.) and the like. Moreover, the SMART RACE cDNA amplification kit (Clontech) and the PCR-based 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23) can be appropriately used for cDNA synthesis and amplification. , 8998-9002; Nucleic Acids Res. (1989) 17(8), 2919-2932). In such a method for synthesizing cDNA, suitable restriction enzyme sites can be inserted at both ends of the cDNA, as described below.

The cDNA fragment of interest is purified from the resulting PCR product and then ligated into vector DNA. Thus, a recombinant vector is constructed and introduced into E. coli and the like. After colony selection, the desired recombinant vector can be obtained from the colony-forming E. coli. It is then examined whether the recombinant vector has a cDNA nucleotide sequence of interest by a known method, such as a dideoxynucleotide chain termination method.

The 5'-RACE method, which uses primers to amplify a variable region gene, is conveniently used to isolate a gene encoding a variable region. First, a 5'-RACE cDNA library is constructed by cDNA synthesis using RNA extracted from hybridoma cells as a template. A commercially available kit, such as the SMART RACE cDNA amplification kit, is appropriately used to synthesize the 5'-RACE cDNA library.

The antibody gene is amplified by a PCR method using the resulting 5'-RACE cDNA library as a template. Primers for amplifying a mouse antibody gene can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary, depending on the immunoglobulin subclass. Thus, it is preferred that the subclass be determined in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

In particular, for example, primers that allow amplification of genes encoding γ1, γ2a, γ2b and γ3 heavy chains and κ and λ light chains are used to isolate genes encoding mouse IgG. Typically, to amplify the gG variable region gene, a primer that hybridizes to a constant region region near the variable region is used as the 3' primer. Meanwhile, the primer supplied with the 5'-RACE cDNA library construction kit is used as the 5' primer.

The PCR products thus amplified are used to reformulate immunoglobulins consisting of a combination of heavy and light chains. Selection of the desired antibody can be carried out using the IL-6R binding activity of the reshaped immunoglobulin as an indicator. For example, when the goal is to isolate an antibody against IL-6R, it is more preferred that the binding of the antibody to IL-6R be specific. Screening for an IL-6R binding antibody can be performed, for example, using the following steps:

(1) contacting an IL-6R expressing cell with an antibody containing the V region encoded by cDNA isolated from the hybridoma;

(2) detection of antibody binding to an IL-6R expressing cell; And

(3) selecting an antibody that binds to an IL-6R expressing cell.

Methods for detecting antibody binding to IL-6R expressing cells are known. In particular, antibody binding to IL-6R expressing cells can be detected using the methods described above, such as FACS. Immobilized samples of IL-6R expressing cells are appropriately used to assess antibody binding activity.

Preferred antibody screening methods that use binding activity as an indicator also include panning methods using phage vectors. Screening methods using phage vectors are advantageous when antibody genes are isolated from libraries of heavy chain and light chain subclasses from a population of cells expressing a polyclonal antibody. Genes encoding heavy chain and light chain variable regions can be linked with an appropriate linker sequence to generate a single chain Fv (scFv). Phages displaying scFv on their surface can be obtained by inserting the gene encoding the scFv into a phage vector. The phages are contacted with the antigen of interest. DNA encoding scFvs having binding activity of interest can then be isolated by harvesting antigen-bound phages. This process can be repeated as needed to increase the content of scFvs having binding activity of interest.

After isolation of the cDNA encoding the V region of the anti-IL-6R antibody of interest, the cDNA is digested with restriction enzymes that recognize the restriction sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and cleave a nucleotide sequence that occurs at low frequency in the nucleotide sequence of an antibody gene. Moreover, preferably, a restriction site for the enzyme that forms the sticky end is introduced into the vector of interest to insert one copy of the cleaved fragment in the correct orientation. The cDNA coding region of the anti-IL-6R antibody V region is digested as described above and inserted into an appropriate expression vector to construct an expression vector for the antibody. In this case, if the gene encoding the constant region of the antibody (C-region) and the gene encoding the above-described V-region are fused in-frame, a chimeric antibody will be obtained. In the context of the present invention, "chimeric antibody" means that the origin of the constant region is different from the variable region. Thus, in addition to heterochimeric mouse/human antibodies, the chimeric antibodies of the present invention include human/human allochimeric antibodies. An expression vector for a chimeric antibody can be constructed by inserting the V region gene into an expression vector that already has a constant region. In particular, for example, a sequence recognized by the first restriction enzyme that cleaves upstream of the V region gene can be appropriately placed at the 5' end of an expression vector containing DNA encoding the desired constant region (C region) of an antibody. An expression vector for a chimeric antibody is constructed by in-frame fusion of two genes cleaved with the same combination of restriction enzymes.

To obtain an anti-IL-6R monoclonal antibody, the antibody genes are inserted into an expression vector such that the genes are expressed under the control of an expression regulating region. The expression control region for antibody expression includes, for example, enhancers and promoters. Moreover, a suitable signal sequence can be attached to the N-terminus so that the expressed antibody is secreted to the outside of the cells. In the examples described below, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as a signal sequence. Meanwhile, other suitable signal sequences can be attached. The expressed polypeptide is cleaved at the C-terminus of the sequence described above and the resulting polypeptide is secreted from the cells as the mature polypeptide. Suitable host cells are then transformed with an expression vector to produce recombinant cells expressing the DNA encoding the IL-6R antibody.

The DNA encoding the heavy chain (H chain) and light chain (L chain) of an antibody are separately inserted into various expression vectors to express the antibody gene. An antibody molecule having H and L chains can be expressed by co-transfection of the same host cell with vectors into which the H chain and L chain genes are respectively inserted. Alternatively, host cells can be transformed with a single expression vector into which DNAs encoding the H and L chains are inserted (see WO 1994/011523).

There are various known host cell/expression vector combinations for producing antibodies by introducing isolated antibody genes into appropriate hosts. All of these expression systems are suitable for isolating the antigen binding domains of the present invention. Suitable eukaryotic cells for use as host cells include animal cells, plant cells and fungal cells. In particular, animal cells include, for example, the following cells.

(1) mammalian cells: CHO, COS, myeloma cells, Chinese hamster kidney (BHK) cells, HeLa, Vero human embryonic kidney (HEK) 293 cells, etc.;

(2) amphibian cells: Xenopus oocytes and the like; And

(3) insect cells: sf9, sf21, Tn5, etc.

Further, as a plant cell, an expression system for antibody genes using cells derived from the genus Nicotiana such as Nicotiana tabacum is known. Cultured callus cells can be appropriately used to transform plant cells.

Moreover, the following cells can be used as fungal cells:

- yeast: the genus Saccharomyces, such as Saccharomyces serevisiae, and the genus Pichia, such as Pichia pastoris; And

- filamentous fungi: the genus Aspergillus, such as Aspergillus niger.

Moreover, expression systems for antibody genes that use prokaryotic cells are also known. For example, when using bacterial cells, E. coli cells, Bacillus subtilis cells, and the like can be suitably used within the scope of the present invention. Expression vectors carrying the antibody genes of interest are introduced into cells by transfection. The transfected cells are cultured in vitro and the desired antibody can be obtained from the culture of the transformed cells.

In addition to the host cells described above, transgenic animals can also be used to produce a recombinant antibody. Therefore, an antibody can be obtained from an animal that has been introduced with a gene encoding the antibody of interest. For example, an antibody gene can be constructed as a fusion gene by inserting in frame into a gene that codes for a protein specifically produced in milk. As the protein secreted in milk, for example, goat β-casein and the like can be used. The DNA fragments containing the fusion gene into which the antibody gene is inserted are injected into a goat embryo, and then the embryo is implanted into a female goat. The desired antibodies can be obtained as a milk fusion protein from the milk produced by a transgenic goat born from a goat that is the recipient of the embryo (or its offspring). In addition, to increase the volume of milk containing the desired antibody produced by the transgenic goat, hormones can be administered to the transgenic goat if necessary, Ebert, K. M. et al., Bio/Technology (1994) 12 (7), 699-702).

When the polypeptide complex described herein is administered to a human, an antigen-binding domain derived from a genetically recombinant antibody that is artificially modified to reduce heterologous antigenicity against a human or the like can be appropriately used as the antigen-binding domain of the complex. Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are suitably prepared by known methods.

The variable region of an antibody used to derive the antigen-binding domain of the polypeptide complex described herein is typically formed by three complementarity-determining regions (CDRs) that are separated by four framework regions (FRs). The CDR is a region that essentially determines the binding specificity of an antibody. The amino acid sequences of the CDRs are highly diverse. On the other hand, FR-forming amino acid sequences often have high identity, even among antibodies with differing binding specificities. Thus, in general, the binding specificity of a particular antibody can be introduced into another antibody by CDR grafting.

A humanized antibody is also referred to as a reshaped human antibody. In particular, humanized antibodies are known that are obtained by grafting the CDR of an antibody from a non-human animal, such as a mouse antibody, into a human antibody, or the like. Also known are standard methods of genetic engineering for the production of humanized antibodies. In particular, for example, overlapped extension PCR is known as a method for transplanting mouse antibody CDRs into human FRs. In overlapped PCR extension, the nucleotide sequence encoding the CDR of the mouse antibody to be transplanted is added to the primers for the synthesis of the FR of the human antibody. Primers are prepared for each of the four FRs. It is generally accepted that when transplanting a mouse CDR into a human FR, selecting a human FR that has a high identity of the mouse FR is advantageous for maintaining CDR function. Thus, it is generally preferable to use a human FR containing an amino acid sequence that has a high identity with the amino acid sequence of the FR adjacent to the CDR of the mouse to be transplanted.

The nucleotide sequences to be ligated are designed to be joined to each other in reading frame. Human FRs are synthesized separately using the appropriate primers. As a result, products are obtained in which the DNA encoding mouse CDRs is linked to individual DNAs encoding FRs. The nucleotide sequences encoding the mouse CDRs of each product are designed to overlap with each other. Complementary chain synthesis is then carried out to hybridize the overlapping CDR regions of the products synthesized using the human antibody gene as a template. With this reaction, human FRs are ligated through mouse CDR sequences.

The full-length V-region gene, in which three CDRs and four FRs are eventually ligated, is amplified using primers that hybridize to its 5' or 3' end, to which the appropriate sequences are added that are recognized by the restriction enzyme. An expression vector for a humanized antibody can be obtained by inserting the DNA prepared as described above and the DNA that encodes the C region of a human antibody into an expression vector such that they are ligated in-frame. After transfection with a recombinant host vector to obtain recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed to obtain a humanized antibody in cell culture (see European Patent Publication No. EP 239400 and International Patent Publication No. WO 1996/002576).

By qualitatively or quantitatively determining and evaluating the antigen-binding activity of the humanized antibody prepared as described above, human FR antibodies can be suitably selected that allow the CDR to form a suitable antigen-binding site when ligated through the CDR. The amino acid residues in the FR can be changed as needed so that the CDRs of the rearranged human antibody form the appropriate antigen binding site. For example, amino acid sequence mutations can be introduced into FR using the PCR method that is used to transplant mouse CDRs into human FRs. More specifically, partial mutations in the nucleotide sequence can be introduced into primers that hybridize to FR. Nucleotide sequence mutations are introduced into FRs synthesized using such primers. Mutant FR sequences having the desired characteristics can be selected by measuring and evaluating the antigen binding activity of the amino acid mutant antibody using the method mentioned above (Cancer Res. (1993) 53: 851-856).

Alternatively, the desired human antibodies can be obtained by immunizing transgenic animals having the full complement of human antibody genes (see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO 1996/034096; WO 1996/033735) by DNA immunization.

Moreover, methods for producing human antibodies by panning using human antibody libraries are also known. For example, the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of a phage by a phage display method. You can select phage expressing scFv that bind to the antigen. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the genes of selected phages. The scFv DNA sequence that binds to the antigen is determined. An expression vector is prepared by fusing the in-frame V-region sequence to the C-region sequence of the desired human antibody, and inserting it into an appropriate expression vector. The expression vector is introduced into cells suitable for expression, such as those described above. A human antibody can be produced by expressing a gene encoding a human antibody in cells. These methods are already known (see WO 1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO 1995/001438; WO 1995/015388).

In addition to the methods described above, B cell cloning methods (identification of each antibody-coding sequence, cloning and isolation of each sequence; use in constructing an expression vector to generate each antibody (IgG1, IgG2, IgG3 or IgG4 , in particular), etc.), as described, for example, Bernasconi et al. (Science (2002) 298: 2199-2202) or WO 2008/081008.

EU numbering system and Kabat numbering system

In accordance with the methods used in the framework of the present invention, the amino acid positions assigned to the CDRs and FRs of antibodies are indicated according to Kabat numbering (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)) . For the purposes of the present invention, when the antigen-binding molecule is an antibody or an antigen-binding fragment, the amino acids of the variable region are indicated according to the Kabat numbering system, while the amino acids of the constant region are indicated according to the EU numbering system based on the Kabat amino acid positions.

Ion Concentration Conditions

Conditions for the concentration of metal ions

In one embodiment of the present invention, the ion concentration refers to the concentration of metal ions. "Metal ions" refers to ions of Group I elements other than hydrogen, such as alkali metals and copper group elements, ions of Group II elements such as alkaline earth metals and zinc group elements, ions of Group III elements other than boron, ions of Group III elements IV, with the exception of carbon and silicon, ions of group VIII elements such as iron group elements and platinum group elements, ions of elements belonging to subgroup A of groups V, VI and VII, and metal elements such as antimony, bismuth and polonium. Metal atoms have the property of releasing valence electrons so that they become cations. This is called the ionization trend. Metals with a high tendency to ionize are considered reactive.

In the context of the present invention, preferred metal ions include, for example, calcium ion. The calcium ion is involved in the modulation of a variety of biological phenomena, including muscle contraction such as skeletal, smooth, and cardiac muscles; activation of movement, phagocytosis, etc. leukocytes; activation of shape change, secretion, etc. platelets; activation of lymphocytes; mast cell activation, including histamine secretion; cellular responses mediated by the catecholamine a receptor or the acetylcholine receptor; exocytosis; the release of substances-transmitters from the endings of neurons; and flow of axoplasm in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and the myosin light chain, which have multiple calcium ion binding sites and are believed to have a common origin in terms of molecular evolution. There are also many known calcium-binding motifs. Such well-known motifs include, for example, cadherin domains, calmodulin EF-hand, protein kinase C C2 domain, coagulation factor IX protein Gla domain, acyaroglycoprotein receptor and mannose-binding receptor C-type lectins, LDL receptor A domains, annexations , thrombospondin type 3 domain, and EGF-like domains.

In the context of the present invention, when the metal ion is a calcium ion, the calcium ion concentration conditions include low calcium ion concentrations and high calcium ion concentrations. "Binding activity varies depending on calcium ion concentrations" means that the antigen-binding activity of an antigen-binding molecule varies due to differences between low and high calcium ion conditions. For example, the antigen-binding activity of an antigen-binding molecule may be higher at a high concentration of calcium ions than at a low concentration of calcium ions. Alternatively, the antigen-binding activity of an antigen-binding molecule may be higher at a low calcium ion concentration than at a high calcium ion concentration.

Within the scope of the present invention, the high concentration of calcium ions is not particularly limited to a specific value; however, the concentration may preferably be selected from values ranging from 100 μM to 10 mM. In another embodiment, the concentration may be selected from values ranging from 200 μM to 5 mm. In an alternative embodiment, the concentration may be selected from values ranging from 400 μM to 3 mm. In another embodiment, the concentration can be selected from values ranging from 200 μm to 2 mm. Moreover, the concentration can be selected from values in the range from 400 μm to 1 mm. In particular, a concentration selected from values ranging from 500 μM to 2.5 mm, which is close to the concentration of calcium ions in plasma (blood) in vivo, is preferred.

Within the scope of the present invention, the low concentration of calcium ions is not particularly limited to a specific value; however, the concentration may preferably be selected from values ranging from 0.1 μM to 30 μM. In another embodiment, the concentration may be selected from values ranging from 0.2 μM to 20 μM. In another embodiment, the concentration may be selected from values ranging from 0.5 μM to 10 μM. In an alternative embodiment, the concentration may be selected from values ranging from 1 μM to 5 μM. Moreover, the concentration can be selected from values ranging from 2 μM to 4 μM. In particular, a concentration selected from values ranging from 1 μM to 5 μM, which is close to the concentration of ionized calcium in early endosomes in vivo, is preferred.

In the context of the present invention, "antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration" means that the antigen-binding activity of an antigen-binding molecule is weaker at a calcium ion concentration selected from values ranging from 0.1 μM to 30 μM than at a calcium ion concentration selected from values ranging from 100 μM to 10 mM. Preferably, this means that the antigen-binding activity of the antigen-binding molecule is weaker at a calcium ion concentration selected from values ranging from 0.5 μM to 10 μM than at a calcium ion concentration selected from values ranging from 200 μM to 5 mM. Particularly preferably, this means that the antigen-binding activity at the calcium ion concentration in the early endosome in vivo is weaker than at the plasma calcium ion concentration in vivo; and in particular, this means that the antigen-binding activity of the antigen-binding molecule is weaker at a calcium ion concentration selected from values ranging from 1 μM to 5 μM than at a calcium ion concentration selected from values ranging from 500 μM to 2, 5 mm.

Whether the antigen-binding activity of an antigen-binding molecule changes with metal ion concentration can be determined, for example, using known measurement methods such as those described in the "Binding Activity" section above. For example, to confirm that the antigen-binding activity of an antigen-binding molecule becomes higher at a high calcium ion concentration than at a low calcium ion concentration, the antigen-binding activity of an antigen-binding molecule at low and high calcium ion concentrations is compared.

Within the scope of the present invention, the expression "antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration" can also be expressed as "the antigen-binding activity of an antigen-binding molecule is higher at a high calcium ion concentration than at a low calcium ion concentration" . In the present description, "antigen-binding activity is lower at low calcium ion concentration than at high calcium ion concentration" is sometimes represented as "antigen-binding capacity is weaker at low calcium ion concentration than at high calcium ion concentration". Also, "the antigen-binding activity at a low calcium ion concentration is reduced so that it is lower than the antigen-binding activity at a high calcium ion concentration" can be represented as "the antigen-binding ability at a low calcium ion concentration is weakened compared to the antigen-binding ability at a high calcium ion concentration" .

When determining antigen-binding activity, conditions other than calcium ion concentration can be appropriately selected by those skilled in the art, and they are not specifically limited. For example, activity can be determined at 37° C. in HEPES buffer. For example, Biacore (GE Healthcare) and the like can be used to determine. When the antigen is a soluble antigen, the antigen-binding activity of the antigen-binding molecule can be assessed by passing the antigen as an analyte over a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane antigen, the binding activity of the antigen-binding molecule to the membrane antigen can be assessed by passing the antigen-binding molecule as an analyte over a chip on which the antigen is immobilized.

Provided that the antigen-binding activity of the antigen-binding molecule of the present invention is weaker at a low calcium ion concentration than at a high calcium ion concentration, the ratio of antigen-binding activity between low and high calcium ion concentrations is not particularly limited. However, the ratio of KD (dissociation constant) of an antigen-binding molecule to antigens at a low calcium ion concentration and KD at a high calcium ion concentration, i. e. the KD (3 μM Ca)/KD (2 mM Ca) value is preferably 2 or more, more preferably 10 or more, and even more preferably 40 or more. The upper limit of the value of KD (3 μM Ca)/KD (2 mM Ca) is not particularly limited, and it can be any value such as 400, 1000 or 10000, provided that the molecule can be obtained by methods known to those skilled in the art.

When the antigen is a soluble antigen, KD (dissociation constant) can be used to display antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, the apparent KD (apparent dissociation constant) can be used to display the activity. KD (dissociation constant) and apparent KD (apparent dissociation constant) can be determined by methods known to those skilled in the art, for example, using Biacore (GE healthcare), a Scatchard plot, or a flow cytometer.

Alternatively, for example, the dissociation rate constant (kd) can preferably also be used as an index to display the ratio of the antigen-binding activity of the antigen-binding molecule of the present invention at low and high calcium concentrations. When the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an index to display the ratio of binding activities, the ratio of dissociation rate constants (kd) at low and high calcium concentrations, i.e. the value of kd (low calcium concentration)/kd (high calcium concentration) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. The upper limit of Kd (low calcium concentration)/kd (high calcium concentration) is not particularly limited, and may be any value such as 50, 100, or 200, as long as the molecule can be obtained by methods known to those skilled in the art.

When the antigen is a soluble antigen, kd (dissociation rate constant) can be used to display antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, the apparent kd (apparent dissociation rate constant) can be used to display the antigen-binding activity. kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be determined by methods known to those skilled in the art, for example using Biacore (GE healthcare) or a flow cytometer. Within the scope of the present invention, when the antigen-binding activity of an antigen-binding molecule is determined at different concentrations of calcium ions, it is preferable to use the same conditions except for the calcium concentrations.

For example, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, including the steps:

(a) determining the antigen-binding activity of the antigen-binding domain or antibody at a low calcium concentration;

(b) determining the antigen-binding activity of the antigen-binding domain or antibody at a high calcium concentration; And

(c) selecting an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium concentration than at a high calcium concentration.

Moreover, an antigen-binding domain or antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies, or a library thereof, including stages:

(a) contacting the antigen with an antigen-binding domain or antibody or library thereof at a high calcium concentration;

(b) incubation at a low calcium concentration of the antigen-binding domain or antibody that has bound to the antigen in step (a); And

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

Moreover, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening antigen-binding domains or antibodies or a library thereof, including the steps :

(a) contacting the antigen with a library of antigen-binding domains or antibodies at a low calcium concentration;

(b) selecting an antigen-binding domain or antibody that did not bind to the antigen in step (a);

(c) allowing the antigen-binding domain or antibody selected in step (c) to bind to the antigen at a high calcium concentration; And

(d) isolating the antigen-binding domain or antibody that has bound to the antigen in step (c).

In addition, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by screening comprising the steps of:

(a) contacting, at a high calcium concentration, a library of antigen-binding domains or antibodies with a column on which the antigen is immobilized;

(b) eluting the antigen-binding domain or antibody that has bound to the column in step (a) from the column at a low calcium concentration; And

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

Moreover, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening process comprising the steps of:

(a) allowing the library of antigen-binding domains or antibodies at low calcium concentration to pass through the column on which the antigen is immobilized;

(b) collecting the antigen-binding domain or antibody that eluted without being bound to the column in step (a);

(c) allowing the antigen-binding domain or antibody assembled in step (b) to bind to the antigen at a high calcium concentration; And

(d) isolating the antigen-binding domain or antibody that has bound to the antigen in step (c).

Moreover, an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium ion concentration than at a high calcium ion concentration, which is one embodiment of the present invention, can be obtained by a screening process comprising the steps of:

(a) contacting the antigen with a library of antigen-binding domains or antibodies at a high calcium concentration;

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

(c) incubation at a low calcium concentration of the antigen-binding domain or antibody obtained in step (b); And

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

The steps described above may be repeated two or more times. Thus, the present invention relates to antigen-binding domains or antibodies, the antigen-binding activity of which is lower at a low concentration of calcium ions than at a high concentration of calcium ions, which are obtained by screening methods that further include the step of repeating two or more times steps (a) - (c) or (a)-(d) in the screening methods described above. The number of cycles of steps (a)-(c) or (a)-(d) is not particularly limited, but is usually 10 or less.

In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody at a low calcium concentration is not particularly limited, as long as it is an antigen-binding activity at an ionized calcium concentration of 0.1 μM to 30 μM, but preferably it is an antigen-binding activity at concentration of ionized calcium from 0.5 μM to 10 μM. More preferably, it is an antigen-binding activity at a concentration of ionized calcium in the early endosome in vivo, in particular from 1 μM to 5 μM. Meanwhile, the antigen-binding activity of the antigen-binding domain or antibody at a high calcium concentration is not particularly limited as long as it is an antigen-binding activity at an ionized calcium concentration of 100 μM to 10 mM, but preferably it is an antigen-binding activity at an ionized calcium concentration of 200 μM. up to 5 mm. More preferably, it is an antigen-binding activity at an in vivo plasma concentration of ionized calcium, in particular from 0.5 mM to 2.5 mM.

The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Conditions other than ionized calcium concentration can be determined by those skilled in the art. The antigen-binding activity of an antigen-binding domain or antibody can be measured as dissociation constant (KD), apparent dissociation constant (apparent KD), dissociation rate constant (kd), apparent dissociation constant (apparent kd), and the like. They can be determined by methods known to those skilled in the art, such as using Biacore (GE healthcare), Scatchard plot, or FACS.

Within the scope of the present invention, the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity is higher at a high calcium concentration than at a low calcium concentration is identical to the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity is lower at a low calcium concentration than at high concentration of calcium.

As long as the antigen-binding activity is higher at a high calcium concentration than at a low calcium concentration, the difference in antigen-binding activity between the high and low concentrations is not particularly limited; however, the antigen-binding activity at a high calcium concentration is preferably two times or more, more preferably 10 times or more, and even more preferably 40 times or more than a low calcium concentration.

The antigen-binding domains or antibodies of the present invention to be screened by the screening methods described above can be any of the antigen-binding domains and antibodies. For example, antigen-binding domains or antibodies as described above can be screened. For example, you can screen for antigen-binding domains or antibodies having natural sequences or sequences with substituted amino acids.

Libraries

In one embodiment, an antigen-binding domain or an antibody of the present invention can be obtained from a library that primarily consists of a plurality of antigen-binding molecules whose sequences differ from each other and whose antigen-binding domains have at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecules, depending on the concentration of ions. Ion concentrations preferably include, for example, a metal ion concentration and a hydrogen ion concentration.

As used herein, a "library" refers to a plurality of antigen-binding molecules or a plurality of fusion polypeptides containing antigen-binding molecules or nucleic acids or polynucleotides encoding their sequences. The sequences of the plurality of antigen-binding molecules or the plurality of antigen-binding molecule-containing fusion polypeptides in the library are not identical, but differ from each other.

In the context of the present invention, the expression "sequences differ from each other" in the expression "a plurality of antigen-binding molecules whose sequences differ from each other" means that the sequences of antigen-binding molecules in the library differ from each other. In particular, in a library, the number of sequences that differ from each other reflects the number of independent clones with different sequences, and may also be referred to as "library size". The library size for a typical phage display library ranges from 10 ⁶ to 10 ¹² . The library size can be increased up to 10 ¹⁴ using known methods such as ribosome display. However, the actual number of phage particles used in panning selection of a phage library is typically 10-10,000 times the size of the library. This excess multiplicity is also referred to as "library equivalents" and means that there are between 10 and 10,000 individual clones that share the same amino acid sequence. Thus, within the scope of the present invention, the expression "sequences differ from each other" means that the sequences of the independent antigen-binding molecules in the library, excluding library equivalents, differ from each other. More specifically, the above means that there are 10 ⁶ to 10 ¹⁴ antigen-binding molecules whose sequences differ from each other, preferably 10 ⁷ to 10 ¹² molecules, more preferably 10 ⁸ to 10 ¹¹ molecules, and particularly preferably 10 ⁸ up to 10 ¹⁰ molecules, the sequences of which differ from each other.

Within the scope of the present invention, the expression "multiple" in the expression "a library mainly consists of a plurality of antigen-binding molecules" in the case of, for example, antigen-binding molecules, fusion polypeptides, polynucleotide molecules, vectors or viruses of the present invention mainly refers to a group of two or more types of substance. For example, when two or more substances differ from each other in a particular characteristic, this means that there are two or more types of substance. Such examples may include, for example, mutant amino acids seen at specific amino acid positions in the amino acid sequence. For example, when there are two or more antigen-binding molecules of the present invention whose sequences are substantially the same, or preferably the same except for non-strict residues or except for specific mutant amino acids at surface-exposed hypervariable positions, there are a plurality of antigen-binding molecules of the present invention. In another example, when there are two or more polynucleotide molecules whose sequences are substantially the same or preferably the same, with the exception of nucleotides encoding non-strict residues or nucleotides encoding mutant amino acids of surface-exposed hypervariable positions, there are a plurality of polynucleotide molecules of the present invention. .

In addition, in the context of the present invention, the expression "mainly consists of" in the expression "a library mainly consists of a plurality of antigen-binding molecules" reflects the number of antigen-binding molecules whose antigen-binding activity varies, depending on the concentration of ions, among independent clones with different sequences in the library . It is particularly preferred that there be at least 10 ⁴ antigen-binding molecules having such binding activity in the library. More preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁵ antigen-binding molecules having such binding activity. Even more preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁶ antigen-binding molecules having such binding activity. Particularly preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁷ antigen-binding molecules having such binding activity. Even more preferably, the antigen-binding domains of the present invention can be obtained from a library containing at least 10 ⁸ antigen-binding molecules having such binding activity. Alternatively, it can also preferably be expressed as the ratio of the number of antigen-binding molecules whose antigen-binding activity varies depending on the concentration of ions and the number of independent clones having different sequences in the library. In particular, the antigen-binding domains of the present invention can be obtained from a library in which the antigen-binding molecules having such binding activity are from 0.1% to 80%, preferably from 0.5% to 60%, more preferably from 1% to 40%. %, even more preferably 2% to 20%, and particularly preferably 4% to 10% independent clones with different sequences in the library. In the case of fused polypeptides, polynucleotide molecules, or vectors, similar expressions may be possible using numbers of molecules or a ratio to the total number of molecules. In the case of viruses, similar expressions using the number of virions or a ratio to the total number of virions may also be possible.

Amino acids that change the antigen-binding activity of antigen-binding domains depending on the concentration of calcium ions

The antigen-binding domains or antibodies of the present invention to be screened by the screening methods described above can be obtained in any way. For example, when the metal ion is a calcium ion, pre-existing antibodies, pre-existing libraries (phage library, etc.), antibodies or libraries derived from hybridomas obtained by immunizing animals or from B cells of immunized animals can be used, antibodies or libraries obtained by introducing calcium-chelating amino acids (such as aspartic acid and glutamic acid) or non-natural amino acid mutations into the antibodies or libraries described above (calcium-chelating amino acids (such as aspartic acid and glutamic acid), libraries with an increased content of non-natural amino acids, libraries obtained by introducing calcium-chelating amino acids (such as aspartic acid and glutamic acid) or unnatural amino acid mutations at specific positions, and the like.

Examples of amino acids that change the antigen-binding activity of antigen-binding molecules depending on the concentration of calcium ions, as described above, can be any type of amino acid, provided that the amino acids form a calcium-binding motif. Calcium binding motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al. (Cell (2000) 102, 275-277); Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490); Moncrief et al (J. Mol. Evol. (1990) 30, 522-562); Chauvaux et al. (Biochem. J. (1990) 265, 261-265); Bairoch and Cox (FEBS Lett. (1990) 269 , 454-456); Davis (New Biol. (1990) 2, 410-419); Schaefer et al. (Genomics (1995) 25, 638-643); Economou et al. (EMBO J. (1990) 9, 349-354); Wurzburg et al. (Structure. (2006) 14, 6, 1049-1058)). In particular, any known calcium-binding motifs, including C-type lectins such as ASGPR, CD23, MBR, and DC-SIGN, can be included in the antigen-binding molecules of the present invention. Preferred examples of such preferred calcium binding motifs also include, in addition to the calcium binding motifs described above, for example, the calcium binding motif in the antigen binding domain of SEQ ID NO: 4.

Moreover, as amino acids that change the antigen-binding activity of the antigen-binding molecules depending on calcium ion concentrations, for example, amino acids having a metal chelating activity can be preferably used. Examples of such metal-chelating amino acids include, for example, serine (Ser(S)), threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)), aspartic acid (Asp(D)) and glutamic acid (Glu(E)).

Positions in the antigen-binding domains containing the amino acids described above are not specifically limited to specific positions, and may be any positions in the heavy chain variable region or light chain variable region that form an antigen-binding domain, provided that they alter the antigen-binding activity of the antigen-binding molecules depending on the concentration of calcium ions. In particular, the antigen-binding domains of the present invention can be derived from a library primarily composed of antigen-binding molecules whose sequences differ from one another and whose heavy chain antigen-binding domains contain amino acids that alter the antigen-binding activity of the antigen-binding molecules depending on calcium ion concentrations. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose heavy chain CDR3 domains contain the amino acids mentioned above. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose heavy chain CDR3 domains contain the above amino acids at positions 95, 96, 100a and/or 101, as indicated according to the Kabat numbering system.

Meanwhile, in one embodiment of the present invention, the antigen-binding domains of the present invention can be obtained from a library mainly consisting of antigen-binding molecules whose sequences differ from each other and whose light chain antigen-binding domains contain amino acids that change the antigen-binding activity of the antigen-binding molecules depending on concentrations of calcium ions. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR1 domains contain the amino acids mentioned above. In another embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR1 domains contain the above amino acids at positions 30, 31 and/or 32, as indicated according to the Kabat numbering system.

In another embodiment, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR2 domains contain the amino acid residues mentioned above. In another embodiment, the present invention relates to libraries primarily composed of antigen-binding molecules whose sequences are different from each other and whose light chain CDR2 domains contain the aforementioned amino acid residues at position 50, as indicated according to the Kabat numbering system.

In another embodiment of the present invention, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR3 domains contain the amino acid residues mentioned above. In an alternative embodiment, the antigen-binding domains of the present invention may be derived from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chain CDR3 domains contain the above-mentioned amino acid residues at position 92, as indicated by the numbering system Kabat.

Moreover, in various embodiments of the present invention, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and in which two or three CDRs selected from the CDR1, CDR2 and CDR3 described above are light chains containing the amino acid residues mentioned above. Moreover, the antigen-binding domains of the present invention can be obtained from a library primarily consisting of antigen-binding molecules whose sequences differ from each other and whose light chains contain the amino acid residues mentioned above at any one or more of positions 30, 31, 32, 50 and /or 92 as specified according to the Kabat numbering system.

In a particularly preferred embodiment, the light chain and/or heavy chain variable region framework sequences of the antigen binding molecule preferably comprise human germline framework sequences. Thus, in one embodiment of the present invention, when the framework sequences are fully human sequences, when such an antigen-binding molecule of the present invention is administered to a human (e.g., for the treatment of diseases), it is expected to induce little or no immunogenic response. In the above context, the expression "comprising a germline sequence" in the context of the present invention means that a portion of the framework sequences of the present invention is identical to a portion of any human germline framework sequences. For example, when the heavy chain FR2 sequence of an antigen binding molecule of the present invention is a combination of heavy chain FR2 sequences of different human germline framework sequences, such molecule is also an antigen binding molecule of the present invention "comprising a germline sequence".

Preferred examples of frameworks include, for example, fully human human framework sequences currently known, which are included on the V-Base website (http://vbase.mrc-cpe.cam.ac.uk/) or others. These framework sequences can be suitably used as the germline sequence contained in the antigen-binding molecule of the present invention. Germline sequences can be categorized according to their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798); Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461); Cox et al. (Nat. Genetics (1994) 7, 162-168)). Appropriate germline sequences can be selected from Vκ, which fall into seven subgroups; Vλ, which are subdivided into ten subgroups; and VH, which are subdivided into seven subgroups.

Fully human VH sequences preferably include, but are not limited to, for example, VH sequences:

VH1 subgroups (eg, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58 and VH1-69);

VH2 subgroups (eg, VH2-5, VH2-26 and VH2-70);

VH3 subgroups (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);

VH4 subgroups (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59 and VH4-61);

subgroups VH5 (VH5-51);

subgroups VH6 (VH6-1); And

subgroups of VH7 (VH7-4 and VH7-81).

They are also described in known documents (Matsuda et al. (J. Exp. Med. (1998) 188, 1973-1975)) and the like, and thus, those skilled in the art can appropriately model antigen-binding molecules according to of the present invention based on the knowledge of these sequences. It is also preferred to use other fully human framework regions or sub-frame regions.

Fully human Vk sequences preferably include, but are not limited to, for example:

A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4, O8, O12, O14 and O18 allocated to the Vk1 subgroup;

A1, A2, A3, A5, A7, A17, A18, A19, A23, O1 and O11 allocated to the Vk2 subgroup;

A11, A27, L2, L6, L10, L16, L20 and L25 allocated to the Vk3 subgroup;

B3 allocated to the Vk4 subgroup;

B2 (also referred to as Vk5-2), allocated to the Vk5 subgroup; And

A10, A14 and A26 allocated to the Vk6 subgroup.

(Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable and Zachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022); Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

Fully human VL sequences preferably include, but are not limited to, 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 allocated to the VL1 subgroup;

V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17 and V2-19 subgrouped VL1;

V3-2, V3-3 and V3-4 allocated to the VL3 subgroup;

V4-1, V4-2, V4-3, V4-4 and V4-6 allocated to the VL4 subgroup; And

V5-1, V5-2, V5-4 and V5-6 subgrouped VL5 (Kawasaki et al. (Genome Res. (1997) 7, 250-261)).

Typically, these framework sequences differ from one another by one or more amino acid residues. These framework sequences can be used in combination with "at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations" of the present invention. Other examples of fully human framework regions used in combination with "at least one amino acid residue that alters the antigen-binding activity of an antigen-binding molecule depending on ion concentrations" of the present invention include, but are not limited to, e.g., KOL, NEWM, REI, EU , TUR, TEI, LAY, and POM (eg, Kabat et al. (1991), supra, Wu et al. (J. Exp. Med. (1970) 132, 211-250)).

Without wishing to be bound by a particular theory, it is believed that one of the reasons to expect that the use of germline sequences will prevent adverse immune responses in most individuals is as follows. As a result of the affinity maturation process during normal immune responses, somatic mutations often occur in the variable regions of immunoglobulins. Such mutations most often occur in the CDR region, the sequences of which are hypervariable, but also affect the remnants of the frame regions. Such framework region mutations do not exist in germline genes, and they are also less likely to be immunogenic in patients. On the other hand, most of the framework sequences expressed from germline genes are present in the normal human population. As a result of immune tolerance, these germline framework regions are expected to have low immunogenicity in patients or no immunogenicity in patients. To maximize the potential for immune tolerance, genes encoding the variable region can be selected from a group of frequently occurring functional germline genes.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl Acad Sci USA (1985) 82, 488-492)) and overlapped extension PCR.

For example, a library that contains a plurality of antigen-binding molecules of the present invention, the sequences of which differ from each other, can be constructed by combining heavy chain variable regions obtained as a randomized library of variable region sequences, where the light chain variable region is selected as a framework sequence, initially containing at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of calcium ions. As a non-limiting example, when the ion concentration is calcium ion concentration, such preferred libraries include, for example, libraries constructed by combining the sequence of the light chain variable region of SEQ ID NO: 4 (Vk5-2) and the heavy chain variable region obtained as randomized variable region sequence library.

Alternatively, the light chain variable region sequence selected as the framework region, initially containing at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule as mentioned above, can be designed to contain different amino acid residues other than the amino acid residues mentioned above. . In the present description, such residues are called non-strict residues. The number and position of non-strict residues is not particularly limited, as long as the antigen-binding activity of the antigen-binding molecule of the present invention varies depending on ion concentrations. In particular, the heavy chain and/or light chain CDR sequences and/or FR sequences may contain one or more non-strict residues. For example, when the ion concentration is calcium ion concentration, non-limiting examples of non-strict residues to be introduced into the light chain variable region sequence of SEQ ID NO: 4 (Vk5-2) include the amino acid residues shown in Tables 1 or 2.

As used herein, non-strict residues refer to the variation in amino acid residues present at hypervariable positions at which several different amino acids are present in the light chain and heavy chain variable regions when the amino acid sequences of known and/or native antibodies or antigen-binding domains are compared. Hypervariable positions are typically located in the CDR regions. In one embodiment, the data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) are useful for determining hypervariable positions in known and/or native antibodies. Moreover, online databases (http://vbase.mrc-cpe.cam.ac.uk/, http://www.bioinf.org.uk/abs/index.html) provide assembled sequences of many light chains and human heavy chains and their positions. Sequence and position information is useful for determining hypervariable positions within the scope of the present invention. In accordance with the present invention, when a certain amino acid position has preferably from about 2 to about 20 possible amino acid residues, preferably from about 3 to about 19, preferably from about 4 to about 18, preferably from 5 to 17, preferably from 6 to 16 , preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, and preferably 10 to 12 possible variants of amino acid residues, this position is hypervariable. In some embodiments, a particular amino acid position may preferably have at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, and preferably about 12 amino acid residue variants.

Alternatively, a library containing a plurality of antigen-binding molecules of the present invention whose sequences differ from each other can be constructed by combining the heavy chain variable regions obtained as a randomized variable region sequence library with light chain variable regions in which at least one amino acid is introduced. a residue that changes the antigen-binding activity of antigen-binding molecules depending on ion concentrations, as mentioned above. When the ion concentration is a calcium ion concentration, non-limiting examples of such libraries preferably include, for example, libraries in which heavy chain variable regions generated as a randomized library of variable region sequences are combined with light chain variable region sequences in which a particular residue (and ) in a germline sequence such as SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3) or SEQ ID NO: 8 (Vk4) is replaced by at least one amino acid residue , which changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of calcium ions. Non-limiting examples of such amino acid residues include amino acid residues in the light chain CDR1. Moreover, non-limiting examples of such amino acid residues include amino acid residues in the light chain CDR2. In addition, non-limiting examples of such amino acid residues also include amino acid residues in the light chain CDR3.

Non-limiting examples of such amino acid residues contained in the light chain CDR1 include residues at positions 30, 31 and/or 32 in the light chain variable region CDR1, as indicated by EU numbering. Moreover, non-limiting examples of such amino acid residues contained in the light chain CDR2 include the amino acid residue at position 50 in the light chain variable region CDR2, as indicated by Kabat numbering. Moreover, non-limiting examples of such amino acid residues contained in the light chain CDR3 include the amino acid residue at position 92 in the light chain variable region CDR3, as indicated by Kabat numbering. These amino acid residues may be present alone or in combination, provided that they form a calcium-binding motif and/or provided that the antigen-binding activity of the antigen-binding molecule varies depending on the concentration of calcium ions. Meanwhile, since troponin C, calmodulin, parvalbumin, and myosin light chain are known, which have multiple calcium ion binding sites and are believed to have a common origin in terms of molecular evolution, it is possible to construct CDR1, CDR2, and/or CDR3 of the light chain such that so that they have their binding motives. For example, domains of cadherin, EF-hand of calmodulin, C2 domain of protein kinase C, Gla domain of coagulation factor IX protein, C-type acyaroglycoprotein receptor and mannose binding receptor domains, LDL receptor A domains, annexia, thrombospondin domain 3 can be used. type, and EGF-like domains as appropriate for the purposes described above.

When heavy chain variable regions obtained as a randomized library of variable region sequences and light chain variable regions in which at least one amino acid residue is introduced that changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of ions, the sequences are combined as described above. light chain variable regions can be designed to contain non-strict residues in a manner similar to that described above. The number and position of such non-strict residues is not specifically limited to specific embodiments, provided that the antigen-binding activity of the antigen-binding molecules of the present invention varies depending on ion concentrations. In particular, the heavy chain and/or light chain CDR sequences and/or FR sequences may contain one or more non-strict residues. When the ion concentration is the calcium ion concentration, non-limiting examples of non-strict residues introduced into the light chain variable region sequence include the amino acid residues shown in Tables 1 and 2.

Preferred heavy chain variable regions to be combined include, for example, randomized variable region libraries. To obtain a randomized library of the variable region, known methods are combined in an appropriate way. In a non-limiting embodiment of the present invention, as a randomized variable region library, it is preferable to use an immune library constructed on the basis of antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infections, individuals with an increased titer of antibodies in the blood as a result of vaccination, patients with cancer or patients with an autoimmune disease.

In another non-limiting embodiment of the present invention, as a randomized variable region library, it is also preferable to use a synthetic library obtained by replacing the CDR sequences of V genes in genomic DNA or functional reshaped V genes with a set of synthetic oligonucleotides containing sequences encoding sets of codons of the appropriate length . In this case, since diversity is observed in the heavy chain CDR3 sequence, only the CDR3 sequence can also be replaced. A criterion for the occurrence of amino acid diversity in the variable region of an antigen-binding molecule is that diversity occurs at amino acid residues at surface-exposed positions of the antigen-binding molecule. A surface-exposed position refers to a position that is considered capable of being exposed to a surface and/or contacting an antigen based on the structure, group of structures, and/or modeled structure of the antigen-binding molecule. Typically, these provisions are CDRs. Preferably, surface exposed positions are determined using coordinates from a 3D model of the antigen binding molecule using a computer program such as the InsightII program (Accelrys). Surface exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Surface exposed positions can be determined using software suitable for protein modeling and the 3D structure information obtained from the antibody. Software that can be used for this purpose preferably includes SYBYL Biopolymer Module software (Tripos Associates). Mostly, or preferably, when the algorithm requires the user to enter a size parameter, the "size" of the sample used in the calculation is set to a radius of about 1.4 Angstroms or less. Moreover, methods for determining surface-exposed areas and areas using personal computer software are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46- 53).

In another non-limiting embodiment of the present invention, as a randomized variable region library, it is also particularly preferable to use a naive library that is constructed from antibody genes derived from lymphocytes of healthy individuals, and whose gene set consists of naive sequences that are antibody sequences with no preferences ( Gejima et al (Human Antibodies (2002) 11, 121-129); Cardoso et al (Scand. J. Immunol. (2000) 51, 337-344)). In the present description, an amino acid sequence containing a naive sequence refers to an amino acid sequence obtained from such a naive library.

In one embodiment of the present invention, the antigen-binding domain of the present invention can be obtained from a library containing a plurality of antigen-binding molecules of the present invention, the sequences of which differ from each other, obtained by combining light chain variable regions, designed as a randomized variable region library, where the variable region heavy chain is selected as a backbone sequence that initially contains "at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule as a function of ion concentrations". When the ion concentration is the calcium ion concentration, non-limiting examples of such libraries preferably include libraries constructed by combining light chain variable regions constructed as a randomized variable region library with the heavy chain variable region sequence of SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1). Alternatively, such a library can be constructed by selecting appropriate light chain variable regions from light chain variable regions that have germline sequences, instead of light chain variable regions constructed as a randomized library of variable region sequences. Such preferred libraries include, for example, libraries in which the heavy chain variable region sequence of SEQ ID NO: 9 (6RL#9-IgG1) or SEQ ID NO: 10 (6KC4-1#85-IgG1) is combined with light chain variable regions, having a germline sequence.

Alternatively, the heavy chain variable region sequence selected as the framework sequence, which initially contains "at least one amino acid residue that alters the antigen-binding activity of the antigen-binding molecule", as mentioned above, can be designed to contain non-strict residues. The number and position of non-strict residues is not particularly limited, as long as the antigen-binding activity of the antigen-binding molecule of the present invention varies depending on ion concentrations. In particular, the heavy chain and/or light chain CDR and/or FR sequences may contain one or more non-strict residues. When the ion concentration is the calcium ion concentration, non-limiting examples of non-strict residues introduced into the heavy chain variable region sequence of SEQ ID NO: 9 (6RL#9-IgG1) include all heavy chain CDR1 and CDR2 amino acid residues and heavy chain CDR3 amino acid residues, except for residues at positions 95, 96 and/or 100a. Alternative non-limiting examples of non-strict residues introduced into the heavy chain variable region sequence of SEQ ID NO: 10 (6KC4-1#85-IgG1) include all heavy chain CDR1 and CDR2 amino acid residues and heavy chain CDR3 amino acid residues, excluding residues at amino acid positions 95 and/or 101.

Alternatively, a library containing a plurality of antigen-binding molecules whose sequences differ from each other can be constructed by combining light chain variable regions designed as a randomized library of variable region sequences or light chain variable regions having germline sequences with heavy chain variable regions, wherein "at least one amino acid residue responsible for an ion concentration-dependent change in the antigen-binding activity of the antigen-binding molecule" has been introduced. When the ion concentration is a calcium ion concentration, non-limiting examples of such libraries preferably include libraries in which light chain variable regions designed as a randomized library of variable region sequences or light chain variable regions having germline sequences are combined with a heavy variable region sequence. a chain in which the specific residue(s) is replaced by at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the concentration of calcium ions. Non-limiting examples of such amino acid residues include heavy chain CDR1 amino acid residues. The following non-limiting examples of such amino acid residues include heavy chain CDR2 amino acid residues. In addition, non-limiting examples of such amino acid residues also include heavy chain CDR3 amino acid residues. Non-limiting examples of such heavy chain CDR3 amino acid residues include amino acids at positions 95, 96, 100a and/or 101 in the heavy chain variable region CDR3, as indicated by Kabat numbering. Moreover, these amino acid residues may be present alone or in combination, provided that they form a calcium-binding motif and/or the antigen-binding activity of the antigen-binding molecule varies depending on the concentration of calcium ions.

When light chain variable regions designed as a randomized library of variable region sequences, or light chain variable regions having a germline sequence, are combined with a heavy chain variable region in which at least one amino acid residue is introduced that alters the antigen-binding activity of the antigen-binding molecule to Depending on the concentration of ions, as mentioned above, the sequence of the heavy chain variable region can also be designed to contain non-strict residues, in a similar way as described above. The number and position of non-strict residues is not particularly limited, as long as the antigen-binding activity of the antigen-binding molecule of the present invention varies depending on ion concentrations. In particular, the heavy chain CDR and/or FR sequences may contain one or more non-strict residues. Moreover, the randomized variable region libraries can preferably be used as the heavy chain variable region CDR1, CDR2 and/or CDR3 amino acid sequences other than the amino acid residues that alter the antigen-binding activity of the antigen-binding molecule. When germline sequences are used as light chain variable regions, non-limiting examples of such sequences include SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3) and SEQ ID NO: 8 (Vk4).

Preferably, any of the amino acids described above that alter the antigen-binding activity of the antigen-binding molecule depending on calcium ion concentrations can be used, provided that they form a calcium-binding motif. In particular, such amino acids include electron donor amino acids. Preferred examples of such electron donor amino acids include serine, threonine, asparagine, glutamic acid, aspartic acid, and glutamic acid.

Conditions for the concentration of hydrogen ions

In one embodiment of the present invention, the ion concentration conditions refer to hydrogen ion concentration conditions or pH conditions. Within the scope of the present invention, the proton concentration, i.e. the nucleus of a hydrogen atom, is used as a synonym for pH (pH). When the activity of hydrogen ions in an aqueous solution is displayed as aH+, the pH is defined as -log10aH+. When the ionic strength of the aqueous solution is low (eg, below 10 ^-3 ), aH+ is substantially equal to the ionic strength of hydrogen. For example, the ionic product of water at 25°C and 1 atmosphere is Kw=aH+aOH=10 ^-14 , and thus, in pure water aH+=aOH=10 ^-7 . In this case, pH=7 is neutral; an aqueous solution whose pH is below 7 is acidic, or whose pH is greater than 7 is alkaline.

In the context of the present invention, when pH conditions are used as ion concentration conditions, the pH conditions include high hydrogen ion concentrations or low pH values, i. acidic pH range, and low hydrogen ion concentrations or high pH values, i.e. range of neutral pH values. "Binding activity varies with pH conditions" means that the antigen-binding activity of an antigen-binding molecule varies due to differences in conditions of high hydrogen ion concentration or low pH values (acidic pH range) and low hydrogen ion concentration or high pH values (neutral pH range). ). This includes, for example, the case where the antigen-binding activity of the antigen-binding molecule is higher in the neutral pH range than in the acidic pH range, and the case where the antigen-binding activity of the antigen-binding molecule is higher in the acidic pH range than in the neutral pH range. pH.

In the present description, the range of neutral pH values is not limited to a specific value, and is preferably selected from the range from pH 6.7 to pH 10.0. In another embodiment, the pH value may be selected from the range of pH 6.7 to pH 9.5. In another embodiment, the pH value may be selected from the range of pH 7.0 to pH 9.0. In another embodiment, the pH value may be selected from the range of pH 7.0 to pH 8.0. A particularly preferred pH value includes a pH of 7.4, which is close to the in vivo plasma (blood) pH.

In the present description, the range of acidic pH values is not limited to a specific value, and is preferably selected from the range from pH 4.0 to pH 6.5. In another embodiment, the pH value may be selected from the range of pH 4.5 to pH 6.5. In another embodiment, the pH value may be selected from the range of pH 5.0 to pH 6.5. In another embodiment, the pH may be selected from the range of pH 5.5 to pH 6.5. A particularly preferred pH includes pH 5.8, which is close to the pH in the early endosome in vivo.

In the context of the present invention, "the antigen-binding activity of an antigen-binding molecule at high hydrogen ion concentration or low pH (acidic pH range) is lower than at low hydrogen ion concentration or high pH (neutral pH range)" means that the antigen-binding activity antigen-binding molecule at a pH value selected from the range from pH 4.0 to pH 6.5 is weaker than at a pH value selected from the range from pH 6.7 to pH 10.0; preferably this means that the antigen-binding activity of the antigen-binding molecule at a pH value selected from the range of pH 4.5 to pH 6.5 is weaker than at a pH selected from the range of pH 6.7 to pH 9.5; more preferably, this means that the antigen-binding activity of the antigen-binding molecule at a pH value selected from the range of pH 5.0 to pH 6.5 is weaker than at a pH value selected from the range of pH 7.0 to pH 9.0 ; even more preferably, this means that the antigen-binding activity of the antigen-binding molecule at a pH value selected from the range of pH 5.5 to pH 6.5 is weaker than at a pH value selected from the range of pH 7.0 to pH 8.0 ; particularly preferably, this means that the antigen-binding activity at the pH of the early endosome in vivo is weaker than the antigen-binding activity at the pH of the plasma in vivo; and specifically, this means that the antigen-binding activity of the antigen-binding molecule at pH 5.8 is weaker than the antigen-binding activity at pH 7.4.

Determination of whether the antigen-binding activity of an antigen-binding molecule changes depending on pH conditions can be carried out, for example, using known measurement methods such as those described in the "Binding Activity" section above. In particular, binding activity is measured under various pH conditions using the measurement methods described above. For example, the antigen-binding activity of an antigen-binding molecule is compared under acidic pH range and neutral pH range conditions to confirm that the antigen-binding activity of the antigen-binding molecule changes such that it is higher under neutral pH range conditions than under acidic pH range conditions.

Moreover, within the scope of the present invention, the expression "antigen-binding activity at high hydrogen ion concentration or low pH value, i.e. in the acidic pH range, is lower than at low hydrogen ion concentration or high pH value, i.e. . in the neutral pH range" can also be expressed as "the antigen-binding activity of the antigen-binding molecule at low hydrogen ion concentration or high pH, i.e. in the neutral pH range, is higher than at high hydrogen ion concentration or low pH , i.e. in the range of acidic pH values". Within the scope of the present invention, "antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the acidic pH range, is lower than at low hydrogen ion concentration or high pH, i.e. in the neutral range pH" can be described as "antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the acidic pH range, is weaker than antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the neutral pH range". Alternatively, "antigen-binding activity at high hydrogen ion concentration or low pH, i.e., in the acidic pH range, is reduced to be lower than at low hydrogen ion concentration or high pH, i.e., in the range neutral pH values" can be described as "antigen-binding activity at high hydrogen ion concentration or low pH value, i.e. in the acidic pH range, is reduced so that it is weaker than antigen-binding activity at low hydrogen ion concentration or high pH values. pH, i.e. in the range of neutral pH values".

Conditions other than hydrogen ion concentration or pH for measurement and antigen-binding activity can be appropriately selected by those skilled in the art and are not specifically limited. The measurement can be carried out, for example, at 37°C using HEPES buffer. The measurement can be carried out, for example, using Biacore (GE Healthcare). When the antigen is a soluble antigen, the antigen-binding activity of the antigen-binding molecule can be determined by evaluating the binding activity of the soluble antigen by applying the antigen as an analyte to a chip on which the antigen-binding molecule is immobilized. When the antigen is a membrane antigen, the binding activity of the membrane antigen can be assessed by applying the antigen-binding molecule as an analyte to a chip on which the antigen is immobilized.

Provided that the antigen-binding activity of the antigen-binding molecule of the present invention at a high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values, is weaker than at a low concentration of hydrogen ions or a high pH value, i.e. in the neutral pH range, the ratio of antigen-binding activity to antigen-binding activity at high hydrogen ion concentration or low pH, i. e. in the range of acidic pH values, and at a low concentration of hydrogen ions or a high pH value, i.e. in the neutral pH range is not particularly limited, and the KD (pH5.8)/KD (pH7.4) value, which is the ratio of the dissociation constant (KD) for an antigen at a high hydrogen ion concentration or a low pH, i.e. in the range of acidic pH values, and KD at low concentration of hydrogen ions or high pH value, i.e. in the neutral pH range, preferably 2 or more; more preferably, the value of KD (pH5.8)/KD (pH7.4) is 10 or more; and even more preferably, the KD (pH5.8)/KD (pH7.4) value is 40 or more. The upper limit of the KD (pH5.8)/KD (pH7.4) value is not particularly limited, and may be any value such as 400, 1000 or 10000, as long as the molecule can be produced by methods known to those skilled in the art.

When the antigen is a soluble antigen, the dissociation constant (KD) can be used as a measure of antigen-binding activity. Meanwhile, when the antigen is a membrane antigen, the apparent dissociation constant (KD) can be used. Dissociation constant (KD) and apparent dissociation constant (KD) can be measured by methods known to those skilled in the art, and Biacore (GE healthcare), Scatchard plot, flow cytometer, and the like can be used.

Alternatively, for example, the dissociation rate constant (kd) can be appropriately used as an indication of the ratio of the antigen-binding activity of an antigen-binding molecule of the present invention between activity at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values, and at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values. When kd (dissociation rate constant) is used as an indicator indicating the ratio of binding activities instead of KD (dissociation constant), the value of kd (in the acidic pH range)/kd (in the neutral pH range), which is the ratio of kd (constant dissociation rate) in relation to the antigen at a high concentration of hydrogen ions or a low pH value, i.e. in the range of acidic values, pH and kd (dissociation rate constant) at a low concentration of hydrogen ions or a high pH value, i.e. in the neutral pH range, preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and even more preferably 30 or more. The upper limit of kd (acidic pH range)/kd (neutral pH range) is not particularly limited, and may be any value such as 50, 100, or 200, as long as the molecule can be produced by methods known in the art. experts in this field.

When the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as the amount of antigen-binding activity, and when the antigen is a membrane antigen, the apparent dissociation rate constant (kd) can be used. Dissociation rate constant (kd) and apparent dissociation rate constant (kd) can be determined by methods known to those skilled in the art, and Biacore (GE healthcare), a flow cytometer, and the like can be used. Within the scope of the present invention, when the antigen-binding activity of an antigen-binding molecule is measured at different concentrations of hydrogen ions, i.e. pH, conditions other than hydrogen ion concentration, i.e. The pH is preferably the same.

For example, an antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which are one of the embodiments contemplated by the present invention, can be obtained by screening antigen-binding domains or antibodies, including the following steps (a)-(c):

(a) obtaining a value of the antigen-binding activity of the antigen-binding domain or antibody in the range of acidic pH values;

(b) obtaining a value of the antigen-binding activity of the antigen-binding domain or antibody in the range of neutral pH values; And

(c) selecting an antigen-binding domain or antibody whose antigen-binding activity is lower in an acidic pH range than in a neutral pH range.

Alternatively, an antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which are one of the embodiments contemplated by the present invention, can be obtained by screening antigen-binding domains or antibodies or their library, including the following steps (a)-(c):

(a) contacting the antigen-binding domain, or antibody, or library thereof, in the neutral pH range, with the antigen;

(b) placing the antigen-binding domain or antibody bound to the antigen in step (a) in an acidic pH range; And

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

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment provided by the present invention, can be obtained by screening antigen-binding domains, or antibodies, or their library, including the following steps (a)-(d):

(a) contacting in the range of acidic pH values of the antigen with a library of antigen-binding domains or antibodies;

(b) selecting an antigen-binding domain or antibody that does not bind to the antigen in step (a);

(c) allowing the antigen-binding domain or antibody selected in step (b) to bind to the antigen in the neutral pH range; And

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

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, ie. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment contemplated by the present invention, can be obtained by a screening method comprising the following steps (a)-(c):

(a) contacting in the range of neutral pH values of the library of antigen-binding domains or antibodies with a column on which the antigen is immobilized;

(b) elution in the acidic pH range from the column of the antigen-binding domain or antibody bound to the column in step (a); And

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

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment contemplated by the present invention, can be obtained by a screening method comprising the following steps (a)-(d):

(a) allowing, in an acidic pH range, a library of antigen-binding domains or antibodies to pass through the column onto which the antigen is immobilized;

(b) collecting the antigen-binding domain or antibody eluted without binding to the column in step (a);

(c) allowing the antigen-binding domain or antibody assembled in step (b) to bind to the antigen in the neutral pH range; And

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

An antigen-binding domain or antibody whose antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values, which is another embodiment contemplated by the present invention, can be obtained by a screening method comprising the following steps (a)-(d):

(a) contacting the antigen with a library of antigen-binding domains or antibodies in the neutral pH range;

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

(c) placing the antigen-binding domain or antibody obtained in step (b) in an acidic pH range; And

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

The steps described above may be repeated two or more times. Thus, the present invention relates to antigen-binding domains and antibodies, the antigen-binding activity of which in the range of acidic pH values is lower than in the range of neutral pH values, which are obtained by a screening method, which further comprises the steps of repeating steps (a) to (c) or (a)-(d) in the screening methods described above. The number of repetitions of steps (a)-(c) or (a)-(d) is not particularly limited; however, usually the number is 10 or less.

In the screening methods of the present invention, the antigen-binding activity of an antigen-binding domain or antibody at high hydrogen ion concentration or low pH, i.e. acidic pH range, is not particularly limited as long as it is antigen-binding activity at pH 4.0 to 6.5, and includes antigen-binding activity at pH 4.5 to 6.6 as preferred pH values. Antigen-binding activity also includes antigen-binding activity at pH 5.0 to 6.5, and antigen-binding activity at pH 5.5 to 6.5 as other preferred pH values. Antigen-binding activity also includes antigen-binding activity at pH in the early endosome in vivo as a more preferred pH, and in particular, antigen-binding activity at pH 5.8. Meanwhile, the antigen-binding activity of an antigen-binding domain or an antibody at low hydrogen ion concentration or high pH, i.e. in the neutral pH range, is not particularly limited as long as it is antigen-binding activity at pH 6.7 to 10, and includes antigen-binding activity at pH 6.7 to 9.5 as preferred pH values. Antigen binding activity also includes antigen binding activity at pH 7.0 to 9.5 and antigen binding activity at pH 7.0 to 8.0 as other preferred pH values. Antigen-binding activity also includes antigen-binding activity at plasma pH in vivo as more preferred pH values, and in particular antigen-binding activity at pH 7.4.

The antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art. Those skilled in the art can appropriately define conditions other than ionized calcium concentration. The antigen-binding activity of an antigen-binding domain or antibody can be estimated based on dissociation constant (KD), apparent dissociation constant (KD), dissociation rate constant (kd), apparent dissociation rate constant (kd), and the like. They can be determined by methods known to those skilled in the art, such as using Biacore (GE healthcare), Scatchard plot, or FACS.

Within the scope of the present invention, the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the range of neutral pH values is higher than at a high concentration of hydrogen ions or a low pH value, i.e. in the acidic pH range, is synonymous with the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity is at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values, is lower than at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values.

Provided that the antigen-binding activity at a low concentration of hydrogen ions or a high pH value, i.e. in the range of neutral pH values is higher than at a high concentration of hydrogen ions or a low pH value, i.e. in the acidic pH range, the difference between antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the range of neutral pH values, and antigen-binding activity at a high concentration of hydrogen ions or a low pH value, i.e. in the range of acidic pH values, not specifically limited; however, antigen-binding activity at low hydrogen ion concentration or high pH, ie. in the neutral pH range, preferably two times or more, more preferably 10 times or more, and even more preferably 40 or more times the antigen-binding activity at high hydrogen ion concentration or low pH, i.e. in the range of acidic pH values.

The antigen-binding domain or antibody of the present invention screened by the screening methods described above may be any antigen-binding domain or antibody, and the above-mentioned antigen-binding domain or antibody can be screened. For example, an antigen-binding domain or antibody having a native sequence can be screened, and an antigen-binding domain or antibody can be screened in which their amino acid sequences have been changed.

The antigen-binding domain or antibody of the present invention to be screened by the screening methods described above can be obtained by any method. For example, conventional antibodies, conventional libraries (phage library, etc.), antibodies or libraries derived from B cells of immunized animals or from hybridomas obtained by immunizing animals, antibodies or libraries (libraries with increased pKa amino acid content) can be used. side chain 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acids, libraries containing amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acid mutations at specific positions, etc.) obtained by introducing amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acid mutations in the antibodies or libraries described above.

Methods for producing an antigen-binding domain or antibody having antigen-binding activity at low hydrogen ion concentration or high pH, i.e. in the range of neutral pH values is higher than at a high concentration of hydrogen ions or a low pH value, i.e. in the acidic pH range, from antigen-binding domains or antibodies derived from hybridomas obtained by immunization of animals or from B-cells of immunized animals, preferably include, for example, an antigen-binding molecule or antibody in which at least one of the amino acids of the antigen-binding domain or antibody is replaced an amino acid with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or a mutation to a non-natural amino acid or antigen-binding domain or antibody into which an amino acid with a side chain pKa of 4.0-8.0 is inserted (for example, histidine and glutamic acid) or a non-natural amino acid such as described in WO 2009/125825.

Mutation sites for amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids are not particularly limited, and may be any position, as long as the antigen-binding activity in the acidic pH range becomes weaker than in the neutral pH range (KD (in the acidic pH range)/KD (in the neutral pH range) or kd (in the acidic pH range)/kd (in the neutral pH range) increases) compared to with the value before replacement or insertion. For example, when the antigen binding molecule is an antibody, the variable region and CDR of the antibody are useful. Those skilled in the art can appropriately determine the number of amino acids to be replaced, or the number of amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or unnatural amino acids to be inserted. Substitution can be made with one amino acid having a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or one unnatural amino acid; insertion of one amino acid having a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or one non-natural amino acid can be carried out; a substitution can be made with two or more amino acids having a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or with two or more unnatural amino acids; and two or more amino acids having a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or two or more unnatural amino acids can be inserted. Alternatively, other amino acids can be removed, added, inserted, and/or substituted in parallel, in addition to substitution for amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, or insertions of amino acids having a side chain pKa chains 4.0-8.0 (eg, histidine and glutamic acid) or non-natural amino acids. Substitution or incorporation of amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or non-natural amino acids can be performed randomly by methods such as histidine scanning, in which alanine in the alanine scanning method known to those skilled in the art areas, replace with histidine. Antigen-binding molecules exhibiting a higher KD (acidic pH range)/KD (neutral pH range) or kd (acidic pH range)/kd (neutral pH range) value than before the mutation can be choose from antigen-binding domains or antibodies that are randomly inserted or mutated to amino acids with a side chain pKa of 4.0-8.0 (eg, histidine and glutamic acid) or unnatural amino acids.

Preferred examples of antigen-binding molecules containing a mutation to amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or non-natural amino acids as described above, and whose antigen-binding activity in the acidic pH range is lower than in the neutral pH range, include antigen-binding molecules whose antigen-binding activity in the neutral pH range after mutation to amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or non-natural amino acids is comparable to the antigen-binding activity before mutation to amino acids with a side chain pKa of 4.0-8.0 (eg histidine and glutamic acid) or unnatural amino acids. Within the scope of the present invention, "an antigen-binding molecule, after mutation to amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid), or non-natural amino acids, has an antigen-binding activity comparable to the antigen-binding activity before mutation to amino acids having a pKa side chain 4.0-8.0 (e.g., histidine and glutamic acid) or non-natural amino acids" means that, upon taking the antigen-binding activity of the antigen-binding molecule prior to mutating to amino acids having a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid), or non-natural amino acids as 100%, the antigen-binding activity of the antigen-binding molecule after mutation to amino acids having a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid), or non-natural amino acids is at least 10 % or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more. Antigen binding activity after mutation to amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids at pH 7.4 may be higher than before mutation to amino acids with a side chain pKa of 4.0 -8.0 (eg, histidine and glutamic acid) or unnatural amino acids at pH 7.4. If the antigen-binding activity of an antigen-binding molecule is reduced due to insertion or substitution with amino acids with a side chain pKa of 4.0-8.0 (e.g., histidine and glutamic acid) or unnatural amino acids, the antigen-binding activity can be made comparable to the antigen-binding activity prior to insertion or substitution with amino acids with side chain pKa 4.0-8.0 (eg, histidine and glutamic acid) or unnatural amino acids by substitution, deletion, insertion and/or insertion of one or more amino acids of the antigen-binding molecule. The present invention also includes antigen-binding molecules whose binding activity is adjusted to be comparable by substitution, deletion, insertion and/or insertion of one or more amino acids after substitution or insertion of an amino acid with a side chain pKa of 4.0-8.0 ( for example, histidine and glutamic acid) or non-natural amino acids.

Meanwhile, when the antigen-binding molecule is a substance containing an antibody constant region, preferred embodiments of antigen-binding molecules whose antigen-binding activity is lower in an acidic pH range than in a neutral pH range include methods in which the antibody constant regions contained in antigen-binding molecules, modified. Specific examples of modified antibody constant regions preferably include the constant regions of SEQ ID NOs: 11, 12, 13 and 14.

Amino acids that alter the antigen-binding activity of the antigen-binding domain depending on hydrogen ion concentration conditions

The antigen binding domains or antibodies of the present invention to be screened by the screening methods described above can be obtained by any method. For example, when the ion concentration conditions are hydrogen ion concentration conditions or pH conditions, conventional antibodies, conventional libraries (phage library, etc.), antibodies or libraries derived from B cells of immunized animals or from hybridomas obtained by immunization can be used. animals, antibodies or libraries (libraries with an increased content of amino acids with a side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or non-natural amino acids, libraries that have been mutated for amino acids with a side chain pKa of 4.0- 8.0 (e.g. histidine and glutamic acid) or unnatural amino acids at specific positions, etc.) obtained by mutating amino acids with a side chain pKa of 4.0-8.0 (e.g. histidine and glutamic acid) or unnatural amino acids in the antibodies or libraries described above.

In one embodiment of the present invention, a library containing a plurality of antigen-binding molecules of the present invention, the sequences of which differ from each other, can also be constructed by combining heavy chain variable regions obtained as a randomized variable region sequence library with light chain variable regions in which introduced "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on the conditions of concentration of hydrogen ions".

Such amino acid residues include, but are not limited to, for example, the amino acid residues contained in the light chain CDR1. Amino acid residues also include, but are not limited to, for example, amino acid residues contained in the light chain CDR2. Amino acid residues also include, but are not limited to, for example, the amino acid residues contained in the light chain CDR3.

The amino acid residues described above contained in the light chain CDR1 include, but are not limited to, for example, amino acid residues at positions 24, 27, 28, 31, 32 and/or 34 according to Kabat numbering in the light chain variable region CDR1. Meanwhile, the amino acid residues contained in the light chain CDR2 include, but are not limited to, for example, the amino acid residues at positions 50, 51, 52, 53, 54, 55 and/or 56 according to Kabat numbering in the light chain variable region CDR2 . Moreover, amino acid residues in the light chain CDR3 include, but are not limited to, for example, amino acid residues at positions 89, 90, 91, 92, 93, 94 and/or 95A according to Kabat numbering, in the CDR3 of the light chain variable region. Moreover, the amino acid residues may be contained alone or they may be contained in a combination of two or more amino acids, provided that they allow the antigen-binding activity of the antigen-binding molecule to vary depending on the concentration of hydrogen ions.

Even when a heavy chain variable region obtained as a randomized variable region sequence library is combined with a light chain variable region described above in which "at least one amino acid residue that changes the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions" is introduced , it can be designed so that non-strict residues are contained in the sequence of the variable region of the light chain in the same way as described above. The number and position of non-strict residues is not specifically limited to a particular embodiment, as long as the antigen-binding activity of the antigen-binding molecule of the present invention changes depending on the hydrogen ion concentration conditions. In particular, the heavy chain and/or light chain CDR and/or FR sequences may contain one or more non-strict residues. For example, the non-strict residues to be introduced in the light chain variable region sequences include, but are not limited to, for example, the amino acid residues shown in Tables 3 and 4. Meanwhile, the amino acid sequences of the light chain variable regions other than the non-strict residues and the amino acid residues that alter antigen-binding activity

antigen-binding molecule depending on the hydrogen ion concentration conditions, respectively include, but are not limited to, germline sequences such as Vk1 (SEQ ID NO: 5), Vk2 (SEQ ID NO: 6), Vk3 (SEQ ID NO: 7) and Vk4 (SEQ ID NO: 8).

(position indicated according to Kabat numbering)

(position indicated according to Kabat numbering)

Any amino acid residue can be suitably used as the above-described amino acid residues that change the antigen-binding activity of the antigen-binding molecule depending on hydrogen ion concentration conditions. In particular, such amino acid residues include amino acids with a side chain pKa of 4.0-8.0. Such electron-donating amino acids preferably include, for example, naturally occurring amino acids such as histidine and glutamic acid, as well as non-natural amino acids such as histidine analogs (US 2009/0035836), m-NO2-Tyr (pKa 7.45), 3.5- Br2-Tyr (pKa 7.21) and 3.5-12-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768). Particularly preferred amino acid residues include, for example, amino acids with a side chain pKa of 6.0-7.0. Such electron-donating amino acid residues preferably include, for example, histidine.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlapped extension PCR can be appropriately used to modify the amino acids of antigen-binding domains. Moreover, various known methods can also be used as an amino acid modification method to replace amino acids with amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci U.S.A. (2003) 100(11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNAs in which an amber suppressor tRNA that is complementary to a UAG codon (amber codon), which is a stop codon, can be appropriately used is linked to a non-natural amino acid.

Preferred heavy chain variable regions that are used in combination include, for example, randomized variable region libraries. Known methods are suitably combined as a method for obtaining a randomized variable region library. In a non-limiting embodiment of the present invention, as a randomized variable region library, an immune library constructed from antibody genes derived from animals immunized with specific antigens, patients with an infection, or individuals with elevated blood antibody titers as a result of vaccination, patients with malignant tumor or lymphocytes from patients with immune diseases.

In another non-limiting embodiment of the present invention, similarly as described above, a synthetic library obtained by replacing the CDR sequences of V genes in genomic DNA or functional reshaped V genes with a set of synthetic oligonucleotides can also be suitably used as a randomized variable region library, containing sequences encoding sets of codons of the appropriate length. In this case, since diversity is observed in the heavy chain CDR3 sequence, only the CDR3 sequence can also be replaced. The basis for obtaining variants of amino acids in the variable region of the antigen-binding molecule is to obtain variants of amino acid residues exposed on the surface of the provisions of the antigen-binding molecule. Surface exposed position refers to a position that is considered capable of surface exposure and/or contact with an antigen based on the structure, group of structures and/or modeled structure of the antigen binding molecule, and typically such positions are CDRs. Preferably, surface exposed positions are determined using coordinates from a 3D model of the antigen binding molecule using a computer program such as the InsightII program (Accelrys). Surface exposed positions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16, 548-558)). Determination of surface exposed positions can be made based on the 3D structure information using software suitable for protein modeling. Software that can be used for this purpose preferably includes SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires the user to enter a size parameter, the "size" of the sample used in the calculation is set to a radius of approximately 1.4 Angstroms or less. Moreover, methods for determining surface-exposed areas and areas using PC software are described by Pacios (Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46-53 ).

In another non-limiting embodiment of the present invention, as a randomized variable region library, it is also particularly useful to use a naive library that is constructed from antibody genes derived from lymphocytes of healthy individuals, and whose gene set consists of naive sequences that are antibody sequences with no preference ( Gejima et al (Human Antibodies (2002) 11, 121-129); Cardoso et al (Scand. J. Immunol. (2000) 51, 337-344)).

FcRn

Unlike the Fcγ receptor, which belongs to the immunoglobulin family, FcRn, in particular human FcRn, is structurally similar to major histocompatibility complex (MHC) class I polypeptides, showing 22%-29% sequence identity with MHC class I molecules (Ghetie et al. Immunol Today (1997) 18 (12): 592-598). FcRn is expressed as a heterodimer consisting of a soluble β- or light chain (β2-microglobulin) complexed with a transmembrane α- or heavy chain. Like the MHC, the FcRn α-chain contains three extracellular domains (α1, α2 and α3) and its short cytoplasmic domain anchors the protein to the cell surface, the α1 and α2 domains interact with the FcRn-binding domain of the antibody Fc region (Raghavan et al. , Immunity (1994) 1:303-315).

FcRn is expressed in the maternal placenta and the mammalian yolk sac and is involved in the transfer of IgG from mother to fetus. Furthermore, in the neonatal rodent small intestine, where FcRn is expressed, FcRn is involved in the transfer of maternal IgG across the brush border epithelium from ingested colostrum or milk. FcRn is expressed in a variety of other tissues and endothelial cell systems of various species. FcRn is also expressed in the adult human endothelium, muscle blood vessels, and sinusoidal capillaries of the liver. FcRn is believed to be involved in maintaining plasma IgG concentration by mediating the recycling of IgG to serum upon binding to IgG. As a rule, FcRn binding to IgG molecules is strongly pH dependent. Optimal binding is observed in the acidic pH range below 7.0.

A human FcRn whose precursor is a polypeptide having the signal sequence of SEQ ID NO: 15 (the signal sequence polypeptide is shown in SEQ ID NO: 16) forms a complex with human β2-microglobulin in vivo. As shown in the Reference Examples described below, soluble human FcRn complexed with β2-microglobulin was produced using conventional recombinant expression methods. The FcRn regions of the present invention can be evaluated for their binding activity to such soluble human FcRn in complex with β2-microglobulin. For the purposes of the present invention, unless otherwise indicated, a human FcRn refers to a form capable of binding to the FcRn binding domain of the present invention. Examples include the complex between human FcRn and human β2-microglobulin.

Fc region

The Fc region contains an amino acid sequence derived from the heavy chain constant region of an antibody. The Fc region is a portion of the antibody heavy chain constant region starting at the N-terminus of the hinge region, which corresponds to the papain cleavage site at amino acid position near position 216 according to the EU numbering system, and contains the hinge region, CH2 and CH3 domains.

The binding activity of the Fc region of the present invention to FcRn, in particular human FcRn, can be measured by methods known to those skilled in the art, as described in the "Binding Activity" section above. Those skilled in the art can define conditions other than pH as appropriate. The antigen-binding activity and the binding activity of the human FcRn of the antigen-binding molecule can be evaluated based on dissociation constant (KD), apparent dissociation constant (KD), dissociation rate constant (kd), apparent dissociation rate constant (kd), and the like. They can be measured by methods known to those skilled in the art. For example, a Biacore (GE healthcare), a Scatchard plot, or a flow cytometer can be used.

When the binding activity of the human FcRn of the Fc region of the present invention is measured, conditions other than pH are not specifically limited and can be appropriately selected by those skilled in the art. Measurements can be made, for example, at 37°C using MES buffer as described in WO 2009125825. Alternatively, the binding activity of the human FcRn Fc region of the present invention can be measured by methods known to those skilled in the art and can be measured using, for example , Biacore (GE Healthcare), etc. The binding activity of an Fc region of the present invention to a human FcRn can be assessed by applying, as an analyte, a human FcRn, an Fc region or an antigen-binding molecule of the present invention containing an Fc region to a chip onto which an Fc region immobilized. a molecule of the present invention containing a human Fc region, or FcRn.

The neutral pH range as the conditions where the Fc region contained in the antigen-binding molecule of the present invention has an FcRn binding activity is generally between pH 6.7 and pH 10.0. Preferably, the neutral pH range is a range indicated by arbitrary pH values from pH 7.0 to pH 8.0, and is preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7 .4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, and especially preferably a pH of 7.4, which is close to the in vivo plasma (blood) pH value. When the binding affinity between the human FcRn binding domain and the human FcRn at pH 7.4 is too low to assess, pH 7.0 can be used instead of pH 7.4. Within the scope of the invention, the range of acidic pH values as conditions where the Fc region contained in the antigen-binding molecule of the present invention has an FcRn binding activity is generally between pH 4.0 and pH 6.5. Preferably, the acidic pH range means pH 5.5 to pH 6.5, particularly preferably pH 5.8 to pH 6.0, which is close to the pH in the early endosome in vivo. with regard to the temperature used as measurement conditions, the binding affinity between the binding domain of human FcRn and human FcRn can be assessed at any temperature from 10°C to 50°C. Preferably, the binding affinity between the human FcRn binding domain and the human FcRn can be determined at 15°C to 40°C. More preferably, the binding affinity between the human FcRn binding domain and the human FcRn can be determined in a similar manner at an arbitrary temperature from 20°C to 35°C, such as any temperature of 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34 and 35°С. In one embodiment of the present invention, the temperature includes, but is not limited to, for example, 25°C.

According to the Journal of Immunology (2009) 182: 7663-7671, the binding activity of natural human IgG1 to human FcRn in the acidic pH range (pH 6.0) is KD 1.7 μM, while this activity is almost undetectable in range of neutral pH values. Thus, in a preferred embodiment, it is possible to screen for antigen binding molecules of the present invention having human FcRn binding activity in the acidic pH range and in the neutral pH range, including antigen binding molecules having a human FcRn binding activity in the acidic pH range of 20 μM (KD) or greater, and the human FcRn binding activity in the neutral pH range is comparable to or greater than the human FcRn binding activity of native human IgG. In a more preferred embodiment, antigen-binding molecules of the present invention can be screened, including antigen-binding molecules that have a human FcRn binding activity in the acidic pH range of 20 μM (KD) or greater and a human FcRn binding activity in the neutral pH range of 40 µM (KD) or stronger. In a further preferred embodiment, antigen-binding molecules of the present invention can be screened, including antigen-binding molecules that have a human FcRn binding activity in the acidic pH range of 0.5 μM (KD) or greater and a human FcRn binding activity in the neutral pH range. is 15 µM (KD) or stronger. The KD values described above are determined by the method described in Journal of Immunology (2009) 182: 7663-7671 (by immobilizing an antigen-binding molecule on a chip and applying a human FcRn as the test sample).

Within the scope of the present invention, preferred Fc regions have human FcRn binding activity in the acidic pH range and in the neutral pH range. When the Fc region initially has human FcRn binding activity in the acidic pH range and in the neutral pH range, it can be used as is. When the Fc region has only little or no human FcRn binding activity in the acidic pH range and/or in the neutral pH range, Fc regions having the desired human FcRn binding activity can be obtained by modifying the amino acids of the antigen binding molecule. Fc regions having the desired human FcRn binding activity in the acidic pH range and/or in the neutral pH range can also be appropriately obtained by modifying the amino acids of the human Fc region. Alternatively, Fc regions having the desired human FcRn binding activity can be obtained by modifying the amino acids of an Fc region that initially has human FcRn binding activity in the acidic pH range and/or in the neutral pH range. Amino acid modifications of the human Fc region that result in such desired binding activity can be identified by comparing the binding activity of human FcRn in the acidic pH range and/or in the neutral pH range before and after the amino acid modification. Those skilled in the art can appropriately modify the amino acids using known methods.

As used herein, "amino acid modification" or "amino acid modification" of an Fc region includes modification to an amino acid sequence that differs from the original Fc region. The parent Fc region can be any Fc region, as long as the variants obtained by modifying the parent Fc region can bind to human FcRn in the neutral pH range.

Moreover, also as the modified Fc region of the present invention, it is preferable to use an Fc region modified from an original Fc region that has already been modified. "Original Fc region" may refer to the polypeptide itself, a composition containing the parent Fc region, or a polynucleotide sequence encoding the parent Fc region. The original Fc-region may contain the Fc-region of a known IgG antibody, obtained by recombination, briefly described in the section "Antibodies". The source of the original Fc regions is not limited, and they can be obtained from a human or any non-human organisms. Such organisms preferably include mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, bovines, horses, camels, and organisms selected from non-human primates. In another embodiment, the parental Fc regions can also be derived from cynomolgus monkeys, marmosets, rhesus monkeys, chimpanzees, or humans. The parent Fc regions can preferably be derived from human IgG1; however, they are not limited to any particular IgG class. This means that a human IgG1, IgG2, IgG3 or IgG4 Fc region can be appropriately used as the parent Fc region, and within the scope of the present invention, it also means that an Fc region of an arbitrary class can preferably be used as the parent Fc region. or an IgG subclass derived from any of the organisms described above. Examples of naturally occurring variants or modified forms of IgG are described in published documents (Curr. Opin. Biotechnol. (2009) 20 (6): 685-91; Curr. Opin. Immunol. (2008) 20 (4), 460-470; Protein Eng Des Sel (2010) 23 (4): 195-202, WO 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338); however, they are not limited to these examples.

Examples of changes include changes with one or more mutations, such as mutations by substituting different amino acid residues for the amino acids of the original Fc regions, by inserting one or more amino acid residues into the original Fc regions, or by deleting one or more amino acids from the original Fc regions. Preferably, the amino acid sequences of the altered Fc regions include at least a portion of the amino acid sequence of the non-native Fc region. Such variants necessarily have less than 100% sequence identity or similarity to their parent Fc region. In a preferred embodiment, the variants have an amino acid sequence identity or similarity of from about 75% to less than 100%, more preferably from about 80% to less than 100%, even more preferably from about 85% to less than 100%, even more preferably, from about 90% to less than 100%, and even more preferably from about 95% to less than 100% with the amino acid sequence of their parent Fc region. In a non-limiting embodiment of the present invention, at least one amino acid differs between the modified Fc region of the present invention and its original Fc region. The amino acid difference between the modified Fc region of the present invention and its original Fc region can also preferably be indicated based on the amino acid differences at the specific amino acid positions described above, according to the EU numbering system.

Fc regions having human FcRn binding activity in the neutral pH range that are contained in the antigen-binding molecules of the present invention can be obtained by any method. In particular, Fc regions having human FcRn binding activity in the neutral pH range can be obtained by modifying the amino acids of an IgG-type human immunoglobulin as the parent Fc region. Fc regions of IgG-type immunoglobulins suitable for modification include, for example, human IgG Fc regions (IgG1, IgG2, IgG3 and IgG4 and their modified forms). Amino acids of any position can be modified to other amino acids, provided that the Fc regions have human FcRn binding activity in the neutral pH range or human FcRn binding activity in the neutral pH range can be increased. When the antigen-binding molecule contains a human IgG1 Fc region as a human Fc region, it is preferable that the resulting Fc region contains a modification that has the effect of enhancing the binding of a human FcRn in the neutral pH range compared to the binding activity of the original human IgG1 Fc region. . Amino acids that allow such modification include, for example, amino acids at positions 221-225, 227, 228, 230, 232, 233-241, 243-252, 254-260, 262-272, 274, 276, 278-289, 291-312, 315-320, 324, 325, 327-339, 341, 343, 345, 360, 362, 370, 375-378, 380, 382, 385-387, 389, 396, 414, 416, 423, 424, 426-438, 440 and 442 according to EU numbering. More specifically, such amino acid modifications include the modifications shown in Table 5. The modification of these amino acids enhances the binding of the IgG-type immunoglobulin Fc region to human FcRn in the neutral pH range.

From the modifications described above, modifications that enhance human FcRn binding in the neutral pH range are appropriately selected for use in the present invention. Particularly preferred amino acids of the modified Fc regions include, for example, amino acids at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 according to the EU numbering system. The binding activity of a human FcRn in the neutral pH range of the Fc region contained in the antigen binding molecule can be increased by replacing at least one amino acid selected from the amino acids described above with another amino acid.

Particularly preferred modifications include, for example:

Met instead of the amino acid at position 237;

Ile instead of the amino acid at position 248;

Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr instead of the amino acid at position 250;

Phe, Trp or Tyr instead of the amino acid at position 252;

Thr instead of amino acid at position 254;

Glu instead of amino acid at position 255;

Asp, Asn, Glu or Gln instead of the amino acid at position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val instead of the amino acid at position 257;

His instead of the amino acid at position 258:

Ala instead of the amino acid at position 265;

Ala or Glu instead of the amino acid at position 286;

His instead of the amino acid at position 289;

Ala instead of the amino acid at position 297;

Ala instead of the amino acid at position 303;

Ala instead of the amino acid at position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr instead of the amino acid at position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr instead of the amino acid at position 308;

Ala, Asp, Glu, Pro, or Arg instead of the amino acid at position 309;

Ala, His or Ile instead of the amino acid at position 311;

Ala or His instead of the amino acid at position 312;

Lys or Arg instead of the amino acid at position 314;

Ala, Asp or His instead of the amino acid at position 315;

Ala instead of the amino acid at position 317;

Val instead of the amino acid at position 332;

Leu instead of the amino acid at position 334;

His instead of the amino acid at position 360;

Ala instead of the amino acid at position 376;

Ala instead of the amino acid at position 380;

Ala instead of the amino acid at position 382;

Ala instead of the amino acid at position 384;

Asp or His instead of the amino acid at position 385;

Pro instead of the amino acid at position 386;

Glu instead of amino acid at position 387;

Ala or Ser instead of the amino acid at position 389;

Ala instead of the amino acid at position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr instead of the amino acid at position 428;

Lys instead of amino acid at position 433;

Ala, Phe, His, Ser, Trp, or Tyr instead of the amino acid at position 434; And

His, Ile, Leu, Phe, Thr or Val instead of the amino acid at position 436 of the Fc region according to EU numbering. Meanwhile, the number of amino acids to be modified is not particularly limited, and it is possible to modify an amino acid in only one region, and it is possible to modify amino acids in two or more regions. Combinations of amino acid modifications at two or more sites include, for example, those described in Table 6.

Antigen binding molecule

In the context of the present invention, the term "antigen-binding molecule" is used in its broadest sense to refer to a molecule containing an antigen-binding domain and an Fc region. In particular, antigen-binding molecules include various types of molecules, so long as they exhibit antigen-binding activity. Molecules in which an antigen binding domain is associated with an Fc region include, for example, antibodies. Antibodies may include single monoclonal antibodies (including agonist and antagonist antibodies), human antibodies, humanized antibodies, chimeric antibodies, and the like. Alternatively, when used as antibody fragments, they preferably include antigen-binding domains and antigen-binding fragments (eg, Fab, F(ab')2, scFv and Fv). Scaffold molecules, where three-dimensional structures, such as the already known stable α/β-barrel protein structure, are used as a scaffold and only some parts of the structures are converted into libraries for constructing antigen-binding domains, are also included in the antigen-binding molecules of the present invention.

The antigen binding molecule of the present invention may contain at least some portions of the Fc region that mediate binding to the FcRn and Fcγ receptor. In a non-limiting embodiment, the antigen-binding molecule includes, for example, antibodies and Fc fusion proteins. A fusion protein refers to a chimeric polypeptide containing a polypeptide having a first amino acid sequence that is linked to a polypeptide having a second amino acid sequence that is not naturally linked. For example, a fusion protein may comprise an amino acid sequence of at least a portion of an Fc region (e.g., a portion of an Fc region responsible for binding to an FcRn or a portion of an Fc region responsible for binding to an Fcγ receptor) and a non-immunoglobulin polypeptide comprising , for example, the amino acid sequence of the ligand-binding domain of the receptor or the receptor-binding domain of the ligand. The amino acid sequences may be in separate proteins that are carried together in a fusion protein, or, as a rule, they may be in the same protein, but they are included in the fusion polypeptide in a new order. Fusion proteins can be generated, for example, by chemical synthesis or genetic recombination techniques to express a polynucleotide encoding the peptide regions in the desired order.

The respective domains of the present invention may be linked together via linkers or directly via polypeptide binding.

Linkers include arbitrary peptide linkers that can be introduced by genetic engineering, synthetic linkers, and linkers as described, for example, in Protein Engineering (1996) 9(3), 299-305. However, peptide linkers are preferred within the scope of the present invention. The length of the peptide linkers is not particularly limited, and may be appropriately selected by those skilled in the art depending on the intended use. The length is preferably five amino acids or more (without particular limitation, the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and 15 amino acids is particularly preferred.

For example, such peptide linkers preferably include:

Ser

Gly⋅Ser

Gly⋅Gly⋅Ser

Ser⋅Gly⋅Gly

Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 17)

Ser⋅Gly⋅Gly⋅Gly (SEQ ID NO: 18)

Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 19)

Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 20)

Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 21)

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 22)

Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 23)

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 24)

(Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 19))n

(Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 20))n

where n is an integer equal to 1 or more.

The length or sequence of the peptide linkers may be appropriately selected by those skilled in the art, depending on the intended use.

Typically, synthetic linkers (chemical crosslinkers) are used to crosslink peptides, and for example:

N-hydroxysuccinimide (NHS),

disuccinimidyl suberate (DSS),

bis(sulfosuccinimidyl)suberate (BS ³ ),

dithiobis(succinimidyl propionate) (DSP),

dithiobis (sulfosuccinimidyl propionate) (DTSSP),

ethylene glycol bis(succinimidyl succinate) (EGS),

ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS),

disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),

bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES),

and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available.

When multiple linkers are used to link respective domains, they may all be of the same type, or they may be of different types.

In addition to the linkers illustrated above, linkers with peptide tags such as His tag, HA tag, myc tag and FLAG tag can also be appropriately used. Moreover, hydrogen bonding, disulfide bonding, covalent bonding, ionic interaction, and bonding properties with each other as a result of their combination can also be appropriately used. For example, the affinity between CH1 and CL of an antibody can be used, and Fc regions derived from the bispecific antibodies described above can also be used to heteroassociate Fc regions. Moreover, disulfide bonds formed between domains can also be appropriately used.

To link the respective domains with a peptide bond, the polynucleotides encoding the domains are linked together in frame. Known methods for linking polynucleotides in frame include methods such as restriction fragment ligation, fusion PCR, and overlap PCR. Such methods may suitably be used alone or in combination to construct the antigen-binding molecules of the present invention. Within the scope of the present invention, the terms "linked" and "fused" or "linked" and "merged" are used interchangeably. These terms mean that two or more elements or components, such as polypeptides, are fused together to form a single structure by any means, including the methods of chemical bonding described above and the methods of genetic recombination. In-frame fusion means, when two or more elements or components are polypeptides, linking two or more in-frame elements to form a continuous longer reading frame while maintaining the correct reading frames of the polypeptides. When two Fab molecules are used as an antigen-binding domain, an antibody can be used as the preferred antigen-binding molecule of the present invention, which is an antigen-binding molecule of the present invention, wherein the antigen-binding domain is linked in-frame to an Fc region via a peptide bond without a linker.

Fcγ receptor

Fcγ receptor (also described as FcγR) refers to a receptor capable of binding to the Fc region of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody and includes all members belonging to the family of proteins essentially encoded by the Fcγ receptor gene. In humans, the family includes FcγRI (CD64), including the isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotype H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including the FcγRIIIa isoform (including the V158 and F158 allotype) and FcγRIIIb (including the FcγRIIIb-NA1 and FcγRIIIb-NA2 allotype); as well as unidentified human FcγRs, FcγR isoforms and their allotypes. However, the Fcγ receptor is not limited to these examples. Without limitation, FcγR includes human, mouse, rat, rabbit and monkey FcγR. FcγR can be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV, CD16-2), as well as unidentified mouse FcγRs, FcγR isoforms, and allotypes thereof. Such preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16) and/or FcγRIIIb (CD16). The polynucleotide sequence and amino acid sequence of FcγRI are shown in SEQ ID NOs: 25 (NM_000566.3) and 26 (NP_000557.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIa (allotype H131) are shown in SEQ ID NO: 27 (BC020823.1) and 28 (AAH20823.1) (allotype R131 is the sequence in which the amino acid at position 166 of SEQ ID NO: 28 is replaced by Arg ), respectively; the polynucleotide sequence and amino acid sequence of FcγIIB are shown in SEQ ID NOs: 29 (BC146678.1) and 30 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcγRIIIa are shown in SEQ ID NOs: 31 (BC033678.1) and 32 (AAH33678.1), respectively; and the polynucleotide sequence and amino acid sequence of FcγRIIIb are shown in SEQ ID NOs: 33 (BC128562.1) and 34 (AAI28563.1), respectively (RefSeq accession number is shown in parentheses in each case).

For example, as described in Reference Example 27 and the like, for FcγRIIIaV, when the V158 allotype is used, unless otherwise indicated, the F158 allotype is used; however, the FcγRIIIa isoform allotype described herein should not be interpreted as being specifically limited.

Whether the Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be assessed by ALPHA screening (Luminescence Proximity Enhanced Assay), Surface Plasmon Resonance (SPR)-based BIACORE method, and others. (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010), in addition to the FACS and ELISA formats described above.

Meanwhile, "Fc ligand" or "effector ligand" refers to a molecule, and preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc/Fc ligand complex. A molecule can originate from any organism. Binding of an Fc ligand to an Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, FcγR, FcγR, FcγR, FcRn, C1q and C3, mannan-binding lectin, mannose receptor, Staphylococcus A protein, Staphylococcus G protein, and viral FcγRs. Fc ligands also include the Fc receptor (FcRH) homologues (Davis et al., (2002) Immunological Reviews 190, 123-136), which are a family of Fc receptors homologous to FcγR. Fc ligands also include unidentified molecules that bind to Fc.

In FcγRI (CD64), including FcγRIa, FcγRIb, and FcγRIc, and FcγRII (CD16), which includes the isoforms FcγRIIIa (including the V158 and F158 allotypes) and FcγRIIIb (including the FcγRIIIb-NA1 and FcγRIIIb-NA2 allotypes), the α-chain that binds with the Fc part of IgG, is associated with a common γ-chain with ITAM responsible for the transmission of an intracellular activating signal. Meanwhile, the cytoplasmic domain of FcγRII (CD32), including FcγRIIa isoforms (including H131 and R131 allotypes) and FcγRIIc, contains ITAM. These receptors are expressed on many cells such as macrophages, mast cells and antigen presenting cells. The activating signal transmitted when these receptors bind to the Fc portion of IgG leads to an increase in the phagocytic activity of macrophages, the production of inflammatory cytokines, degranulation of mast cells, and an increase in the function of antigen-presenting cells. Fcγ receptors having the ability to transmit an activating signal as described above are also referred to as activating Fcγ receptors.

Meanwhile, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM responsible for inhibitory signaling. Binding between FcγRIIb and the B cell receptor (BCR) on B cells suppresses the activating signal from the BCR, resulting in suppression of antibody production via the BCR. Binding of FcγRIII and FcγRIIb on macrophages inhibits phagocytic activity and production of inflammatory cytokines. Fcγ receptors having the ability to transmit an inhibitory signal as described above are also referred to as an inhibitory Fcγ receptor.

ALPHA screening is carried out using ALPHA technology based on the principle described below, where two types of beads are used: donor and acceptor. The luminescent signal is only detected when the molecules bound to the donor beads biologically interact with the molecules bound to the acceptor beads and the two types of beads are close to each other. Excited by the laser beam, the photosensitizer in the donor granules converts the oxygen around the granule into excited singlet oxygen. When singlet oxygen propagates among donor beads and reaches nearby acceptor beads, a chemiluminescent reaction is induced in the acceptor beads. This eventually results in the emission of light. When the molecules bound to the donor beads do not interact with the molecules bound to the acceptor beads, the oxygen produced by the donor beads does not reach the acceptor beads and the chemiluminescent reaction does not occur.

For example, a biotin-labeled antigen-binding molecule containing an Fc region is immobilized on donor beads, and an Fcγ receptor labeled with glutathione S-transferase (GST) is immobilized on acceptor beads. In the absence of an antigen-binding molecule containing a competing Fc region polypeptide variant, the Fcγ receptor interacts with the polypeptide complex containing the wild-type Fc region, resulting in signal induction in the 520-620 nm region. An antigen-binding molecule having an unlabeled variant Fc region competes with an antigen-binding molecule containing a native Fc region to interact with the Fcγ receptor. Relative binding affinity can be determined by quantifying the reduction in fluorescence due to competition. Methods for biotinylating antigen-binding molecules such as antibodies using sulfo-NHS-biotin and the like are known. Appropriate methods for adding a GST tag to the Fcγ receptor include methods that involve fusing polypeptides encoding Feγ and GST in-frame, expressing the fused gene using cells into which a vector to which the gene is operably linked is introduced, and then purifying using a column. with glutathione. The induced signal can preferably be analyzed, for example, by fitting it to a one-way competition model based on non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

When observing their interaction, one of the substances is immobilized as a ligand on a thin gold film on a sensor chip. When light is incident on the rear surface of the sensor chip, so that the overall reflection occurs on the contact surface between the thin gold film and the glass, the intensity of the reflected light is partially reduced in a certain area (SPR signal). The other of the substances, when an interaction is detected, is injected as an analyte onto the surface of the sensor chip. When an analyte binds to a ligand, the mass of the immobilized ligand molecule increases. A change in the refractive index causes a shift in the position of the SPR signal (on the other hand, dissociation causes the signal to shift back to its original position). In the Biacore system, the shift amount mentioned above (i.e., mass change on the surface of the sensor chip) is plotted on the vertical axis, and thus the mass change over time is shown as measurement data (sensogram). The kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensogram curves, and affinity (KD) is determined from the ratio of these two constants. The BIACORE method preferably uses an inhibition assay. Examples of such an inhibition assay are described in Proc. Natl. Acad. Sci USA (2006) 103(11): 4005-4010.

Heterocomplex containing four elements from: two FcRn molecules and one molecule of the activating Fcγ receptor

Crystallographic studies of FcRn and IgG antibodies have demonstrated that the FcRn-IgG complex consists of one IgG molecule per two FcRn molecules, and it is believed that these two molecules bind near the contact surface of the CH2 and CH3 domains located on both sides of the Fc region. IgG (Burmeister et al. (Nature (1994) 372, 336-343)). Meanwhile, as shown in Example 3 below, it was demonstrated that the Fc region of an antibody is capable of forming a four-element complex of: two FcRn molecules and one activating Fcγ receptor molecule (FIG. 48). The formation of this heterocomplex is a phenomenon that was revealed as a result of analysis of the properties of antigen-binding molecules containing an Fc region with FcRn binding activity under neutral pH conditions.

Without being bound by a particular principle, in vivo administered antigen binding molecules may be considered to cause the effects described below on the in vivo pharmacokinetics (plasma retention) of antigen binding molecules and the immune response (immunogenicity) to administered antigen binding molecules by forming heterocomplexes containing the four elements of: Fc region contained in antigen-binding molecules, two FcRn molecules and one Fcγ activating receptor molecule. As described above, in addition to various types of activating Fcγ receptor, FcRn is expressed on immune cells, and the formation of such four-part complexes by antigen-binding molecules on immune cells indicates that affinity for immune cells increases and that cytoplasmic domains assemble, which leads to amplification of the internalization signal and accelerated incorporation into immune cells. The same also applies to antigen-presenting cells, and it is possible that the formation of four-part complexes on the cell membrane of antigen-presenting cells allows for easy incorporation of antigen-binding molecules into antigen-presenting cells. Typically, antigen-binding molecules incorporated into antigen-presenting cells are degraded in the lysosomes of antigen-presenting cells and presented to T cells. As a result, since incorporation of antigen-binding molecules into antigen-presenting cells is mediated by the formation of the four-part complexes described above on the cell membrane of antigen-presenting cells, plasma retention of antigen-binding molecules may be impaired. Similarly, an immune response can be induced (amplified).

For this reason, it is understood that when an antigen-binding molecule having an impaired ability to form such four-part complexes is administered to the body, the plasma retention of the antigen-binding molecules can be increased and the induction of an immune response in the body can be suppressed. Preferred embodiments of such antigen-binding molecules that inhibit the formation of these complexes on immune cells, including antigen-presenting cells, include the three embodiments described below.

(Embodiment 1) An antigen-binding molecule containing an Fc region having an FcRn binding activity under neutral pH range conditions, and whose binding activity for an activating FcγR is lower than the binding activity of a native Fc region for an activating FcγR.

The antigen-binding molecule of Embodiment 1 forms a three-part complex by binding to two FcRn molecules; however, it does not form any complex containing an activating FcγR (FIG. 49). An Fc region whose binding activity to an activating FcγR is lower than the binding activity of a native Fc region to an activating FcγR can be obtained by modifying the amino acids of the native Fc region as described above. Whether the binding activity of the modified Fc region to the activating FcγR is lower than the binding activity to the activating FcγR of the native Fc region can be appropriately examined using the methods described in the "Binding Activity" section above.

Examples of preferred activating Fcγ receptors include FcγRI (CD64), which includes FcγRIa, FcγRIb and FcγRIc; FcγRIIa (including R131 and H131 allotypes); and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes VI58 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2).

For the pH conditions for measuring the binding activity of the Fc region and the Fcγ receptor contained in the antigen-binding molecule of the present invention, conditions in the acidic pH range or in the neutral pH range can be suitably used. The neutral pH range as conditions for measuring the binding activity of the Fc region and Fcγ receptor contained in the antigen-binding molecule of the present invention is generally referred to as pH 6.7 to pH 10.0. Preferably, it is a range, denoted by arbitrary pH values from pH 7.0 to pH 8.0; and preferably, it is selected from 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 and pH 8.0; and especially preferably it is pH 7.4, which is close to the pH of plasma (blood) in vivo. In the context of the present invention, the range of acidic pH values, as a condition for the presence of Fc region and Fcγ receptor binding activity, which the antigen-binding molecule of the present invention has, is generally referred to as pH 4.0 to pH 6.5. Preferably, it is referred to as pH 5.5 to pH 6.5, and particularly preferably it is referred to as pH 5.8 to pH 6.0, which is close to the pH in the early endosome in vivo. With regard to the temperature used as measurement conditions, the binding affinity between the Fc region and the human Fcγ receptor can be assessed at any temperature from 10°C to 50°C. Preferably, a temperature of 15°C to 40°C is used to determine the binding affinity between the human Fc region and the Fcγ receptor. More preferably, similarly, a temperature of 20°C to 35°C, such as any of 20°C, 21°C, 22°C, 23°C, 24°C, is used to determine the binding affinity between the Fc region and the Fcγ receptor. C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C. In one embodiment of the present invention, a non-limiting example is a temperature of 25°C.

In the context of the present invention, "the binding activity of the Fc region variant to the activating Fcγ receptor is lower than the binding activity of the native Fc region to the activating Fcγ receptor" means that the binding activity of the Fc region variant to any of the human Fcγ receptors of FcγRI, FcγRIIa, FcγRIIIa and/or FcγRIIIb is lower than the binding activity of the native Fc region to these human Fcγ receptors. For example, this means that, based on the assay method described above, the binding activity of an antigen-binding molecule containing a variant Fc region is 95% or less, preferably 90% or less, 85% or less, 80% or less, 75% or less, particularly 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 compared to the binding activity of an antigen-binding molecule containing a native Fc region as a control. The native Fc region can be used as the native Fc region, and the Fc regions of wild-type antibodies of various isotypes can also be used.

Meanwhile, the binding activity of the native form to the activating FcγR is preferably the binding activity to the Fcγ receptor for human IgG1. In addition to the modifications described above, the isotype can also be changed to human IgG2, human IgG3, or human IgG4 to reduce Fcγ receptor binding activity. Alternatively, in addition to making the modifications described above, Fcγ receptor binding activity can also be reduced by expressing an antigen-binding molecule containing an Fc region having Fcγ receptor binding activity in hosts that do not add sugar chains, such as Escherichia coli.

As an antigen-binding molecule containing an Fc region to be used as a control, antigen-binding molecules having an Fc region of an IgG monoclonal antibody can be suitably used. The structures of these Fc regions are shown in SEQ ID NO: 1 (A added at the N-terminus of RefSeq accession no. AAC82527.1), SEQ ID NO: 2 (A added at the N-terminus of RefSeq accession no. AAB59393. 1), SEQ ID NO: 3 (RefSeq Accession No. CAA27268.1), and SEQ ID NO: 4 (A added at the N-terminus of RefSeq Accession No. AAB59394.1). In addition, when an antigen-binding molecule containing the Fc region of a specific antibody isotype is used as a test substance, the effect of the binding activity of the antigen-binding molecule containing the Fc region on the Fcγ receptor is examined using the antigen-binding molecule having the Fc region as a control. monoclonal IgG antibody of that particular isotype. Thus, antigen-binding molecules containing an Fc region that has been shown to have high Fcγ receptor binding activity are appropriately selected.

In a non-limiting embodiment of the present invention, preferred examples of Fc regions whose binding activity to an activating FcγR is lower than the binding activity of a native Fc region to an activating FcγR include Fc regions in which one or more amino acids at any of positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, 328 and 329, as indicated by EU numbering, are modified to amino acids that differ from those of the native Fc region, among the amino acids of the Fc region described above. Modifications in the Fc region are not limited to the example described above, and may be, for example, modifications such as deglycosylation (N297A and N297Q), IgG1-L234A/L235A, IgG1-A325A/A330S/P331S, IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A IgG1-L234F/L235E/P331S IgG1-S267E/L328F IgG2-V234A/G237A gG4-L235A/G237A/E318A and IgG4-L236E described in Current Opinion in Biotechnology (2009) 20 (6), 685-691; modifications such as G236R/L328R, L235G/G236R, N325A/L328R and N325LL328R described in WO 2008/092117; amino acid insertions at positions 233, 234, 235 and 237 according to EU numbering; and modifications in the positions described in WO 2000/042072.

In a non-limiting embodiment of the present invention, examples of a suitable Fc region include Fc regions having one or more of the following modifications, as indicated by EU numbering, in the Fc region mentioned above:

the amino acid at position 234 is any of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, or Trp;

the amino acid at position 235 is any of Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg;

the amino acid at position 236 is any of Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, or Tyr;

the amino acid at position 237 is any of Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg;

the amino acid at position 238 is any of Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg;

the amino acid at position 239 is any of Gln, His, Lys, Phe, Pro, Trp, Tyr, or Arg;

the amino acid at position 265 is any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val;

the amino acid at position 266 is any of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr;

the amino acid at position 267 is any of Arg, His, Lys, Phe, Pro, Trp, or Tyr;

the amino acid at position 269 is any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

the amino acid at position 270 is any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

the amino acid at position 271 is any of Arg, His, Phe, Ser, Thr, Trp, or Tyr;

the amino acid at position 295 is any of Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, or Tyr;

the amino acid at position 296 is any of Arg, Gly, Lys, or Pro;

the amino acid at position 297 is any of Ala;

the amino acid at position 298 is any of Arg, Gly, Lys, Pro, Trp, or Tyr;

the amino acid at position 300 is any of Arg, Lys, or Pro;

the amino acid at position 324 is any of Lys or Pro;

the amino acid at position 325 is any of Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val;

the amino acid at position 327 is any of Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

the amino acid at position 328 is any of Arg, Asn, Gly, His, Lys, or Pro;

the amino acid at position 329 is any of Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or Arg;

the amino acid at position 330 is any of Pro or Ser;

the amino acid at position 331 is any of Arg, Gly or Lys; or

the amino acid at position 332 is any of Arg, Lys, or Pro.

(Embodiment 2) An antigen-binding molecule comprising an Fc region having an FcRn-binding activity under neutral pH range conditions, and whose binding activity to an inhibitory FcγR is greater than the binding activity to an activating Fcγ receptor

By linking two FcRn molecules and one inhibitory FcγR molecule, the antigen-binding molecule according to Embodiment 2 can form a complex containing these four elements. However, since one antigen-binding molecule can only bind one FcγR molecule, an antigen-binding molecule in a state associated with an inhibitory FcγR cannot bind to other activating FcγRs (FIG. 50). Moreover, it has been described that antigen-binding molecules that are incorporated into cells in a state associated with an inhibitory FcγR are recycled to the cell membrane and thus escape intracellular degradation (Immunity (2005) 23, 503-514). Thus, it is believed that antigen-binding molecules having selective binding activity for an inhibitory FcγR are unable to form heterocomplexes containing an activating FcγR and two FcRn molecules that elicit an immune response.

Examples of preferred activating Fcγ receptors include FcγRI (CD64) which includes FcγRIa, FcγRIb and FcγRIc; FcγRIIa (including R131 and H131 allotypes); and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). Meanwhile, examples of preferred inhibitory Fcγ receptors include FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

In the context of the present invention, "the binding activity to an inhibitory FcγR is higher than the binding activity to an activating Fcγ receptor" means that the binding activity of the Fc region variant to FcγRIIb is higher than the binding activity to any of the human Fcγ receptors: FcγRI , FcγRIIa, FcγRIIIa and/or FcγRIIIb. For example, this means that, based on the assay method described above, the FcγRIIb binding activity of an antigen-binding molecule containing a variant Fc region is 105% or more, preferably 110% or more, 120% or more, 130% or more, 140 % or more, particularly preferably 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 450% or more, 500% or more, 750% or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more compared to activity binding to any of the human Fcγ receptors of FcγRI, FcγRIIa, FcγRIIIa and/or FcγRIIIb.

Most preferably, the FcγRIIb binding activity is higher than each of the FcγRIa, FcγRIIa (including R131 and H131 allotypes) and FcγRIIIa (including V158 and F158 allotypes) binding activities. FcγRIa shows markedly high affinity for native IgG1; thus, the binding is expected to be saturated in vivo due to the presence of a large amount of endogenous IgG1. For this reason, inhibition of complex formation may be possible even if the FcγRIIb binding activity is higher than the FcγRIIa and FcγRIIIa binding activity and lower than the FcγRIa binding activity.

As an antigen-binding molecule containing an Fc region to be used as a control, antigen-binding molecules having an Fc region of an IgG monoclonal antibody can be suitably used. The structures of these Fc regions are shown in SEQ ID NO: 11 (A added to the N-terminus of RefSeq Accession No. AAC82527.1), SEQ ID NO: 12 (A added to the N-terminus of RefSeq Accession No. AAB59393.1), SEQ ID NO: 13 (RefSeq Accession No. CAA27268.1) and SEQ ID NO: 14 (A added to the N-terminus of RefSeq Accession No. AAB59394.1). In addition, when an antigen-binding molecule containing the Fc region of a specific antibody isotype is used as a test substance, the effect of binding activity of the antigen-binding molecule containing the Fc region to the Fey receptor is examined using the antigen-binding molecule having the Fc region of a monoclonal as a control. IgG antibodies of that particular isotype. Thus, antigen-binding molecules containing an Fc region that has been shown to have high binding activity to the Fcγ receptor are appropriately selected.

In a non-limiting embodiment of the present invention, preferred examples of Fc regions having the ability to selectively bind to an inhibitory FcγR include Fc regions in which, among the amino acids of the Fc region described above, the amino acid at position 328 or 329, as indicated by EU numbering, is modified to an amino acid that is different from the native Fc region amino acid. Moreover, as Fc regions having a selective binding activity for an inhibitory Fcγ receptor, the Fc regions or modifications described in US 2009/0136485 can be appropriately selected.

In another non-limiting embodiment of the present invention, a preferred example is an Fc region having one or more of the following modifications, as indicated by EU numbering, in the above Fc region: the amino acid at position 238 is Asp; or the amino acid at position 328 is Glu.

In another non-limiting embodiment of the present invention, examples of a suitable Fc region include Fc regions having one or more of the following modifications:

replacement of Pro at position 238 in EU numbering with Asp, amino acid in position 237 in EU numbering is Trp, amino acid in position 237 in EU numbering is Phe, amino acid in position 267 in EU numbering is Val, amino acid in position 267 in EU numbering EU is Gln, amino acid at position 268 according to EU numbering is Asn, amino acid at position 271 according to EU numbering is Gly, amino acid at position 326 according to EU numbering is Leu, amino acid at position 326 according to EU numbering is Gln, amino acid EU amino acid at position 326 is Glu, EU amino acid at position 326 is Met, EU amino acid at position 239 is Asp, EU amino acid at position 267 is Ala, EU amino acid at position 234 is Trp, EU amino acid at position 234 is Tyr, EU amino acid at position 237 is Ala, EU amino acid at position 237 is Asp, EU amino acid at position 237 is Glu, amino acid at EU amino acid at position 237 is Leu, EU amino acid at position 237 is Met, EU amino acid at position 237 is Tyr, EU amino acid at position 330 is Lys, EU amino acid at position 330 is is Arg, the amino acid at position 233 according to EU numbering is Asp, the amino acid at position 268 according to EU numbering is Asp, the amino acid at position 268 according to EU numbering is Glu, the amino acid at position 326 according to EU numbering is Asp, the amino acid at position EU 326 is Ser, EU 326 is Thr, EU 323 is He, EU 323 is Leu, EU 323 is Met, the EU amino acid at position 296 is Asp, the EU amino acid at position 326 is Ala, the EU amino acid at position 326 is Asn, and the EU amino acid at position 330 is Met.

(Embodiment 3) An antigen-binding molecule containing an Fc region in which one of the two polypeptides constituting the Fc region has FcRn binding activity under neutral pH range conditions and the other has no FcRn binding activity under neutral pH range conditions.

By binding to one FcRn molecule and one FcγR molecule, the antigen-binding molecule according to Embodiment 3 can form a three-part complex; however, it does not form any heterocomplex containing four elements from two FcRn molecules and one FcγR molecule (Fig. 51). As an Fc region in which one of the two polypeptides constituting the Fc region has FcRn binding activity under neutral pH range conditions and the other does not have any FcRn binding activity under neutral pH range conditions contained in the antigen binding molecule according to embodiment 3, Fc regions derived from bispecific antibodies can be appropriately used. Bispecific antibodies are two types of antibodies that have specificity for different antigens. Bispecific IgG-type antibodies can be secreted from hybridomas (quadromes) obtained by fusing two types of IgG-producing hybridomas (Milstein et al. (Nature (1983) 305, 537-540).

When the antigen-binding molecule according to Embodiment 3 described above is produced using recombination methods such as those described in the Antibody section above, a method can be used in which genes encoding polypeptides that constitute two types of Fc regions of interest , are introduced into cells for their co-expression. However, the resulting Fc regions will be a mixture in which the following components will exist in a molecular ratio of 2:1:1: Fc regions in which one of the two polypeptides forming the Fc region has FcRn binding activity under neutral range conditions pH and the other polypeptide does not have any FcRn binding activity under neutral pH range conditions; Fc regions in which both of the two polypeptides constituting the Fc region have FcRn binding activity under neutral pH range conditions; and Fc regions, in which both of the two polypeptides forming the Fc region do not have any FcRn binding activity under neutral pH range conditions. It is difficult to purify antigen-binding molecules containing the desired combination of Fc regions from the three IgG types.

When preparing antigen-binding molecules according to Embodiment 3 using such recombinant methods, antigen-binding molecules containing a heteromeric combination of Fc regions can preferably be secreted by adding appropriate amino acid substitutions to the CH3 domains constituting the Fc regions.

Specifically, this method is carried out by substituting an amino acid having a larger side chain (protrusion (meaning "raised")) for an amino acid in the CH3 domain of one of the heavy chains, and replacing an amino acid having a smaller side chain (an indentation (meaning "raised")). cavity")) of an amino acid in the CH3 domain of another heavy chain, so that the protrusion is located in the cavity. This allows the formation of a heteromeric H chain and simultaneously inhibits the formation of a homomeric H chain (WO 1996027011; Ridgway et al., Protein Engineering (1996) 9, 617-621; Merchant et al., Nature Biotechnology (1998) 16, 677-681 ).

Moreover, there are also known methods for producing a bispecific antibody using methods for controlling association of polypeptides or association of heteromeric multimers formed by a polypeptide to associate between two polypeptides that form an Fc region. In particular, methods for controlling association of polypeptides can be used to produce a bispecific antibody (WO 2006/106905) in which the amino acid residues forming the contact surface between two polypeptides that form an Fc region are altered to inhibit association between Fc regions having the same sequence, and to ensure the formation of polypeptide complexes formed by two Fc regions with different sequences. Such methods can be used to obtain an antigen-binding molecule according to embodiment 3 of the present invention.

In a non-limiting embodiment of the present invention, the two polypeptides constituting the Fc region derived from the bispecific antibody described above can be suitably used as the Fc region. More specifically, two polypeptides constituting an Fc region can be appropriately used in which, in the amino acid sequence of one of the polypeptides, the amino acid at position 349, as indicated by EU numbering, is Cys and the amino acid at position 366 is Trp, and in amino acid sequence of other polypeptides amino acid at position 356, as indicated by EU numbering, is Cys, amino acid at position 366 is Ser, amino acid at position 368 is Ala, and amino acid at position 407 is Val.

In another non-limiting embodiment of the present invention, two polypeptides constituting an Fc region can be suitably used as the Fc region, in which, in the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to EU numbering is Asp, and in the amino acid sequence of the other polypeptide, the amino acid at position 399 according to EU numbering is Lys. In the above embodiment, the amino acid at position 409 may be Glu instead of Asp, and the amino acid at position 399 may be Arg instead of Lys. Moreover, in addition to the amino acid Lys at position 399, Asp can be appropriately added as the amino acid at position 360, or Asp can be appropriately added as the amino acid at position 392.

In another non-limiting embodiment of the present invention, two polypeptides constituting the Fc region can be suitably used as the Fc region, in which in the amino acid sequence of one of the polypeptides, the amino acid at position 370 according to EU numbering is Glu, and in the amino acid sequence of the other of the polypeptides the amino acid at position 357 according to EU numbering is Lys.

In another non-limiting embodiment of the present invention, two polypeptides constituting the Fc region can be suitably used as the Fc region, in which in the amino acid sequence of one of the polypeptides, the amino acid at position 439 according to EU numbering is Glu, and in the amino acid sequence of the other of the polypeptides the amino acid at position 356 according to EU numbering is Lys.

In another non-limiting embodiment of the present invention, any of the embodiments below, in which the above is combined, can be suitably used as the Fc region:

two polypeptides constituting an Fc region in which, in the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to EU numbering is Asp and the amino acid at position 370 is Glu, and in the amino acid sequence of the other of the polypeptides, the amino acid at position 399 according to EU numbering is is Lys and the amino acid at position 357 is Lys (in this embodiment, the amino acid at position 370 according to EU numbering may be Asp instead of Glu, and the amino acid Asp at position 392 according to EU numbering can be used instead of the amino acid Glu at position 370 according to EU numbering) ;

two polypeptides constituting an Fc region in which, in the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to EU numbering is Asp and the amino acid at position 439 is Glu, and in the amino acid sequence of the other of the polypeptides, the amino acid at position 399 according to EU numbering is is Lys and the amino acid at position 356 is Lys (in this embodiment, the amino acid Asp at position 360 according to EU numbering, the amino acid Asp at position 392 according to EU numbering, or the amino acid Asp at position 439 according to EU numbering can be used instead of the amino acid Glu at position 439 according to EU numbering);

two polypeptides constituting an Fc region in which, in the amino acid sequence of one of the polypeptides, the amino acid at position 370 according to EU numbering is Glu and the amino acid at position 439 is Glu, and in the amino acid sequence of the other of the polypeptides, the amino acid at position 357 according to EU numbering is is Lys and the amino acid at position 356 is Lys; And

two polypeptides constituting an Fc region in which, in the amino acid sequence of one of the polypeptides, the amino acid at position 409 according to EU numbering is Asp, the amino acid at position 370 is Glu, and the amino acid at position 439 is Glu, and in the amino acid sequence of the other of polypeptides, the amino acid at position 399 according to EU numbering is Lys, the amino acid at position 357 is Lys, and the amino acid at position 356 is Lys (in this embodiment, the amino acid at position 370 according to EU numbering may not be replaced by Glu, and, moreover, when the amino acid at position 370 is not replaced by Glu, the amino acid at position 439 may be Asp instead of Glu, or the Asp amino acid at position 392 may be used instead of the Glu amino acid at position 439).

In addition, in another non-limiting embodiment of the present invention, two polypeptides constituting an Fc region can also be appropriately used, in which in the amino acid sequence of one of the polypeptides, the amino acid at position 356 according to EU numbering is Lys, and in the amino acid sequence of the other of the polypeptides the amino acid at position 435 according to EU numbering is Arg; and the amino acid at position 439 is Glu.

In another non-limiting embodiment of the present invention, two polypeptides constituting an Fc region can be suitably used, in which, in the amino acid sequence of one of the polypeptides, the amino acid at position 356 according to EU numbering is Lys and the amino acid at position 357 is Lys, in the amino acid sequence of the other of the polypeptides, the amino acid at position 370 according to EU numbering is Glu, the amino acid at position 435 is Arg, and the amino acid at position 439 is Glu.

These antigen-binding molecules of Embodiments 1-3 are expected to be able to reduce immunogenicity and increase plasma retention compared to antigen-binding molecules capable of forming four-part complexes.

Decreased immune response (decreased immunogenicity)

Whether the immune response against the antigen-binding molecule of the present invention can be modified can be assessed by measuring the response in an organism to which a pharmaceutical composition containing the antigen-binding molecule as an active ingredient has been administered. The body's responses include mainly two immune responses: cellular immunity (induction of cytotoxic T cells that recognize peptide fragments of antigen-binding molecules associated with MHC class I) and humoral immunity (induction of the production of antibodies that bind to antigen-binding molecules). With respect to proteinaceous pharmaceuticals in particular, the production of antibodies against administered antigen-binding molecules is referred to as immunogenicity. There are two types of immunogenicity assessment methods: in vivo antibody production assessment methods and in vitro immune cell response assessment methods.

The immune response (immunogenicity) in vivo can be assessed by measuring the antibody titer after administration of antigen-binding molecules into the body. For example, antibody titers are measured after administration of antigen-binding molecules A and B to mice. When the antibody titer for antigen-binding molecule A exceeds the antibody titer for antigen-binding molecule B, or when after administration to several mice, when antigen-binding molecule A is administered, the occurrence of increased antibody titer in mice is higher, then A is considered to have higher immunogenicity than B. Antibody titers can be measured using methods for detecting molecules that specifically bind to introduced molecules using ELISA, ECL or SPR, which are known to those skilled in the art (J. Pharm. Biomed. Anal. (2011) 55 (5), 878-888 ).

Methods for assessing the body's immune response to antigen-binding molecules (immunogenicity) in vitro include methods consisting in reacting in vitro human peripheral blood mononuclear cells isolated from donors (or fractionated cells thereof) with antigen-binding molecules and measuring the number of cells or the percentage of helper T cells and the like, which react or proliferate, or the amount of cytokines produced (Clin. Immunol. (2010) 137 (1), 5-14; Drug R D. (2008) 9 (6), 385-396). For example, when evaluating antigen-binding molecules A and B with such in vitro immunogenicity tests, when the response for antigen-binding molecule A is greater than that for antigen-binding molecule B, or when multiple donors are evaluated and the positivity rate for antigen-binding molecule A is higher, A is then considered to be more immunogenic than B.

Without wishing to be bound by a particular theory, since antigen-binding molecules having FcRn-binding activity in the neutral pH range can form tetrameric heterocomplexes containing two FcRn molecules and one FcγR molecule on the cell membrane of antigen-presenting cells, it is believed that an immune response is easily induced due to enhanced incorporation into antigen presenting cells. There are phosphorylation sites in the intracellular domains of FcγR and FcRn. Typically, phosphorylation of the intracellular domains of receptors expressed on the cell surface occurs upon assembly of the receptors, and their phosphorylation causes internalization of the receptors. Assembly of intracellular FcγR domains does not occur even though native IgG1 forms a dimeric FcγR/IgG1 complex on antigen-presenting cells. However, in the case where an IgG molecule having FcRn binding activity under conditions of the neutral pH range forms a complex containing four FcγR elements/two FcRn/IgG molecules, assembly of the three intracellular domains of FcγR and FcRn occurs, and it is possible that as a result, internalization of a heterocomplex containing four elements from FcγR/two FcRn/IgG molecules. It is believed that heterocomplexes containing four elements from FcγR/two FcRn/IgG molecules are formed on antigen-presenting cells co-expressing FcγR and FcRn, and it is possible that the number of antibody molecules included in antigen-presenting cells thereby increases, resulting in a decrease in immunogenicity. It is believed that by inhibiting the above-described complex formation on antigen-presenting cells using any of the methods according to embodiments 1, 2, or 3 found within the scope of the present invention, incorporation into antigen-presenting cells can be reduced and therefore immunogenicity can be increased.

Improved pharmacokinetics

Without being bound by a particular principle, the reasons why the number of antigens that one antigen-binding molecule can bind increases and why the decrease in plasma antigen concentration accelerates after ensuring incorporation into the cells of the body when administered to the body, for example, an antigen-binding molecule containing an Fc region, having human FcRn binding activity under neutral pH range conditions, and an antigen-binding domain whose antigen-binding activity varies with ion concentration conditions such that antigen-binding activity under acidic pH range conditions is lower than antigen-binding activity in the neutral pH range , can be explained, for example, as follows.

For example, when the antigen-binding molecule is an antibody that binds to a membrane antigen, the antibody introduced into the body binds to the antigen and is then taken up by internalization into endosomes in cells along with the antigen while the antibody continues to be bound to the antigen. The antibody is then transferred to lysosomes while the antibody continues to be bound to the antigen, and the antibody is degraded by the lysosome along with the antigen. Internalization-mediated elimination from plasma is referred to as antigen-dependent elimination, and such elimination has been described for numerous antibody molecules (Drug Discov Today (2006) 11(1-2): 81-88). When one IgG antibody molecule binds to antigens in a bivalent manner, one antibody molecule is internalized, the antibody continues to be associated with two antigen molecules, and is degraded in the lysosome. Thus, in the case of conventional antibodies, one IgG antibody molecule cannot bind to three or more antigen molecules. For example, one IgG antibody molecule having neutralizing activity cannot neutralize three or more antigen molecules.

The relatively prolonged retention (slow elimination) of IgG molecules in plasma is a consequence of the function of the human FcRn, which is known as a rescue receptor for IgG molecules. When taken up into endosomes by pinocytosis, IgG molecules bind to human FcRn expressed in endosomes under the acidic conditions of the endosomes. While IgG molecules that have not bound to human FcRn are transported to lysosomes where they are degraded, IgG molecules that are bound to human FcRn are transported to the cell surface and re-enter plasma by dissociation from human FcRn under neutral conditions. in plasma.

Alternatively, when the antigen binding molecule is an antibody that binds to a soluble antigen, the antibody introduced into the body binds to the antigen and then is taken up into cells while the antibody continues to be bound to the antigen.

Most antibodies incorporated into cells bind to FcRn in endosomes and translocate to the cell surface. Antibodies dissociate from human FcRn under neutral plasma conditions and are released into the external environment of the cells. However, antibodies having conventional antigen-binding domains, the antigen-binding activity of which does not change depending on ion concentration conditions such as pH, are released into the external environment of the cells while remaining associated with antigens; thus, they are unable to re-bind with antigens. Thus, similar to antibodies that bind to membrane antigens, one conventional IgG antibody molecule, whose antigen-binding activity does not change depending on ion concentration conditions such as pH, is unable to bind to three or more antigen molecules.

Antibodies that bind to antigens in a pH-dependent manner, that bind strongly to antigens under conditions of the neutral pH range of plasma and dissociate from antigens under conditions of the acidic pH range of endosomes (antibodies that bind to antigens under conditions of the neutral pH range and dissociate under conditions of the acidic pH range), and antibodies that bind to antigens in a calcium ion concentration-dependent manner that bind strongly to antigens under conditions of high plasma calcium ions and dissociate from antigens under conditions of low endosome calcium ions (antibodies , which bind to antigens under conditions of high calcium ion concentration and dissociate under conditions of low calcium ion concentration) can dissociate from antigens in endosomes. Antibodies that bind to antigens in a pH dependent manner, or antibodies that bind to antigens in a calcium ion concentration dependent manner, are able to bind again to antigens after they have dissociated from the antigens and are recycled to plasma by the FcRn. Thus, one antibody molecule can repeatedly bind to several antigen molecules. Meanwhile, antigens bound to antigen-binding molecules dissociate from antibodies in endosomes and are degraded in lysosomes without being recycled to plasma. By introducing such antigen-binding molecules into organisms, the incorporation of antigens into cells is accelerated and the plasma concentration of antigen can be reduced.

Incorporation into cells of antigens to which antigen-binding molecules bind is further accelerated by conferring the ability to bind human FcRn under conditions of the neutral pH range (pH 7.4) to antibodies that bind to antigens in a pH-dependent manner that bind strongly to antigens under conditions of the pH range. neutral plasma pH and dissociate from antigens under acidic pH range conditions in endosomes (antibodies that bind to antigens under neutral pH range conditions and dissociate under acidic pH range conditions), and antibodies that bind to antigen in a concentration dependent manner calcium ions in a manner that binds strongly to antigens under conditions of high plasma calcium ions and dissociates from antigens under conditions of low calcium ions in endosomes (antibodies that bind to antigens under conditions of high calcium ions and dissociate under conditions of low calcium ions) ). Thus, by administering such antigen-binding molecules to organisms, antigen elimination is accelerated and plasma concentrations of antigens can be reduced. Conventional antibodies that lack the ability to bind to antigens in a pH-dependent manner or the ability to bind to antigens in a calcium ion concentration-dependent manner, as well as their antigen-antibody complexes, are incorporated into cells by nonspecific endocytosis, transported to the cell surface after binding to FcRn in acidic conditions in endosomes, and are recycled into plasma after dissociation from FcRn under neutral conditions on the cell surface. For this reason, when an antibody that binds to an antigen in a pH-dependent manner sufficiently (which binds under the conditions of the neutral pH range and dissociates under the conditions of the acidic pH range), or an antibody that binds to the antigen in a calcium ion concentration-dependent manner sufficiently (which binds under conditions of high concentration of calcium ions and dissociates under conditions of low concentration of calcium ions) binds to the antigen in plasma and dissociates in endosomes from the antigen to which it is associated, the rate of elimination of the antigen will be equivalent to the rate of incorporation into cells through non-specific endocytosis an antibody or its antigen-antibody complex. When pH dependence or calcium ion concentration dependence in binding between antibodies and antigens is sufficient, antigens that have not dissociated from antibodies in endosomes will be recycled into plasma along with the antibodies. However, when pH dependence or calcium ion concentration dependence is sufficient, the rate of incorporation into cells by non-specific endocytosis will be rate-limiting for the rate of antigen elimination. Meanwhile, since FcRn transports antibodies from endosomes to the cell surface, it is believed that part of the FcRn is also present on the cell surface.

Typically, an IgG-type immunoglobulin, which is an embodiment of an antigen-binding molecule, exhibits little or no FcRn binding activity in the neutral pH range. The present inventors concluded that an IgG-type immunoglobulin having FcRn binding activity in the neutral pH range can bind to FcRn on the cell surface and will be incorporated into cells in an FcRn-dependent manner by binding to FcRn on the cell surface. The rate of FcRn-mediated incorporation into cells is higher than the rate of incorporation into cells by non-specific endocytosis. Thus, the present inventors have concluded that the rate of antigen elimination by antigen-binding molecules can be further increased by imparting the ability to bind FcRn in the neutral pH range. In particular, antigen-binding molecules that have the ability to bind FcRn in the neutral pH range can target antigens to cells more rapidly than native IgG-type immunoglobulins, release antigens in endosomes, recycle back to the cell surface or plasma, and re-bind with antigens there. and re-incorporate into cells via FcRn. The rate of this cycle can be increased by increasing the ability to bind FcRn in the neutral pH range; thus, the rate of elimination of antigens from plasma increases. Moreover, the rate of antigen elimination from plasma can be further increased by reducing the antigen-binding activity in the acidic pH range of the antigen-binding molecule compared to the antigen-binding activity in the neutral pH range. In addition, it is believed that the number of antigen molecules to which one antigen-binding molecule can bind will increase due to the increase in the number of cycles, which leads to an acceleration of this cycle. The antigen-binding molecules of the present invention comprise an antigen-binding domain and an FcRn-binding domain, and the FcRn-binding domain does not affect antigen binding. Moreover, due to the mechanism described above, they do not depend on the type of antigens. Thus, by reducing the antigen-binding activity (ability to bind) of an antigen-binding molecule under conditions of an acidic pH range or ion concentration, such as a low calcium ion concentration, compared to the antigen-binding activity (binding activity) under conditions of a neutral pH range or ion concentration, such as a high concentration of calcium ions, and/or by increasing FcRn binding activity at plasma pH, can accelerate the incorporation of antigens into cells via antigen-binding molecules and can accelerate antigen elimination.

As used herein, "incorporation of an antigen into cells" by means of antigen-binding molecules means that antigens are incorporated into cells by endocytosis. Moreover, as used herein, "accelerate incorporation into cells" indicates that the rate of incorporation into cells of antigen-binding molecules that bind antigens in plasma is increased and/or the amount of incorporated antigens that are recycled into plasma is reduced. In this case, the rate of incorporation into cells of an antigen-binding molecule that has a human FcRn-binding activity in the neutral pH range, or an antigen-binding molecule that has this human FcRn-binding activity and whose antigen-binding activity in the acidic pH range is lower than in the pH range neutral pH should be increased compared to an antigen-binding molecule that has no human FcRn binding activity in the neutral pH range, or an antigen-binding molecule whose antigen-binding activity is lower in the acidic pH range than in the neutral pH range. In another embodiment, the rate of incorporation into cells of the antigen-binding molecule of the present invention is preferably increased relative to the rate for native human IgG, and particularly preferably it is increased than that for native human IgG. Thus, within the scope of the present invention, whether incorporation of antigens into cells is accelerated by antigen-binding molecules can be determined based on whether the rate of incorporation of antigen into cells is increased. The rate of incorporation of antigens into cells can be measured, for example, by adding antigen-binding molecules and antigens to a culture medium containing cells expressing human FcRn and measuring the decline over time in the concentration of antigens in the medium, or by measuring the amount of antigens incorporated into cells over time. expressing human FcRn. Using the methods of accelerating the incorporation of antigens into cells mediated by the antigen-binding molecules of the present invention, for example, by administering antigen-binding molecules, the rate of elimination of antigen from plasma can be increased. Thus, whether incorporation by antigen-binding molecules of antigens in cells increases can also be assessed, for example, by measuring whether the rate of elimination of antigens present in plasma increases, or measuring whether the total plasma antigen concentration decreases after administration of antigen-binding molecules. .

As used herein, "native human IgG" refers to unmodified human IgG and is not limited to a specific IgG subclass. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as "native human IgG", provided that they are able to bind to human FcRn in the acidic pH range. Preferably, "native human IgG" may be human IgG1.

As used herein, "the ability to eliminate antigens from plasma" refers to the ability to eliminate antigens present in plasma from plasma following in vivo administration of antigen-binding molecules or in vivo secretion of antigen-binding molecules. Thus, as used herein, "the ability of antigen-binding molecules to eliminate antigens from plasma is increased" means that when antigen-binding molecules are administered, the activity of antigen-binding molecules to bind human FcRn in the neutral pH range is increased, or that, in addition to this increase in binding activity FcRn human, the rate of elimination of antigen from plasma increases compared to the rate of elimination of antigen from plasma to reduce antigen-binding activity in the range of acidic pH values compared with antigen-binding activity in the range of neutral pH values. Whether the ability of an antigen-binding molecule to eliminate antigens from plasma is increased can be assessed, for example, by administering soluble antigens and the antigen-binding molecule in vivo and measuring the plasma concentration of soluble antigens after administration. If the plasma concentration of soluble antigens decreases after the administration of soluble antigens and antigen-binding molecules, after an increase in the activity of antigen-binding molecules in binding human FcRn in the neutral pH range, or, in addition to an increase in this activity in human FcRn binding, after a decrease in antigen-binding activity in the acidic range pH compared to antigen-binding activity in the neutral pH range, then the ability of antigen-binding molecules to eliminate antigens in plasma is considered to be increased. A soluble antigen can be an antigen that binds to an antigen-binding molecule or an antigen that does not bind to an antigen-binding molecule, and its concentration can be defined as "plasma concentration of antigen bound to antigen-binding molecules" or as "plasma concentration of antigen which is not bound to antigen-binding molecules", respectively (the latter is synonymous with "plasma free antigen concentration"). "Total plasma antigen concentration" means the sum of the concentration of antigen bound or not bound by an antigen-binding molecule, or "free plasma antigen concentration", which is the concentration of antigen not bound to an antigen-binding molecule. Thus, the concentration of soluble antigen can be defined as "total plasma antigen concentration".

Various methods for measuring "total plasma antigen concentration" or "free plasma antigen concentration" are well known in the art, as described hereinafter.

In the present description, "enhancing pharmacokinetics", "improving pharmacokinetics" and "superior pharmacokinetics" can be reformulated as "enhancing plasma (blood) retention", "improving plasma (blood) retention", "superior plasma (blood) retention", and "prolonged retention in plasma (blood)". These terms are synonyms.

As used herein, "improvement in pharmacokinetics" means not only prolonging the period of time until elimination from plasma (for example, until the antigen-binding molecule is degraded intracellularly, etc. and can return to plasma) after administration of the antigen-binding molecule to a human or non-human animal, such as mice, rats, monkeys, rabbits and dogs, but also prolongation of plasma retention of antigen-binding molecules in a form that allows antigen binding (eg, antigen-free form of an antigen-binding molecule) during the period of administration until elimination due to degradation. Human IgG having a wild-type Fc region can bind to FcRn from non-human animals. For example, a mouse can be preferably used for administration in order to confirm the property of an antigen-binding molecule of the invention, since human IgG having a wild-type Fc can bind more strongly to mouse FcRn than to human FcRn (Int Immunol. (2001) 13(12): 1551 -1559). As another example, also for administration in order to confirm the property of the antigen-binding molecule of the invention described below, it is preferable to use a mouse in which its native FcRn genes are disrupted and contain a transgene for the human FcRn gene (Methods Mol Biol. 2010; 602: 93- 104). In particular, "improved pharmacokinetics" also includes prolonging the time to elimination due to degradation of the non-antigen-bound antigen-binding molecule (antigen-free form of the antigen-binding molecule). An antigen-binding molecule in plasma cannot bind to a new antigen if the antigen-binding molecule is already bound to the antigen. Thus, the longer the period of time when an antigen-binding molecule is not bound to an antigen, the longer is the period of time when it can bind to a new antigen (higher probability of binding to another antigen). This provides for a reduction in the period of time that the antigen is free from the antigen-binding molecule in vivo and an extension of the period of time that the antigen is bound to the antigen-binding molecule. The plasma concentration of the antigen-free form of the antigen-binding molecule can be increased and the period of time that the antigen is bound to the antigen-binding molecule can be prolonged by accelerating the elimination of the antigen from plasma by administration of the antigen-binding molecule. Specifically, as used herein, "improving the pharmacokinetics of an antigen-binding molecule" includes improving the pharmacokinetic parameter of the antigen-free form of the antigen-binding molecule (any of prolonging plasma half-life, prolonging mean plasma retention time, and decreasing plasma clearance), prolonging the period of time when the antigen associated with the antigen-binding molecule, after administration of the antigen-binding molecule, and accelerating the antigen-binding molecule-mediated elimination from plasma. Improvement in the pharmacokinetics of an antigen-binding molecule can be assessed by determining any of the parameters: plasma half-life, mean plasma retention time, and plasma clearance, for the antigen-binding molecule or its antigen-free form ("Pharmacokinetics: Enshu-niyoru Rikai (Understanding through practice)" Nanzando). For example, the plasma concentration of an antigen-binding molecule or an antigen-free form thereof is determined after administration of the antigen-binding molecule to mice, rats, monkeys, rabbits, dogs, or humans. Then, each parameter is determined. When the plasma half-life or mean plasma retention time is prolonged, the plasma pharmacokinetics of the antigen-binding molecule can be considered to be increased. The parameters can be determined by methods known to those skilled in the art. Parameters can be assessed as appropriate, for example, by non-compartmental analysis using WinNonlin pharmacokinetic analysis software (Pharsight) according to the instructions for use. The plasma concentration of an antigen-free antigen-binding molecule can be determined by methods known to those skilled in the art, for example, using the assay method described in Clin Pharmacol. 2008 Apr; 48(4): 406-417.

Within the scope of the present invention, "improving pharmacokinetics" also includes prolonging the period when an antigen is bound to an antigen-binding molecule after administration of an antigen-binding molecule. Assessing whether the period when the antigen is bound to the antigen-binding molecule is prolonged after administration of the antigen-binding molecule can be made by determining the plasma concentration of free antigen. Prolongation can be inferred from a given plasma concentration of free antigen or the period of time required to increase the ratio of free antigen to total antigen.

The concentration of free antigen not bound to an antigen-binding molecule, or the ratio of free antigen to total antigen, can be determined by methods known to those skilled in the art, such as the method used in Pharm Res. 2006 Jan; 23(1):95-103. Alternatively, when an antigen exhibits a particular function in vivo, whether the antigen is bound to an antigen-binding molecule that neutralizes the function of the antigen (an antagonist molecule) can be determined by examining whether the function of the antigen is neutralized. Whether antigen function is neutralized can be assessed by in vivo analysis of a marker that reflects antigen function. Whether an antigen is bound to an antigen-binding molecule that activates antigen function (agonist molecule) can be assessed by in vivo analysis of a marker that reflects antigen function.

Determining the plasma concentration of free antigen and the ratio of the amount of free antigen in plasma and the amount of total antigen in plasma, in vivo marker analysis and similar detection methods are not particularly limited; however, the assays are preferably performed after a certain period of time has elapsed after administration of the antigen-binding molecule. Within the scope of the present invention, the period following administration of the antigen-binding molecule is not specifically limited; those skilled in the art can determine the appropriate period depending on properties and the like. administered antigen-binding molecule. Such periods include, for example, one day after administration of the antigen-binding molecule, three days after administration of the antigen-binding molecule, seven days after administration of the antigen-binding molecule, 14 days after administration of the antigen-binding molecule, and 28 days after administration of the antigen-binding molecule. As used herein, "plasma antigen concentration" includes both "total plasma antigen concentration", which is the sum of the concentration of antigen bound and not bound by an antigen-binding molecule, and "free plasma antigen concentration", which is the concentration of antigen not bound by an antigen-binding molecule.

By administering the antigen-binding molecule of the present invention, the total plasma antigen concentration can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or even greater than when a reference antigen-binding molecule containing a wild type IgG Fc region is administered as a reference antigen-binding molecule, or when a molecule with an antigen-binding domain of the present invention is not administered.

The molar ratio of antigen/antigen binding molecule can be calculated as shown below;

value A: Molar concentration of antigen at each time point

value B: Molar concentration of the antigen-binding molecule at each time point

C value: Molar concentration of antigen per molar concentration of antigen-binding molecule (molar ratio of antigen/antigen-binding molecule) at each time point

C=A/B.

A lower C value indicates a higher antigen elimination efficiency per antigen binding molecule, while a higher C value indicates a lower antigen elimination efficiency per antigen binding molecule.

The molar ratio of antigen/antigen binding molecule can be calculated as described above.

The molar ratio of antigen/antigen-binding molecule can be reduced by administering the antigen-binding molecule of the present invention by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or even higher compared to the introduction of a reference antigen-binding molecule containing the wild-type human IgG Fc region as the human FcRn-binding domain.

In the present specification, wild-type human IgG1, IgG2, IgG3, or IgG4 is preferably used as wild-type human IgG to compare with antigen-binding molecules in terms of their human FcRn binding activity or in vivo binding activity. Preferably, a reference antigen-binding molecule containing the same antigen-binding domain as the antigen-binding molecule of interest and a wild-type human IgG Fc region as the human FcRn-binding domain can be suitably used. More preferably, unaltered human IgG1 is used as a reference wild-type human IgG to compare with antigen-binding molecules for their human FcRn binding activity or in vivo activity.

The reduction in total plasma concentration or antigen/antibody molar ratio can be assessed as described in Examples 4, 5 and 12. More specifically, using a transgenic mouse line with human FcRn 32 or 276 (Jackson Laboratories, Methods Mol Biol. 2010; 602: 93-104), they can be assessed using either an antigen-antibody co-injection model or a stationary infusion model where the antigen-binding molecule does not cross-react with the mouse antigen analog. When an antigen-binding molecule cross-reacts with a mouse analog, they can be assessed by simply injecting the antigen-binding molecule into transgenic mouse mice with human FcRn 32 or 276 (Jackson Laboratories). In the mouse co-injection model, a mixture of antigen-binding molecule and antigen is injected. In the stationary infusion model, mice are implanted with an infusion pump containing an antigen solution to achieve a constant plasma concentration of antigen, and then the mice are injected with an antigen-binding molecule. The antigen-binding molecule to be tested is administered at the same dosage. Total plasma antigen concentration, plasma free antigen concentration, and plasma concentration of antigen-binding molecule are measured at the appropriate time point using a method known to those skilled in the art.

Plasma total or free antigen concentration and antigen/antigen binding molecule molar ratio can be measured 2, 4, 7, 14, 28, 56, or 84 days after administration to assess the long-term effect of the present invention. In other words, long-term plasma antigen concentration is determined by measuring the concentration of total or free antigen in plasma and the molar ratio of antigen/antigen-binding molecule after 2, 4, 7, 14, 28, 56, or 84 days after administration of the antigen-binding molecule to evaluate the property of the antigen-binding molecule by the present invention. Whether a decrease in plasma antigen concentration or antigen/antigen binding molecule molar ratio is achieved by an antigen binding molecule of the present invention can be determined by assessing the decrease at any one or more of the time points described above.

Plasma total or free antigen concentration and antigen/antigen-binding molecule molar ratio can be measured 15 minutes, 1, 2, 4, 8, 12, or 24 hours after administration to assess the short-term effect of the present invention. In other words, short-term plasma antigen concentration is determined by measuring the total or free antigen plasma concentration and the antigen/antigen-binding molecule molar ratio at 15 min, 1, 2, 4, 8, 12, or 24 hours after administration of the antigen-binding molecule to evaluate the property of the antigen-binding molecule. according to the present invention.

The route of administration of the antigen-binding molecule of the present invention can be selected from intradermal, intravenous, intravitreal, subcutaneous, intraperitoneal, parenteral and intramuscular injection.

Within the scope of the present invention, improvement in the pharmacokinetics of an antigen-binding molecule in humans is preferred. Where human plasma retention is difficult to determine, it can be predicted from plasma retention in mice (eg healthy mice, transgenic mice expressing human antigen, transgenic mice expressing human FcRn) or monkeys (eg cynomolgus monkeys).

As used herein, "improvement in pharmacokinetics and prolonged plasma retention of antigen-binding molecules" means an improvement in any parameter of pharmacokinetics (any of prolongation of plasma half-life, prolongation of mean plasma retention, reduction in plasma clearance and bioavailability) following in vivo administration of an antigen-binding molecule, or an increase plasma concentrations of the antigen-binding molecule over an appropriate period of time following administration. It can be determined by measuring any parameter such as plasma half-life, mean plasma retention time, plasma clearance, and bioavailability of the antigen-binding molecule (Pharmacokinetics: Enshu-niyoru Rikai (Understanding through practice), (Nanzando)). For example, when

the antigen-binding molecule is administered to mice (normal mice and transgenic mice with human FcRn), rats, monkeys, rabbits, dogs, humans, etc., and the plasma concentration of the antigen-binding molecule is determined and each of the parameters is calculated, it can be considered that the pharmacokinetics of the antigen-binding molecule improves when plasma half-life and mean plasma retention time are prolonged. These parameters can be determined by methods known to those skilled in the art. For example, parameters can be appropriately assessed using a non-compartmental assay using WinNonlin pharmacokinetic analysis software (Pharsight) according to the accompanying instructions.

Without wishing to be bound by a particular theory, since an antigen-binding molecule that has FcRn binding activity in the neutral pH range can form a tetrameric complex containing two FcRn molecules and one FcγR molecule on the cell membrane of antigen-presenting cells, incorporation into antigen-presenting cells is accelerated, and thus , it is believed that the plasma retention time is reduced and the pharmacokinetics are degraded. There are phosphorylation sites in the cytoplasmic domains of FcγR and FcRn. Typically, phosphorylation of the cytoplasmic domain of a cell-surface-expressed receptor occurs upon assembly of the receptor, and phosphorylation induces receptor internalization. Even if native IgG1 forms a dimeric FcγR/IgG1 complex on antigen-presenting cells, assembly of FcγR cytoplasmic domains does not occur. However, when an IgG molecule having FcRn binding activity under the conditions of the neutral pH range forms a heteromeric tetrameric complex containing FcγR/two FcRn/IgG molecules, the three cytoplasmic domains of FcγR and FcRn will assemble, and thereby internalization of the heteromeric tetrameric complex containing FcγR/two FcRn/IgG molecules. It is believed that the formation of heteromeric tetrameric complexes containing FcγR/two FcRn/IgG molecules occurs on antigen-presenting cells co-expressing FcγR and FcRn, and therefore the number of antibody molecules incorporated into antigen-presenting cells may increase and, as a result, pharmacokinetics may deteriorate. Thus, by inhibiting the formation of the above-described complex on antigen-presenting cells using any of the methods of embodiments 1, 2, and 3 found within the scope of the present invention, incorporation into antigen-presenting cells can be reduced, and as a result, pharmacokinetics can be improved.

Method for producing antigen-binding molecules whose binding activity varies depending on ion concentration conditions

In a non-limiting embodiment of the present invention, after isolation of a polynucleotide encoding an antigen-binding domain whose binding activity varies depending on the conditions selected as described above, the polynucleotide is inserted into an appropriate expression vector. For example, when the antigen-binding domain is the variable region of an antibody, after obtaining a cDNA encoding the variable region, the cDNA is cleaved with restriction enzymes that recognize the restriction sites inserted at both ends of the cDNA. Preferably, the restriction enzymes recognize and cleave a nucleotide sequence that occurs at low frequency in the nucleotide sequence constituting the gene of the antigen binding molecule. Moreover, restriction enzymes are preferably inserted that provide sticky ends to insert a single copy of the cleaved fragment into the vector in the correct orientation. cDNA encoding the variable region of the antigen-binding molecule, cleaved as described above, is inserted into an appropriate expression vector to obtain an expression vector for the antigen-binding molecule of the present invention. At this time, the gene encoding the constant region (C-region) of the antibody can be fused in frame with the gene encoding the variable region.

To obtain an antigen-binding molecule of interest, a polynucleotide encoding an antigen-binding molecule is inserted, so that it is operably linked to a regulatory sequence, into an expression vector. Regulatory sequences include, for example, enhancers and promoters. Moreover, an appropriate signal sequence can be linked to the N-terminus so that the expressed antigen-binding molecule is secreted into the external environment of the cells. As a signal sequence, for example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3); however, other appropriate signal sequences may also be attached. The expressed polypeptide is cleaved at the C-terminus of the sequence described above, and the cleaved polypeptide is secreted as the mature polypeptide into the external environment of the cells. Appropriate host cells are then transformed with this expression vector so that recombinant cells can be obtained expressing a polynucleotide encoding the antigen-binding molecule of interest. Antigen-binding molecules of the present invention can be obtained from recombinant cells by the methods described above in the section on antibodies.

In a non-limiting embodiment of the present invention, after isolation of a polynucleotide encoding an antigen-binding molecule as described above, the binding activity of which varies depending on the selected conditions, the polynucleotide variant is inserted into an appropriate expression vector. Such variants preferably include those obtained by humanization based on a polynucleotide sequence encoding an antigen-binding molecule of the present invention, obtained by screening as a randomized variable region library of an engineered synthetic library or an immune library derived from non-human animals. The same methods as described above for making the humanized antibodies described above can be used as a way to make humanized variants of the antigen-binding molecule.

In another embodiment, such variants preferably include variants obtained by introducing a change that increases the antigen affinity (affinity maturation) of an antigen-binding molecule of the present invention to an isolated polynucleotide sequence for a molecule obtained by screening using a synthetic library or a naive library as randomized. variable region libraries. Such variants can be generated by various known affinity maturation methods, including CDR mutagenesis (Yang et al. (J. Mol. Biol. (1995) 254, 392-403)), chain shuffling (Marks et al. (Bio/Technology (1992) 10, 779-783)), use of E. coli mutant strains (Low et al. (J. Mol. Biol. (1996) 250, 359-368)), DNA shuffling (Patten et al. (Curr. Opin. Biotechnol (1997) 8, 724-733)), phage display (Thompson et al. (J. Mol. Biol. (1996) 256, 77-88)) and sex PCR (Clameri et al. (Nature (1998) 391 , 288-291)).

As described above, antigen-binding molecules that are obtained by the preparation methods of the present invention include antigen-binding molecules having an Fc region. Various variants can be used as Fc regions. In one embodiment, the variants of the present invention preferably comprise polynucleotides encoding antigen-binding molecules having a heavy chain, wherein the polynucleotide encoding the Fc region variant as described above is linked in-frame to a polynucleotide encoding the antigen-binding molecule described above whose binding activity is varies depending on the selected conditions.

In a non-limiting embodiment of the present invention, Fc regions preferably include, for example, Fc constant regions of antibodies such as IgG1 SEQ ID NO: 11 (Ala added to the N-terminus of AAC82527.1), IgG2 SEQ ID NO: 12 (Ala added to N -end of AAB59393.1), IgG3 SEQ ID NO: 13 (CAA27268.1) and IgG4 SEQ ID NO: 14 (Ala added to the N-terminus of AAB59394.1). The plasma retention time of IgG molecules is relatively long (elimination from plasma is slow) because FcRn, in particular human FcRn, functions as a rescue receptor for IgG molecules. IgG molecules incorporated into endosomes by pinocytosis bind under the acidic conditions of endosomes to FcRn, in particular human FcRn expressed in endosomes. IgG molecules that cannot bind to FcRn, in particular human FcRn, are transferred to and degraded in lysosomes. Meanwhile, IgG molecules bound in FcRn, in particular human FcRn, are transferred to the cell surface and then returned to plasma by dissociation from FcRn, in particular human FcRn, under neutral conditions in plasma.

Since antibodies containing a typical Fc region do not have binding activity to FcRn, in particular human FcRn, under conditions of the neutral pH range, typical antibodies and antibody-antigen complexes are incorporated into cells by non-specific endocytosis and transferred to the cell surface by binding to FcRn, in particular human FcRn, under endosomal acidic pH range conditions. FcRn, in particular human FcRn, transports antibodies from the endosome to the cell surface. Thus, some of the FcRn, in particular human FcRn, are also believed to be present on the cell surface. However, antibodies are recycled to plasma because they dissociate from FcRn, in particular human FcRn, under conditions of the neutral pH range at the cell surface.

Fc regions having human FcRn binding activity in the neutral pH range that are included in the antigen-binding molecules of the present invention can be obtained by any method. In particular, Fc regions having human FcRn binding activity in the neutral pH range can be obtained by changing the amino acids of human IgG-type immunoglobulin as the original Fc region. Preferred human IgG-type immunoglobulin Fc regions include, for example, human IgG Fc regions (IgG1, IgG2, IgG3 and IgG4, and variants thereof). Amino acids at any positions can be changed to other amino acids, provided that the resulting regions have human FcRn binding activity in the neutral pH range or increased human FcRn binding activity in the neutral range. When the antigen-binding molecule contains a human IgG1 Fc region as a human Fc region, it is preferable that the resulting region contains a change that has the effect of enhancing human FcRn binding in the neutral pH range compared to the binding activity of the original human IgG1 Fc region. Amino acids that allow such changes include, for example, amino acids at positions 221-225, 227, 228, 230, 232, 233-241, 243-252, 254-260, 262-272, 274, 276, 278-289, 291-312, 315-320, 324, 325, 327-339, 341, 343, 345, 360, 362, 370, 375-378, 380, 382, 385-387, 389, 396, 414, 416, 423, 424, 426-438, 440 and 442 (listed according to EU numbering). More specifically, such amino acid changes include the changes shown in Table 5. Changing these amino acids enhances binding to human FcRn of the IgG-type immunoglobulin Fc region in the neutral pH range.

Among those described above, suitable modifications are selected for use in the present invention that enhance human FcRn binding in the neutral pH range. Particularly preferred amino acids for such Fc region variants include, for example, amino acids at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 EU ). The human FcRn binding activity of the Fc region included in the antigen binding molecule can be increased in the neutral pH range by replacing at least one amino acid with another amino acid.

Particularly preferred changes in the Fc region include, for example, substitutions:

Met instead of the amino acid at position 237;

Ile instead of the amino acid at position 248;

Ala, Phe, He, Met, Gin, Ser, Val, Trp, or Tyr instead of the amino acid at position 250;

Phe, Trp or Tyr instead of the amino acid at position 252;

Thr instead of amino acid at position 254;

Glu instead of amino acid at position 255;

Asp, Asn, Glu or Gln instead of the amino acid at position 256;

Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val instead of the amino acid at position 257;

His instead of the amino acid at position 258;

Ala instead of the amino acid at position 265;

Ala or Glu instead of the amino acid at position 286;

His instead of the amino acid at position 289;

Ala instead of the amino acid at position 297;

Ala instead of the amino acid at position 303;

Ala instead of the amino acid at position 305;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr instead of the amino acid at position 307;

Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr instead of the amino acid at position 308;

Ala, Asp, Glu, Pro, or Arg instead of the amino acid at position 309;

Ala, His or Ile instead of the amino acid at position 311;

Ala or His instead of the amino acid at position 312;

Lys or Arg instead of the amino acid at position 314;

Ala, Asp or His instead of the amino acid at position 315;

Ala instead of the amino acid at position 317;

Val instead of the amino acid at position 332;

Leu instead of the amino acid at position 334;

His instead of the amino acid at position 360;

Ala instead of the amino acid at position 376;

Ala instead of the amino acid at position 380;

Ala instead of the amino acid at position 382;

Ala instead of the amino acid at position 384;

Asp or His instead of the amino acid at position 385;

Pro instead of amino acid a at position 386;

Glu for instead of amino acid at position 387;

Ala or Ser instead of the amino acid at position 389;

Ala instead of the amino acid at position 424;

Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr instead of the amino acid at position 428;

Lys instead of amino acid at position 433;

Ala, Phe, His, Ser, Trp, or Tyr instead of the amino acid at position 434; And

His, Ile, Leu, Phe, Thr or Val instead of the amino acid at position 436 in the EU numbering system. Meanwhile, the number of altered amino acids is not specifically limited; such amino acid changes include a single amino acid change and an amino acid change at two or more sites. Combinations of amino acid changes at two or more sites include, for example, combinations of changes described in Table b.

The present invention is not limited to a specific theory, but provides methods for producing antigen-binding molecules, which include not only the above-described change, but also the change of the Fc region so that a tetrameric heterocomplex is not formed, consisting of an Fc region included in an antigen-binding molecule, two FcRn molecules and an activating Fcγ receptor. Preferred embodiments of such antigen-binding molecules include the three embodiments described below.

(Embodiment 1) Antigen-binding molecules which contain an Fc region having FcRn binding activity under neutral pH range conditions and whose binding activity to activating FcγRs is lower than that of the native Fc region

The antigen-binding molecules of Embodiment 1 form trimeric complexes by binding to two FcRn molecules; however, they do not form an activating FcγR complex (FIG. 49). Fc regions whose binding activity to an activating FcγR is lower than that of the native Fc region can be obtained by altering the amino acids of the native Fc region as described above. Whether the binding activity of the altered Fc region to the activating FcγR is lower than that of the native Fc region can be appropriately examined using the methods described in the "Binding Activity" section above.

Within the scope of the present invention, that the binding activity of the altered Fc region to the activating Fcγ receptor is lower than that of the native Fc region means that the binding activity of the altered Fc region to any of the human Fcγ receptors: FcγRIa, FcγRIIa, FcγRIIIa and/or FcγRIIIb is lower than that of the native Fc region and, for example, means that when compared based on the analytical method described above, the binding activity of an antigen-binding molecule having an Fc region variant is 95% or less, preferably 90% or less, 85% or less, 80% or less, 75% or less, particularly 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, 1% or less compared to the binding activity of a control antigen-binding molecule having a native Fc region. Such native Fc regions include the original Fc region and Fc regions from wild-type antibodies of various isotypes.

Suitable antigen-binding molecules having an Fc region as a control include antigen-binding molecules having an Fc region from an IgG monoclonal antibody. The structures of these Fc regions are shown in SEQ ID NOs: 11 (A added to the N-terminus of RefSeq accession no. AAC 82527.1), 12 (A added to the N-terminus of RefSeq accession no. RefSeq Accession No. CAA27268.1) and 14 (A added to the N-terminus of RefSeq Accession No. AAB 59394.1). Meanwhile, when an antigen-binding molecule having an Fc region from an antibody of a certain isotype is used as a test substance, the activity of an antigen-binding molecule having an Fc region to bind an Fcγ receptor can be examined using an antigen-binding molecule having an Fc as a control. -region from a monoclonal IgG antibody of the same isotype. It is necessary to select an antigen-binding molecule containing an Fc region that has been shown to have high Fcγ receptor binding activity as described above.

In a non-limiting embodiment of the present invention, preferred Fc regions whose binding activity to an activating FcγR is lower than that of a native Fc region include, for example, Fc regions in which one or more amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325 and 329 (listed according to EU numbering) among the amino acids of the Fc region described above are replaced by other amino acids of the native Fc region. Such Fc region changes are not limited to the changes described above and include, for example, changes such as deglycosized chains (N297A and N297Q), IgG1-L234A/L235A, IgG1-A325A/A330S/P331S, IgG1-C226S/C229S, IgG1-C226S /C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S, IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S, IgG4-L235A/ G237A/E318A and IgG4-L236E described in Current Opinion in Biotechnology (2009) 20 (6), 685-691; variations such as G236R/L328R, L235G/G236R, N325A/L328R and N325LL328R described in WO 2008/0 92117; amino acid insertions at positions 233, 234, 235 and 237 (listed according to EU numbering); and changes in the regions described in WO 2000/042072.

Moreover, in a non-limiting embodiment of the present invention, preferred Fc regions include Fc regions modified to have one or more of:

substitutions for Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, or Trp amino acids at position 234;

substitutions for Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val or Arg amino acids at position 235;

substitutions for Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro or Tyr amino acids at position 236;

substitutions for Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr or Arg of the amino acid at position 237;

substitutions for Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp or Arg amino acids at position 238;

substitutions for Gln, His, Lys, Phe, Pro, Trp, Tyr or Arg amino acids at position 239;

substitutions for Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val amino acids at position 265;

substitutions for Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp or Tyr amino acids at position 266;

substitutions for Arg, His, Lys, Phe, Pro, Trp or Tyr amino acids at position 267;

substitutions for Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val amino acids at position 269;

substitutions for Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val amino acids at position 270;

substitutions for Arg, His, Phe, Ser, Thr, Trp or Tyr amino acids at position 271;

substitutions for Arg, Asn, Asp, Gly, His, Phe, Ser, Trp or Tyr amino acids at position 295;

substitutions for Arg, Gly, Lys or Pro amino acids at position 296;

substitutions for Ala amino acids at position 297;

substitutions for Arg, Gly, Lys, Pro, Trp or Tyr amino acids at position 298;

substitutions for Arg, Lys or Pro amino acids at position 300;

substitutions for Lys or Pro amino acids at position 324;

substitutions for Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr or Val amino acids at position 325;

substitutions for Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val amino acids at position 327;

substitutions for Arg, Asn, Gly, His, Lys, or Pro amino acids at position 328;

substitutions for Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or Arg amino acids at position 329;

substitutions for Pro or Ser amino acids at position 330;

substitutions for Arg, Gly or Lys amino acids at position 331; And

substitutions for Arg, Lys or Pro amino acids at position 332 according to the EU numbering system in the Fc region.

(Embodiment 2) Antigen binding molecules which contain an Fc region having an FcRn binding activity under neutral pH range conditions and whose binding activity to an inhibitory FcγR is higher than the binding activity to an activating Fcγ receptor

The antigen-binding molecules of Embodiment 2 can form a tetrameric complex by binding to two FcRn molecules and one inhibitory FcγR molecule. However, since one antigen-binding molecule can only bind to one FcγR molecule, an antigen-binding molecule bound to an inhibitory FcγR cannot further bind to an activating FcγR (FIG. 50). Moreover, antigen-binding molecules incorporated into cells in an inhibitory FcγR state have been described to be recycled to the cell membrane and thus elude intracellular degradation (Immunity (2005) 23, 503-514). In particular, it is contemplated that antigen-binding molecules having selective binding activity for an inhibitory FcγR cannot form a heteromeric complex containing an activating FcγR that is responsible for an immune response and two FcRn molecules.

In the context of the present invention, "binding activity to an inhibitory FcγR is higher than binding activity to an activating Fcγ receptor" means that the binding activity of the Fc region variant to FcγRIIb is higher than the binding activity to any human Fcγ receptor: FcγRI , FcγRIIa, FcγRIIIa and/or FcγRIIIb. For example, this means that, based on the above analytical method, the FcγRIIb binding activity of an antigen-binding molecule having an Fc region variant is 105% or more, preferably 110% or more, 120% or more, 130% or more, 140% or more, particularly preferably 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% or more, 300% or more, 350% or more , 400% or more, 450% or more, 500% or more, 750% or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more exceeds the activity of binding to any human Fcγ receptors: FcγRI, FcγRIIa, FcγRIIIa and/or FcγRIIIb.

As control antigen-binding molecules having an Fc region, antigen-binding molecules having an Fc region from an IgG monoclonal antibody can be suitably used. The structures of these Fc regions are shown in SEQ ID NO: 11 (A added at the N-terminus of RefSeq accession no. AAC 82527.1), 12 (A added at the N-terminus of RefSeq accession no. RefSeq Accession No. CAA 27268.1) and 14 (A added at the N-terminus of RefSeq Accession No. AAB 59394.1). Meanwhile, when an antigen-binding molecule which has an Fc region from an antibody of a certain isotype is used as a test substance, the activity of an antigen-binding molecule having an Fc region to bind an Fcγ receptor can be examined using an antigen-binding molecule having an Fcγ receptor as a control. Fc region of a monoclonal IgG antibody of the same isotype. As described above, an antigen-binding molecule containing an Fc region that has been shown to have high Fcγ receptor binding activity is appropriately selected.

In a non-limiting embodiment of the present invention, preferred Fc regions having selective binding activity for an inhibitory FcγR include, for example, Fc regions in which the amino acid at position 238 or 328 (listed according to EU numbering) among the amino acids of the Fc region described above is changed to a different amino acid of the native Fc region. Moreover, as Fc regions having selective binding activity to an inhibitory FcγR, Fc regions or changes from those described in US 2009/0136485 can also be appropriately selected.

In another non-limiting embodiment of the present invention, preferred Fc regions include Fc regions in which any one or more of: the amino acid at position 238 (indicated according to EU numbering) is replaced by Asp and the amino acid at position 328 (indicated according to EU numbering) is replaced by Glu in the Fc region described above.

In another non-limiting embodiment of the present invention, preferred Fc regions include the substitution of Asp for Pro at position 238 (listed according to EU numbering), and the following substitutions:

replacement with Trp amino acid at position 237 (specified according to EU numbering),

a change to Phe for the amino acid at position 237 (listed according to EU numbering),

a change to Val for the amino acid at position 267 (listed according to EU numbering),

a change to Gln amino acid at position 267 (listed according to EU numbering),

substitution with Asn for the amino acid at position 268 (listed according to EU numbering),

amino acid change to Gly at position 271 (listed according to EU numbering),

a change to Leu for the amino acid at position 326 (listed according to EU numbering),

amino acid change to Gln at position 326 (listed according to EU numbering),

amino acid change to Glu at position 326 (listed according to EU numbering),

a change to Met for the amino acid at position 326 (listed according to EU numbering),

substitution for Asp amino acid at position 239 (listed according to EU numbering),

a change to Ala amino acid at position 267 (listed according to EU numbering),

replacement with Trp amino acid at position 234 (specified according to EU numbering),

substitution for Tyr amino acid at position 234 (listed according to EU numbering),

a change to Ala amino acid at position 237 (listed according to EU numbering),

substitution with Asp for the amino acid at position 237 (listed according to EU numbering),

amino acid change to Glu at position 237 (listed according to EU numbering),

a change to Leu for the amino acid at position 237 (listed according to EU numbering),

substitution for Met amino acid at position 237 (listed according to EU numbering),

substitution for Tyr amino acid at position 237 (listed according to EU numbering),

a change to Lys for the amino acid at position 330 (listed according to EU numbering),

substitution for Arg of the amino acid at position 330 (specified according to EU numbering),

substitution for Asp amino acid at position 233 (listed according to EU numbering),

substitution with Asp for the amino acid at position 268 (listed according to EU numbering),

amino acid change to Glu at position 268 (listed according to EU numbering),

substitution for Asp amino acid at position 326 (listed according to EU numbering),

substitution for Ser of the amino acid at position 326 (specified according to EU numbering),

a change to Thr amino acid at position 326 (specified according to EU numbering),

substitution for Ile amino acid at position 323 (listed according to EU numbering),

a change to Leu for the amino acid at position 323 (listed according to EU numbering),

a Met substitution for the amino acid at position 323 (listed according to EU numbering),

substitution with Asp for the amino acid at position 296 (listed according to EU numbering),

a change to Ala amino acid at position 326 (listed according to EU numbering),

a substitution with Asn for the amino acid at position 326 (listed according to EU numbering), and

substitution for Met amino acid at position 330 (indicated according to EU numbering).

(Embodiment 3) Antigen binding molecules comprising an Fc region wherein one of the two polypeptides constituting the Fc region has FcRn binding activity under neutral pH range conditions and the other has no FcRn binding activity under neutral pH range conditions

The antigen-binding molecule according to Embodiment 3 can form trimeric complexes by binding to one FcRn molecule and one FcγR molecule; however, they do not form a tetrameric heterocomplex containing two FcRn molecules and one FcγR molecule (Fig. 51). Fc regions derived from bispecific antibodies can be suitably used as Fc regions in which one of the two polypeptides constituting the Fc region has FcRn binding activity under conditions of the neutral pH range and the other does not have FcRn binding activity at neutral pH range conditions that are included in the antigen-binding molecule of Embodiment 3. A bispecific antibody refers to two types of antibodies that have specificity for different antigens. Bispecific IgG-type antibodies can be secreted from hybridomas (quadromes) obtained by fusing two types of IgG-producing hybridomas (Milstein et al. (Nature (1983) 305, 537-540).

When the antigen-binding molecule according to Embodiment 3 described above is produced using recombination methods such as those described in the Antibody section above, a method can be used in which genes encoding polypeptides that constitute two types of Fc regions of interest , are introduced into cells for their co-expression. However, the resulting Fc region is a mixture that contains, in a 2:1:1 molar ratio, an Fc region in which one of the two polypeptides constituting the Fc region has FcRn binding activity under neutral pH range conditions and the other does not. has FcRn binding activity under conditions of the neutral pH range, an Fc region in which both polypeptides constituting the Fc region have FcRn binding activity under conditions of the neutral pH range, and an Fc region in which both polypeptides constituting the Fc region , do not have FcRn binding activity under neutral pH range conditions. It is difficult to purify antigen-binding molecules containing the desired combination of Fc regions from the three IgG types.

When preparing antigen-binding molecules according to Embodiment 3 using recombination methods such as those described above, antigen-binding molecules containing a heterocombination of Fc regions can preferably be secreted by changing the CH3 domain that constitutes the Fc region using appropriate amino acid substitutions. Specifically, this method is a method of enhancing hetero-H chain formation and inhibiting homo-H chain formation by replacing an amino acid side chain in one heavy chain CH3 domain with a larger side chain (protrusion (meaning "bulge")) while replacing the amino acid side chain in the other heavy chain CH3 domain with a smaller side chain (deep (meaning "cavity")) so that the "protrusion" fits within the "depression" (WO 1996027011, Ridgway et al. (Protein Engineering (1996) ) 9, 617-621), Merchant et al (Nat. Biotech. (1998) 16, 677-681)).

Moreover, known methods for producing bispecific antibodies include methods that use means to regulate association of polypeptides or association to form heteromeric multimers formed by polypeptides to associate a pair of polypeptides that constitute an Fc region. In particular, to obtain bispecific antibodies, methods can be used to regulate the association of polypeptides by changing the amino acid residues that form the contact surface between a pair of polypeptides that make up the Fc region so that a complex is formed of two polypeptides with different sequences that make up the Fc region, while inhibiting the association of polypeptides. having an identical sequence, which constitute the Fc region (WO 2006/106905). Such methods can be used to prepare the antigen-binding molecules of the present invention described in Embodiment 3.

In a non-limiting embodiment of the present invention, the pair of polypeptides that constitute the Fc region described above, derived from a bispecific antibody, can be suitably used as the Fc region. More specifically, preferably, the Fc regions are a pair of polypeptides that make up the Fc region, one of which has an amino acid sequence in which the amino acids at positions 349 and 366 (listed according to EU numbering) are Cys and Trp, respectively, and the other has an amino acid sequence in which the amino acid at position 356 (listed according to EU numbering) is Cys, the amino acid at position 366 (shown according to EU numbering) is Ser, the amino acid at position 368 is Ala, and the amino acid at position 407 (shown according to EU numbering) EU numbering) is Val.

In another non-limiting embodiment of the present invention, preferably, the Fc regions are a pair of polypeptides that constitute an Fc region, one of which has an amino acid sequence in which the amino acid at position 409 (listed according to EU numbering) is Asp and the other has amino acid sequence in which the amino acid at position 399 (listed according to EU numbering) is Lys. In the above embodiment, the amino acid at position 409 may be Glu instead of Asp, and the amino acid at position 399 may be Arg instead of Lys. Alternatively, it is preferred that when the amino acid at position 399 is Lys, additionally the amino acid at position 360 may be Asp, or the amino acid at position 392 may be Asp.

In another non-limiting embodiment of the present invention, the Fc regions are preferably a pair of polypeptides that constitute an Fc region, one of which has an amino acid sequence in which the amino acid at position 370 (indicated according to EU numbering) is Glu and the other has an amino acid the sequence in which the amino acid at position 357 (listed according to EU numbering) is Lys.

In another non-limiting embodiment of the present invention, the Fc regions are preferably a pair of polypeptides that make up the Fc region, one of which has the amino acid sequence in which the amino acid at position 439 (indicated according to EU numbering) is Glu and the other has the amino acid the sequence in which the amino acid at position 356 (listed according to EU numbering) is Lys.

In another non-limiting embodiment of the present invention, such preferred Fc regions include Fc regions as a combination of any of the embodiments described above, such as:

a pair of polypeptides that make up the Fc region, one of which has an amino acid sequence in which the amino acids at positions 409 and 370 (listed according to EU numbering) are Asp and Glu, respectively, and the other of which has an amino acid sequence in which both of amino acids at positions 399 and 357 (indicated according to EU numbering) are Lys (in this embodiment, the amino acid at position 370 (indicated according to EU numbering) may be Asp instead of Glu, or the amino acid at position 392 may be Asp instead of Glu at position amino acids 370);

a pair of polypeptides that make up the Fc region, one of which has an amino acid sequence in which the amino acids at positions 409 and 439 (listed according to EU numbering) are Asp and Glu, respectively, and the other has an amino acid sequence in which both of the amino acids in positions 399 and 356 (indicated according to EU numbering) are Lys (in this embodiment, instead of Glu at amino acid position 439 (indicated according to EU numbering), the amino acid at position 360 may be Asp, the amino acid at position 392 may be Asp or the amino acid at position 439 may be Asp);

a pair of polypeptides that make up the Fc region, one of which has an amino acid sequence in which both of the amino acids at positions 370 and 439 (listed according to EU numbering) are Glu, and the other has an amino acid sequence in which both of the amino acids at positions 357 and 356 (listed according to EU numbering) are Lys; And

a pair of polypeptides that make up the Fc region, one of which has an amino acid sequence in which the amino acids at positions 409, 370, and 439 (listed according to EU numbering) are Asp, Glu, and Glu, respectively, and the other has an amino acid sequence in which all amino acids at positions 399, 357, and 356 (listed according to EU numbering) are Lys (in this embodiment, the amino acid at position 370 may not be replaced by Glu, and furthermore, when the amino acid at position 370 is not replaced by Glu, the amino acid at position 439 may be Asp instead of Glu, or the amino acid at position 439 may be Asp instead of Glu at amino acid position 392).

In another non-limiting embodiment of the present invention, a pair of polypeptides are preferably used that make up the Fc region, one of which has an amino acid sequence in which the amino acid at position 356 (indicated according to EU numbering) is Lys, and the other has an amino acid sequence in which the amino acids at positions 435 and 439 (listed according to EU numbering) are Arg and Glu, respectively.

These antigen-binding molecules of Embodiments 1-3 are expected to have reduced immunogenicity and increased plasma retention time compared to antigen-binding molecules capable of forming a tetrameric complex.

Suitable known methods can be used to alter the amino acids of Fc regions, such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlapped extension PCR. Moreover, various known methods can also be used as a method of changing amino acids to replace amino acids with amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci U.S.A. (2003) 100(11), 6353-6357). For example, it is also preferable to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which the non-natural amino acid is linked to an amber suppressor tRNA that is complementary to the UAG (amber codon) stop codon.

In one embodiment of the embodiments of the present invention, polynucleotides encoding antigen-binding molecules that have a heavy chain, wherein the polynucleotide encoding the Fc region is modified to have an amino acid mutation as described above linked in frame to a polynucleotide encoding the above an antigen-binding molecule, the binding activity of which varies depending on the selected conditions.

The present invention relates to methods for producing antigen-binding molecules, including the collection of antigen-binding molecules from cell culture media, into which vectors are introduced, in which a polynucleotide encoding an Fc region is operably linked in frame to a polynucleotide encoding an antigen-binding domain, the binding activity of which varies depending on on the conditions of ion concentration. Moreover, the present invention also relates to methods for producing antigen-binding molecules, comprising collecting antigen-binding molecules from cell culture media, in which vectors are introduced, constructed by functionally linking a polynucleotide encoding an antigen-binding domain, the binding activity of which varies depending on ion concentration conditions, with a polynucleotide coding for an Fc region that is previously operably linked to the vector.

Pharmaceutical compositions

When a conventional neutralizing antibody against a soluble antigen is administered, plasma retention of the antigen is expected to be prolonged due to binding to the antibody. As a rule, antibodies have a long half-life (from one week to three weeks), while the half-life of an antigen is usually short (one day or less). Meanwhile, antibody-bound antigens have a significantly longer plasma half-life compared to when the antigens are present alone. For this reason, the introduction of an existing neutralizing antibody leads to an increase in the plasma concentration of the antigen. Such cases have been described for various neutralizing antibodies that target soluble antigens, including, for example, IL-6 (J. Immunotoxicol. (2005) 3, 131-139), amyloid-beta (mAbs (2010) 2 (5), 1 -13), MCP-1 (ARTHRITIS & RHEUMATISM (2006) 54, 2387-2392), hepcidin (AAPS J. (2010) 4, 646-657) and sIL-6 receptor (Blood (2008) 112 (10), 3959-64). It is described that the introduction of existing neutralizing antibodies increases the total concentration of antigen in plasma approximately 10-1000 times (the level of increase varies, depending on the antigen) from the initial level. In the context of the present invention, the total plasma antigen concentration refers to the concentration as the total amount of antigen in the plasma, i. e. the sum of the concentrations of antibody-bound and non-antibody-bound antigen. An increase in total plasma antigen concentration is undesirable for such antibody pharmaceuticals that target soluble antigen. The reason for this is that the antibody concentration must be higher than at least the total plasma concentration of the antigen in order to neutralize the soluble antigen. In particular, "total plasma antigen concentration is increased 10-1000-fold" means that in order to neutralize the antigen, the plasma concentration of antibody (i.e., antibody dose) must be 10-1000 times higher than when the increase the total concentration of antigen in plasma does not occur. Conversely, if the total plasma concentration of the antigen can be reduced by 10-1000 times compared to the existing neutralizing antibody, the dose of the antibody can also be reduced to a similar extent. Thus, antibodies capable of reducing total plasma antigen concentration by eliminating soluble antigen from plasma are highly useful compared to existing neutralizing antibodies.

The present invention is not limited to a particular theory, but it can be explained, for example, as follows why the number of antigens to which single antigen-binding molecules can bind increases, and why the elimination of an antigen from plasma is accelerated when antigen-binding molecules that have an antigen-binding domain whose antigen-binding activity varies with ion concentration conditions so that the antigen-binding activity in the acidic pH range is lower than in the neutral pH range, and further have an FcRn binding domain such as an antibody constant region exhibiting human FcRn binding activity under conditions of the neutral pH range, is administered in vivo and cell uptake is enhanced in vivo.

For example, when an antibody that binds to a membrane antigen is administered in vivo, after binding to the antigen, the antibody in the antigen-bound state is incorporated into the endosome by intracellular internalization. The antibody is then transferred to the lysosome, remaining bound to the antigen, and degraded there along with the antigen. Internalization-mediated elimination from plasma is referred to as antigen-dependent elimination and has been described for many antibody molecules (Drug Discov Today (2006) 11(1-2), 81-88). When one IgG antibody molecule binds to antigens in a bivalent fashion, one antibody molecule is internalized while remaining bound to two antigens and degraded in the lysosome. In the case of typical antibodies, therefore, one IgG antibody molecule cannot bind to three or more antigen molecules. For example, one IgG antibody molecule with neutralizing activity cannot neutralize three or more antigen molecules.

The plasma retention time of the IgG molecule is relatively long (elimination is slow) because the human FcRn, which is known as the rescue receptor for the IgG molecule, functions. IgG molecules incorporated into endosomes by pinocytosis bind under the acidic conditions of the endosomes to human FcRn expressed in the endosomes. IgG molecules that cannot bind to human FcRn are transported to and degraded by lysosomes. Meanwhile, IgG molecules bound to human FcRn are transferred to the cell surface. IgG molecules dissociate from human FcRn under neutral conditions in plasma and are recycled back into plasma.

Alternatively, when the antigen-binding molecules are antibodies that bind to a soluble antigen, the antibodies administered in vivo bind to the antigens and then the antibodies are incorporated into cells while remaining bound to the antigens. Most antibodies incorporated into cells bind to FcRn in the endosome and then are transferred to the cell surface. Antibodies dissociate from human FcRn under neutral plasma conditions and are released into the external environment of the cells. However, antibodies having typical antigen-binding domains, whose antigen-binding activity does not vary depending on ionic concentration conditions such as pH, are released into the external environment of the cells, remaining bound to the molecules, and thus they cannot bind again to the antigen. Thus, like antibodies that bind to membrane antigens, a single typical IgG antibody molecule, whose antigen-binding activity does not vary depending on ion concentration conditions such as pH, cannot bind three or more antigen molecules.

Antibodies that bind to antigens in a pH-dependent manner that bind strongly to antigens under conditions of the neutral pH range and dissociate from antigens under conditions of the acidic pH range in the endosome (antibodies that bind under conditions of the neutral pH range and dissociate under conditions of the pH range acidic pH values), and antibodies that bind to antigens in a calcium ion concentration dependent manner that bind strongly to antigens under conditions of high plasma calcium ions and dissociate from antigens under conditions of low endosome calcium ions (antibodies that bind to antigens under conditions of high concentration of calcium ions and dissociate under conditions of low concentration of calcium ions) can dissociate from the antigen in the endosome. Antibodies that bind to antigens in a pH-dependent or calcium ion concentration-dependent manner, when recycled to plasma via FcRn, can re-bind to the antigen after dissociation from the antigens. Thus, such a single antibody molecule can repeatedly bind to several antigen molecules. Meanwhile, the antigen bound to the antigen-binding molecules is dissociated from the antibody in the endosome and degraded in the lysosome without being recycled to the plasma. By administering such antigen-binding molecules in vivo, the uptake of antigen into cells is accelerated and it is possible to reduce the plasma concentration of antigen.

The uptake of antigens bound by antigen-binding molecules into cells is further accelerated by conferring human FcRn binding activity under neutral pH range conditions (pH 7.4) to antibodies that bind to antigens in a pH dependent manner that bind strongly to antigens under neutral pH range conditions. in plasma and dissociate from antigens under acidic pH range conditions in the endosome (antibodies that bind to antigens under neutral pH range conditions and dissociate under acidic pH range conditions), and antibodies that bind to antigens in a calcium ion concentration dependent manner , which bind strongly to antigens under conditions of high plasma calcium ions and dissociate from antigens under conditions of low calcium ions in the endosome (antibodies that bind to antigens under conditions of high calcium ions and dissociate under conditions of low calcium ions). In particular, by administering such antigen-binding molecules in vivo, the elimination of the antigen is accelerated, and it is possible to lower the plasma concentration of the antigen. Exemplary antibodies that do not have the ability to bind antigens in a pH dependent or calcium ion concentration dependent manner and antigen-antibody complexes of such antibodies are incorporated into cells by non-specific endocytosis and transported to the cell surface by binding to FcRn under the acidic conditions of endosomes. They dissociate from FcRn under neutral conditions on the cell surface and are recycled into plasma. Thus, when an antibody that binds to an antigen in a fully pH dependent manner (which binds under conditions of the neutral pH range and dissociates under conditions of the acidic pH range) or in a completely calcium ion concentration dependent manner (which binds under conditions of high calcium ion concentration and dissociates under conditions of low concentration of calcium ions) binds to the antigen in plasma and dissociates from the antigen in the endosome, the rate of elimination of the antigen is considered equal to the rate of capture of the antibody or antigen-antibody complex into cells through nonspecific endocytosis. When pH or calcium ion dependence of antigen-antibody binding is insufficient, antigens that have not dissociated from antibodies in the endosome are recycled in plasma along with the antibodies. On the other hand, when the dependence on pH or calcium ion concentration is high enough, the rate-limiting step of antigen elimination is cellular uptake by non-specific endocytosis. Meanwhile, FcRn transports antibodies from the endosome to the cell surface, and it is expected that part of the FcRn is also distributed on the cell surface.

Typically, an IgG-type immunoglobulin, which is one embodiment of antigen-binding molecules, has little FcRn binding activity in the neutral pH range. The present inventors concluded that an IgG-type immunoglobulin having FcRn binding activity in the neutral pH range can bind to FcRn on the cell surface and is incorporated into cells in an FcRn-dependent manner by binding to FcRn on the cell surface. The rate of FcRn-mediated cellular uptake is faster than cellular uptake by non-specific endocytosis. Thus, the authors of the present invention suggested that the rate of elimination of antigen antigennegative molecules can be further increased by making the antigennegative molecules the ability to bind FcRn in the range of neutral pH values. In particular, antigen-binding molecules that have the ability to bind FcRn in the neutral pH range deliver antigens to cells faster than native IgG-type immunoglobulin; molecules dissociate from antigens in the endosome and recycle to the cell surface or plasma; and re-bind there with antigens and are incorporated into cells via FcRn. The rate of cycling can be increased by increasing the ability to bind FcRn in the neutral pH range, which results in faster elimination of the antigen from plasma. Moreover, the rate of elimination of antigen from plasma can be further increased by reducing the antigen-binding activity of the antigen-binding molecule at acidic pH values relative to activity in the neutral pH range. In addition, the number of antigen molecules to which one antigen-binding molecule can bind is predicted to increase due to the increase in the number of cycles resulting from the increase in the cycling rate. The antigen-binding molecules of the present invention comprise an antigen-binding domain and an FcRn-binding domain. Since the FcRn-binding domain does not affect antigen binding and is independent of antigen type, based on the mechanism described above, antigen-binding molecule-mediated uptake of antigen into cells can be increased to accelerate the rate of antigen elimination by reducing the antigen-binding activity (ability to bind) of the antigen-binding molecule so to be lower under ion concentration conditions, such as an acidic pH range or low calcium ion concentration, than under ion concentration conditions, such as a neutral pH range or high calcium ion concentration, and/or by increasing FcRn binding activity at plasma pH. Thus, the antigen binding molecules of the present invention are expected to exhibit better effects than conventional therapeutic antibodies in terms of reducing antigen side effects, increasing antibody dose, improving in vivo antibody dynamics, and so on.

In FIG. 1 shows the mechanism by which soluble antigens are eliminated from plasma upon administration of an antibody with pH dependent antigen binding that has increased FcRn binding activity at neutral pH compared to a conventional neutralizing antibody. After binding to a soluble antigen in plasma, the existing neutralizing antibody, which does not have the ability to pH-dependently bind the antigen, is slowly incorporated into cells through a non-specific interaction with the cells. The complex between the neutralizing antibody and the soluble antigen incorporated into the cell is transported into the acidic endosome and then recycled to plasma via the FcRn. Meanwhile, a pH-dependent antigen-binding antibody that has increased FcRn-binding activity under neutral conditions, after binding to a soluble plasma antigen, is rapidly incorporated into cells expressing FcRn on their cell membrane. Then, the soluble antigen bound to the antibody with pH dependent antigen binding dissociates from the antibody in the acidic endosome due to the pH dependent binding ability. The soluble antigen dissociated from the antibody is transferred to the lysosome and degraded by proteolytic activity. Meanwhile, the antibody dissociated from the soluble antigen is recycled to the cell membrane and then released back into the plasma. Free antibody, recycled as described above, can again bind to other soluble antigens. By repeating this cycle: FcRn-mediated uptake into cells; dissociation and degradation of soluble antigen; and antibody recycling, such antibodies with pH dependent antigen binding as described above, having increased FcRn binding activity under neutral conditions, can carry a large amount of soluble antigen into the lysosome and thereby reduce the total plasma antigen concentration.

In particular, the present invention also relates to pharmaceutical compositions containing antigen-binding molecules of the present invention, antigen-binding molecules obtained by the modification methods of the present invention, or antigen-binding molecules obtained by the preparation methods of the present invention.

The antigen-binding molecules of the present invention or the antigen-binding molecules obtained by the preparation methods of the present invention are useful as pharmaceutical compositions because they, when administered, have a potent antigen-lowering effect in plasma compared to typical antigen-binding molecules, and exhibit improved in vivo immune response, pharmacokinetics and so on. in animals to which the molecules are administered. Pharmaceutical compositions of the present invention may contain pharmaceutically acceptable carriers.

Within the scope of the present invention, pharmaceutical compositions generally refer to agents for the treatment or prevention or research and diagnosis of diseases.

Pharmaceutical compositions of the present invention may be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions, including water or other pharmaceutically acceptable liquid. For example, such compositions can be formulated by mixing in the form of a unit dose required by generally approved drug manufacturing practice, by combining as appropriate with pharmacologically acceptable carriers or vehicles, in particular, with sterile water, saline, vegetable oil, emulsifier, suspension, a surfactant, a stabilizer, a flavoring agent, an excipient, a carrier, a preservative, a binder, and the like. In such formulations, the amount of the active ingredient is adjusted so as to obtain an appropriate amount within the predetermined range.

Sterile injectable compositions may be formulated using carriers such as distilled water for injection in accordance with standard formulation practice.

Aqueous injections include, for example, physiological saline and isotonic solutions containing dextrose or other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, and sodium chloride). They can also be used in combination with appropriate solubilizers such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), non-ionic surfactants (polysorbate 80(TM), HCO-50 and etc.).

Oils include sesame oil and soybean oils. In combination with solubilizers, benzyl benzoate and/or benzyl alcohol can be used. Buffers (eg, phosphate buffer, sodium acetate buffer), sedatives (eg, procaine hydrochloride), stabilizers (eg, benzyl alcohol and phenol), and/or antioxidants can also be combined. The resulting injectables are filled into suitable ampoules.

The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, compositions are administered in dosage form for injection, transnasal, pulmonary or transdermal administration. For example, they can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.

Routes of administration can be appropriately selected taking into account the age and symptoms of the patient. The dose of the pharmaceutical composition containing the antigen binding molecule may be, for example, from 0.0001 to 1.000 mg/kg for each administration. Alternatively, the dose may be, for example, from 0.001 to 100,000 mg per patient. However, the present invention is not limited to the numerical values described above. Doses and routes of administration vary depending on the patient's body weight, age, symptoms, and the like. Those skilled in the art can determine suitable doses and routes of administration, taking into account the factors described above.

Moreover, the present invention relates to kits for use in the methods of the present invention, which contain at least an antigen-binding molecule of the present invention. In addition to those described above, pharmaceutically acceptable carriers, media, instructions describing the use of the method, and the like may be packaged in kits.

Moreover, the present invention relates to agents for improving the pharmacokinetics of antigen-binding molecules or agents for reducing the immunogenicity of antigen-binding molecules, which contain, as an active ingredient, an antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention.

The present invention also relates to methods for treating immune inflammatory diseases, which include the step of administering to individuals (study subjects) an antigen-binding molecule of the present invention or an antigen-binding molecule obtained by the production method of the present invention.

The present invention also relates to the use of antigen-binding molecules of the present invention or antigen-binding molecules obtained by the production methods of the present invention, for obtaining agents for improving the pharmacokinetics of antigen-binding molecules or agents for reducing the immunogenicity of antigen-binding molecules.

In addition, the present invention relates to antigen-binding molecules of the present invention and antigen-binding molecules obtained by the production methods of the present invention, for use in the methods of the present invention.

The amino acids contained in the amino acid sequences of the present invention may be post-translationally modified (eg, modification of the N-terminal glutamine to pyroglutamic acid by pyroglutamylation is well known to those skilled in the art). Naturally, such post-translationally modified amino acids are included in the amino acid sequences of the present invention.

All prior art documents cited in the specification are incorporated herein by reference.

[Examples]

Below, the present invention is specifically described by means of examples, however, it should not be considered limited to them.

[Example 1] Effect of enhancing binding to human FcRn under neutral conditions on plasma retention time and immunogenicity of a human antibody with pH dependent human IL-6 receptor binding

For an FcRn binding domain, such as the Fc region of antigen binding molecules such as antibodies that interact with FcRn (Nat. Rev. Immunol. (2007) 7(9), 715-25), it is important to have an FcRn binding activity in the range neutral pH values to eliminate soluble antigen from plasma. As indicated in Reference Example 5, an FcRn binding domain mutant (amino acid substitution) that has an FcRn binding domain activity of binding to FcRn in the neutral pH range was tested. F1-F600, which were produced as Fc mutants, were evaluated for their FcRn binding activity in the neutral pH region, and it was confirmed that antigen elimination from plasma was accelerated by enhancing the FcRn binding activity in the neutral pH region. For the development of these Fc mutants as pharmaceuticals, in addition to having the preferred pharmacological properties (such as accelerating the elimination of antigen from plasma by enhancing FcRn binding), it is also preferable to have improved stability and purity of antigen-binding molecules, increased plasma retention time of antigen-binding molecules in the body and low immunogenicity.

Plasma retention of an antibody is known to be impaired by binding to FcRn under neutral conditions. If an antibody binds to an FcRn under neutral conditions, even if the antibody returns to the cell surface by binding to an FcRn under acidic conditions in endosomes, the IgG antibody does not recycle to plasma until the IgG antibody dissociates from the FcRn in plasma under neutral conditions, so thereby, on the contrary, causing a deterioration in plasma confinement. For example, plasma retention of an antibody is described to be impaired when the antibody is administered to mice for which binding to mouse FcRn was observed under neutral conditions (pH 7.4) by introducing an amino acid substitution in IgG1 (Non-Patent Document 10). However, on the other hand, it has also been described that when the antibody was administered to cynomolgus monkeys that were observed to bind to human FcRn under neutral conditions (pH 7.4), there was no improvement in plasma retention of the antibody, and no change in retention in plasma was observed. plasma (non-patent documents 10, 11 and 12).

In addition, it has been described that FcRn is expressed in antigen-presenting cells and is involved in antigen presentation. In a report describing the assessment of the immunogenicity of a protein (hereinafter referred to as MBP-Fc) obtained by fusion of the mouse IgG1 Fc region with myelin basic protein (MBP), although not an antigen-binding molecule, T cells that specifically react with MBP -Fc undergo activation and proliferation as a result of cultivation in the presence of MBP-Fc. T cell activation is known to be enhanced in vitro by increasing incorporation into antigen presenting cells mediated by FcRn expressed in antigen presenting cells by introducing a modification to the Fc region of MBP-Fc that causes increased FcRn binding. However, since plasma retention is impaired by the introduction of a modification that causes an increase in FcRn binding, it has been described that T cell activation is, on the contrary, reduced in vivo (Non-Patent Document 43).

Thus, the effect of enhancing FcRn binding under neutral conditions on plasma retention and immunogenicity of antigen-binding molecules has not been sufficiently investigated. In the case of developing antigen-binding molecules as pharmaceuticals, the plasma retention of these antigen-binding molecules is preferably as long as possible, and the immunogenicity is preferably as low as possible.

(1-1) Production of human antibodies binding human IL-6 receptor

Thus, in order to assess the plasma retention of antigen-binding molecules that contain an FcRn-binding domain having the ability to bind to human FcRn under neutral pH conditions, and to assess the immunogenicity of these antigen-binding molecules, human IL-6 receptor-binding antibodies having binding activity to human FcRn under neutral pH conditions in the form of Fv4-IgG1 consisting of VH3-IgG1 (SEQ ID NO: 35) and VL3-CK (SEQ ID NO: 36), Fv4-IgG1-F1 consisting of VH3 -IgG1-F1 (SEQ ID NO: 37) and VL3-CK, Fv4-IgG1-F157, consisting of VH3-IgG1-F157 (SEQ ID NO: 38) and VL3-CK, Fv4-IgG1-F20, consisting of VH3 -IgG1-F20 (SEQ ID NO: 39) and VL3-CK, and Fv4-IgG1-F21, consisting of VH3-IgG1-F21 (SEQ ID NO: 40) and VL3-CK, according to the methods shown in the reference example 1 and reference example 2.

(1-2) Kinetic analysis of mouse FcRn binding

Antibodies containing VH3-IgG1 or VH3-IgG1-F1 as heavy chain and L(WT)-CK (SEQ ID NO: 41) as light chain were prepared using the method of Reference Example 2, and the binding activity to Mouse FcRn, as described below.

Binding between antibody and mouse FcRn was analyzed kinetically using Biacore T100 (GE Healthcare). Sufficient protein L (ACTIGEN) was immobilized on a CM4 sensor chip (GE Healthcare) via the amine addition method and the antibodies of interest were allowed to be captured on the chip. Then, diluted FcRn solutions and running buffer (as a reference solution) were injected to allow interaction of the mouse FcRn with the antibody captured on the sensor chip. The running buffer used contained 50 mmol/l sodium phosphate, 150 mmol/l NaCl and 0.05% (w/v) Tween20 (pH 7.4). FcRn was diluted using each buffer. The sensor chip was regenerated using 10 mmol/l glycine-HCl (pH 1.5). Analyzes were performed exclusively at 25°C. The association rate constant ka (1/Ms) and the dissociation rate constant k _d (1/s), each of which are kinetic parameters, were calculated based on the sensograms obtained in the assays, and the KD (M) of each antibody was determined from these values. for mouse FcRn. Each parameter was calculated using Biacore T100 Evaluation software (GE Healthcare).

As a result, although the KD(M) of IgG1 was not detected, the KD(M) of the produced IgG1-F1 was 1.06E-06(M). This indicates that the binding activity of the resulting IgG1-F1 to mouse FcRn is enhanced under conditions of the neutral pH region (pH 7.4).

(1-3) PK in vivo study using normal mice

The PK study was performed using the method below using normal mice producing human antibodies with pH dependent binding to the human IL-6 receptor, Fv4-IgG1 and Fv4-IgG1-F1. Anti-human IL-6 receptor antibody was administered at a dose of 1 mg/kg as a single injection into the tail vein and under the back skin of normal mice (C57BL/6J mouse, Charles River Japan). Blood sampling was carried out 5 minutes, 7 hours and 1, 2, 4, 7, 14, 21 and 28 days after administration of antibodies against human IL-6 receptor. Plasma was obtained by immediate centrifugation of the taken blood for 15 minutes at 4°C and 15,000 rpm. The separated plasma was stored in a freezer set at -20° C. or lower until the time of measurement.

(1-4) ELISA determination of anti-human IL-6 receptor antibody concentration in plasma

The concentration of anti-human IL-6 receptor antibody in mouse plasma was determined by ELISA. First, anti-human IgG (γ-chain specific) F(ab') 2 fragment (SIGMA) was dispensed into Nunc-Immuno Plate, MaxiSoup (Nalge nunc International) and allowed to stand overnight at 4°C to give tablets with anti-human IgG antibody in solid phase. Samples for a calibration curve were prepared having plasma concentrations of anti-human IgG antibody of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μg/mL, and mouse plasma samples for measurement, diluted 100 times or more. Then mixtures prepared by adding 200 µl of soluble human IL-6 receptor at 20 ng/ml to 100 µl of standard curve samples and measurement plasma samples were allowed to stand intact for 1 hour at room temperature. The anti-human IgG solid phase plate to each well of which the mixtures were dispensed was then allowed to stand intact for 1 hour at room temperature. Then, the reaction liquid obtained by reacting for one hour with a biotinylated anti-human IL-6 R antibody (R&D) at room temperature and further reacting for one hour with Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) at room temperature was carried out. TMB One Component HRP Microwell Substrate (BioFX Laboratories) as substrate. After stopping the reaction with 1 N sulfuric acid (Showa Chemical), the absorbance of the reaction liquids in each well at 450 nm was measured on a microplate reader. Plasma antibody concentrations in mice were calculated from absorbance values on the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

The concentrations of antibodies with pH-dependent binding to the human IL-6 receptor following intravenous or subcutaneous administration of human antibodies with pH-dependent binding of the human IL-6 receptor to normal mice are shown in FIG. 2. Based on the results presented in FIG. 2, compared with intravenously administered Fv4-IgG1, plasma retention was shown to be impaired by intravenous administration of Fv4-IgG1-F1, in which case binding to mouse FcRn under neutral conditions was enhanced. On the other hand, although subcutaneously administered Fv4-IgG1 showed comparable plasma retention to intravenous plasma retention, in the case of subcutaneously administered Fv4-IgG1-F1, a sudden decrease in plasma concentration was observed 7 days after administration, which was believed to was a consequence of the production of mouse antibodies against Fv4-IgG1-F1, and on the 14th day after administration, Fv4-IgG1-F1 was not detected in plasma. Based on this result, it was confirmed that plasma retention and immunogenicity deteriorated by enhancing the binding of antigen-binding molecules to FcRn under neutral conditions.

[Example 2] Obtaining a mouse antibody binding to human IL-6 receptor having mouse FcRn binding activity under neutral pH conditions

A mouse antibody having mouse FcRn binding activity under neutral pH conditions was obtained by the method below.

(2-1) Obtaining a mouse antibody that binds to the human IL-6 receptor

The amino acid sequence of a mouse antibody capable of binding to human IL-6R, Mouse PM-1 (Sato, K., et al., Cancer Res. (1993) 53 (4), 851-856) was used as the variable region of the mouse antibody. . In the description below, the Mouse PM-1 heavy chain variable region is referred to as mPM1H (SEQ ID NO: 42), while the light chain variable region is referred to as mPM1L (SEQ ID NO: 43).

In addition, a naturally occurring mouse IgG1 (SEQ ID NO: 44, hereinafter referred to as mIgG1) was used as the heavy chain constant region, while a naturally occurring mouse kappa sequence (SEQ ID NO: 45, hereinafter referred to as mk1) was used as the light chain constant region.

An expression vector having the base sequences of mPM1H-mIgG1 heavy chain (SEQ ID NO: 46) and mPM1L-mk1 light chain (SEQ ID NO: 47) was obtained by the method of Reference Example 1. In addition, mPM1-mIgG1, which is a mouse antibody binding human IL-6R, consisting of mPM1H-mIgG1 and mPM1L-mk1, was obtained according to the method of Reference Example 2.

(2-2) Production of an mPM1 antibody capable of binding to mouse FcRn under neutral pH conditions

The resulting mPM1-mIgG1 is a mouse antibody that contains a naturally occurring mouse Fc region and lacks mouse FcRn binding activity under neutral pH conditions. Thus, an amino acid modification was made to the mPM1-mIgG1 heavy chain constant region to confer mouse FcRn binding activity under neutral pH conditions.

More specifically, mPH1H-mIgG1-mF3 (SEQ ID NO: 48) was obtained by introducing an amino acid substitution obtained by substituting Tyr for Thr at position 252 of mPH1H-mIgG1, as indicated by EU numbering, an amino acid substitution obtained by substituting Glu for Thr at position 256 (EU numbering), an amino acid substitution obtained by substituting Lys for His at position 433 (EU numbering), and an amino acid substitution obtained by substituting Phe for Asn at position 434 (EU numbering).

Similarly, mPH1H-mIgG1-mF14 (SEQ ID NO: 49) was generated by introducing the amino acid substitution obtained by substituting Tyr for Thr at position 252 (EU numbering) of mPH1H-mIgG1, the amino acid substitution obtained by substituting Glu for Thr at position 256 ( EU numbering), and an amino acid substitution obtained by substituting Lys for His at position 433 (EU numbering).

Moreover, mPM1H-mIgG1-mF38 (SEQ ID NO: 50) was obtained by introducing an amino acid substitution obtained by substituting Tyr for Thr at position 252 (EU numbering) of mPH1H-mIgG1, an amino acid substitution obtained by substituting Glu for Thr at position 256 (EU numbering), and an amino acid substitution obtained by substituting Trp for Asn at position 434 (EU numbering).

A mouse IgG1 antibody capable of binding to mouse FcRn under neutral pH conditions, mPM1-mIgG1-mF3, which is composed of mPM1H-mIgG1-mF3 and mPM1L-mk1, was obtained using the method of Reference Example 2.

(2-3) Confirmation of mouse FcRn binding activity using Biacore

Received antibodies that contained mPM1-mIgG1 or mPM1-mIgG1-mF3 as a heavy chain and L(WT)-CK (SEQ ID NO: 41) as a light chain and measured the binding activity of these antibodies to mouse FcRn at pH 7.0 (dissociation constant KD). The results are presented in table 5 below.

[Example 3] Binding experiment of antigen-binding molecules having an Fc region to FcRn and FcγR

In Example 1, it was confirmed that plasma retention and immunogenicity deteriorated as a result of increased binding of antigen-binding molecules to FcRn under neutral conditions. Because naturally occurring IgG1 does not have human FcRn binding activity in the neutral region, it was believed that plasma retention and immunogenicity would be impaired by imparting FcRn binding ability under neutral conditions.

(3-1) FcRn binding domain and FcγR binding domain

In the Fc region of an antibody, there is an FcRn binding domain and an FcγR binding domain. The FcRn binding domain is present in two regions within the Fc region, and it has previously been described that two FcRn molecules are able to simultaneously bind to the Fc region of a single antibody molecule (Nature (1994) 372 (6504), 379-383). On the other hand, although the FcγR binding domain is also present in two regions in the Fc region, it is believed that two FcγR molecules cannot bind simultaneously. This is a consequence of the fact that the second FcγR molecule is unable to bind due to structural changes in the Fc region that occur due to the binding of the first FcγR molecule to the Fc region (J. Biol. Chem. (2001) 276 (19), 16469-16477).

As previously described, active FcγR is expressed on the cell membranes of numerous immune cells such as dendritic cells, NK cells, macrophages, neutrophils, and adipocytes. Moreover, in humans, FcRn has been described to be expressed in immune cells such as antigen presenting cells, eg dendritic cells, macrophages and monocytes (J. Immunol. (2001) 166 (5), 3266-3276). Because normal naturally occurring IgG1 is unable to bind to FcRn in the neutral pH region and is only able to bind to FcγR, naturally occurring IgG1 binds to antigen presenting cells by forming an FcγR/IgG1 binary complex. Phosphorylation sites are found in the intracellular domains of FcγR and FcRn. Typically, phosphorylation of the intracellular domains of receptors expressed on cell surfaces occurs by receptor conjugation, and as a result of this phosphorylation, the receptors are internalized. Even if naturally occurring IgG1 forms a binary FcγR/IgG1 complex on antigen-presenting cells, conjugation of the intracellular FcγR domain does not occur. However, when hypothetically an IgG molecule with FcRn binding activity under neutral pH conditions forms a four-component complex: FcγR/two FcRn/IgG molecules, internalization of a four-component heterocomplex consisting of FcγR/two FcRn/IgG molecules can be induced as a result. FcRn/IgG, since conjugation of the three intracellular domains of FcγR and FcRn occurs. It is believed that the formation of a heterocomplex containing four components, consisting of FcγR/two FcRn/IgG molecules, occurs on antigen-presenting cells expressing both FcγR and FcRn, and it was believed that as a result of this, the retention in plasma of antibody molecules included in antigen-presenting cells , worsens and the possibility of worsening immunogenicity was also taken into account.

However, there are no reports supporting the pathway by which antigen-binding molecules containing an FcRn-binding domain, such as an Fc region having FcRn-binding activity under conditions of the neutral pH region, bind to immune cells, such as antigen-presenting cells expressing FcγR and FcRn. together.

Whether a four-component complex of FcγR/two FcRn/IgG molecules can be formed can be determined by whether an antigen-binding molecule containing an Fc region having FcRn binding activity under conditions of the neutral pH region can simultaneously bind to FcγR and FcRn. Thus, an experiment was carried out on simultaneous binding to FcRn and FcγR of the Fc region contained in the antigen-binding molecule according to the method below.

(3-2) Assessing co-binding to FcRn and FcγR using Biacore

Whether a human or mouse FcRn and a human or mouse FcγR simultaneously bind to an antigen-binding molecule was assessed using the Biacore T100 or T200 System (GE Healthcare). The antigen-binding molecule of interest was captured with human or mouse FcRn immobilized on a CM4 sensor chip (GE Healthcare) by the amine addition method. Diluted human or mouse FcγRs and running buffer as blank were then injected to allow the human or mouse FcγR to interact with the antigen-binding molecule bound to the FcRn on the sensor chip. A buffer consisting of 50 mmol/l sodium phosphate, 150 mmol/l NaCl and 0.05% (w/v) Tween 20 (pH 7.4) was used as a running buffer, and this buffer was also used to dilute FcγR . 10 mmol/L Tris-HCl (pH 9.5) was used to regenerate the sensor chip. All binding measurements were performed at 25°C.

(3-3) Simultaneous binding experiment of human IgG, human FcRn, human FcγR or mouse FcγR

Whether Fv4-IgG1-F157 prepared according to Example 1, which is a human antibody capable of binding to human FcRn under neutral pH range conditions, binds to different types of human FcγR or different types of FcγR while binding to human FcRn.

The result showed that Fv4-IgG1-F157 was able to bind to human FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb and FcγRIIIa(F) concurrently with binding to human FcRn (FIGS. 3, 4, 5, 6 and 7 ). In addition, Fv4-IgG1-F157 was shown to be able to bind to FcγRI, FcγRIIb, FcγRIII and FcγRIV simultaneously with binding to FcRn (FIGS. 8, 9, 10 and 11).

Based on the above, it has been shown that human antibodies having the ability to bind to human FcRn under neutral pH conditions are able to bind to various types of human FcγR and various types of mouse FcγR, such as FcγRIa, FcγRIIa(R), FcγRIIa(H) , FcγRIIb and FcγRIIIa(F) human, as well as FcγRI, FcγRIIb, FcγRIII and FcγRIV mouse simultaneously with binding to human FcRn.

(3-4) Simultaneous binding experiment of human IgG, mouse FcRn and mouse FcγR

It was assessed whether the Fv4-IgG1-F20 prepared according to Example 1, which is a human antibody having mouse FcRn binding activity under neutral pH conditions, binds to different types of mouse FcγR simultaneously with binding to mouse FcRn.

The result showed that Fv4-IgG1-F20 was able to bind to mouse FcγRI, FcγRIIb, FcγRIII, and FcγRIV at the same time as binding to mouse FcRn (FIG. 12).

(3-5) Simultaneous binding experiment of mouse IgG, mouse FcRn and mouse FcγR

It was assessed whether mPM1-mIgG1-mF3 prepared according to Example 2, which is a mouse antibody having mouse FcRn binding activity under neutral pH conditions, binds to different types of mouse FcγR simultaneously with binding to mouse FcRn.

The result showed that mPM1-mIgG1-mF3 was able to bind to mouse FcγRIIb and FcγRIII simultaneously with binding to mouse FcRn (FIG. 13). Based on the report that the mouse IgG1 antibody does not have the ability to bind to mouse FcγRI and FcγRIV (J. Immunol. (2011) 187 (4), 1754-1763), the result that binding to mouse FcγRI and FcγRIV was not confirmed is considered plausible result.

Based on these data, it was shown that human antibodies and mouse antibodies having binding activity to mouse FcRn under neutral pH conditions are also able to bind to different types of mouse FcγR simultaneously with binding to mouse FcRn.

The above data indicate the possibility of the formation of a heterocomplex containing one Fc molecule, two FcRn molecules and one FcγR molecule without any mutual interference, although in the Fc region of human and mouse IgG there is an FcRn-binding region and an FcγR-binding region.

This property of the Fc region of an antibody to be able to form such a heterocomplex has not been previously described and was determined for the first time within the scope of the present invention. As previously described, various types of active FcγR and FcRn are expressed on antigen presenting cells, and the formation of this type of four-component complex on antigen presenting cells is believed to increase the affinity of antigen presenting molecules while further accelerating incorporation into antigen presenting cells by enhancing internalization signals via intracellular domain conjugation. Typically, antigen-binding molecules incorporated into antigen-presenting cells are degraded in the lysosomes of antigen-presenting cells and then presented to T cells.

In other words, antigen-binding molecules having FcRn-binding activity in the neutral pH region form a heterocomplex containing four components, including one active FcγR molecule and two FcRn molecules, and this is believed to be the result of incorporation into antigen-presenting cells, thereby impairing retention in plasma and further impairing immunogenicity.

Therefore, in the case of mutating an antigen-binding molecule having FcRn-binding activity in the neutral pH region to produce an antigen-binding molecule in which the ability to form such a four-component complex is reduced, and introducing this antigen-binding molecule into the body, the plasma retention time of this antigen-binding molecule increases, and the induction of an immune response in the body can be inhibited (namely, immunogenicity can be reduced). Examples of preferred embodiments of antigen-binding molecules incorporated into cells without such complex formation include the three types shown below.

(Embodiment 1) Antigen binding molecules which have FcRn binding activity under neutral pH conditions and whose binding activity to active FcγR is lower than the binding activity of the native FcγR binding domain.

The antigen-binding molecules of Embodiment 1 form a three-component complex by binding to two FcRn molecules, but do not form an active FcγR complex.

(Embodiment 2) Antigen-binding molecules that have an FcRn binding activity under neutral pH conditions and have an inhibitory FcγR selective binding activity

The antigen-binding molecules of Embodiment 2 are capable of forming a four-component complex by binding to two FcRn molecules and one inhibitory FcγR molecule. However, since an antigen-binding molecule is only able to bind to one FcγR molecule, one antigen-binding molecule is unable to bind to another active FcγR while it is bound to an inhibitory FcγR. Moreover, it has been reported that antigen-binding molecules that are incorporated into cells while still bound to inhibitory FcγRs are recycled to the cell membrane to avoid degradation in cells (Immunity (2005) 23, 503-514). That is, it is believed that antigen-binding molecules having the activity of selective binding to an inhibitory FcγR are unable to form a complex containing an active FcγR that elicits an immune response.

(Embodiment 3) Antigen binding molecules in which only one of the two polypeptides constituting the FcRn binding domain has FcRn binding activity under neutral pH conditions while the other has no FcRn binding activity under neutral pH conditions pH

Although the antigen-binding molecules of Embodiment 3 are capable of forming a ternary complex by binding to one FcRn molecule and one FcγR molecule, they do not form a four-component heterocomplex comprising two FcRn molecules and one FcγR molecule.

The antigen-binding molecules of Embodiments 1-3 are expected to be able to increase plasma retention and reduce immunogenicity compared to antigen-binding molecules that are capable of forming four-component complexes comprising two FcRn molecules and one FcγR molecule.

[Example 4] Evaluation of plasma retention of human antibodies that have binding activity to human FcRn in the neutral pH region and whose binding activity to human and mouse FcγR is lower than the binding activity of the native FcγR binding domain

(4-1) Obtaining an antibody whose binding activity to human FcγR is lower than the binding activity of the native FcγR binding domain and which binds to the human IL-6 receptor in a pH dependent manner

Antigen-binding molecules according to Embodiment 1 among the three embodiments shown in Example 3, namely, antigen-binding molecules having FcRn binding activity under neutral pH conditions and whose binding activity to active FcγR is lower than the binding activity of native FcγR binding domain was obtained in the same way as described below.

Fv4-IgG1-F21 and Fv4-IgG1-F157 prepared according to Example 1 are antibodies that have binding activity to human FcRn under neutral pH conditions and bind to the human IL-6 receptor in a pH dependent manner. Variants were generated in which binding to mouse FcγR was reduced by an amino acid substitution in which Lys replaced Ser at position 239 (EU numbering) in their amino acid sequences. More specifically, VH3-IgG1-F140 (SEQ ID NO: 51) was obtained in which Lys replaced Ser at position 239 (EU numbering) of the VH3-IgG1-F21 amino acid sequence. In addition, VH3-IgG1-F424 (SEQ ID NO: 52) was obtained in which Lys replaced Ser at position 239 (EU numbering) of the amino acid sequence of VH3-IgG1-F157.

Fv4-IgG1-F140 and Fv4-IgG1-F424 containing these heavy chains and VL3-CK light chain were prepared using the method of Reference Example 2.

(4-2) Confirmation of binding activity to human FcRn and mouse FcγR

Binding activity (dissociation constant KD) to human FcRn at pH 7.0 and binding activity to mouse FcγR at pH 7.4 of antibodies containing heavy chains from derived VH3-IgG1-F21, VH3-IgG1-F140, VH3-IgG1-F157 or VH3-IgG1-F424 and light chain from L(WT)-CK were measured using the method below.

(4-3) Kinetic analysis of binding to human FcRn

Kinetic analysis of binding between human FcRn and the above antibodies was performed using Biacore T100 or T200 (GE Healthcare). Antibodies of interest were captured on a CM4 sensor chip (GE Healthcare) on which an appropriate amount of L protein (ACTIGEN) was appropriately immobilized by the amine coupling method. Next, the diluted human FcRn and running buffer used as a blank were injected to allow interaction between the human FcRn and the antibody captured on the sensor chip. A buffer consisting of 50 mmol/L sodium phosphate, 150 mmol/L NaCl and 0.05% (w/v) Tween 20 (pH 7.0 or pH 7.4) was used as running buffer, and each buffer also used to dilute human FcRn. To regenerate the sensor chip, 10 mmol/L glycine-HCl (pH 1.5) was used. All binding measurements were performed at 25°C. The K _D (M) of each human FcRn antibody was calculated based on the kinetic parameters, i.e. association rate constants ka (1/Ms) and dissociation rate constants kd (1/s) calculated from the sensorgram obtained by measurement. Biacore T100 or T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

The results are presented in table 6 below.

Mouse FcγR binding activity at pH 7.4 was measured using the method below.

(4-4) Evaluation of mouse FcγR binding activity

The binding activity between antibodies and mouse FcγRI, FcγRII, FcγRIII and FcγRIV (R&D Systems, Sino Biological) (hereinafter referred to as mouse FcγR) was evaluated using Biacore T100 or T200 (GE Healthcare). Antibodies of interest were captured with protein L (ACTIGEN) which was immobilized in appropriate amounts on a CM4 sensor chip (GE Healthcare) by the amine coupling method. Diluted mouse FcγRs and running buffer, used as a blank, were then injected to ensure interaction with the antibody captured on the sensor chip. A buffer consisting of 20 mmol/L ACES, 150 mmol/L NaCl and 0.05% (w/v) Tween 20 (pH 7.4) was used as running buffer and this buffer was also used to dilute mouse FcγR . To regenerate the sensor chip, 10 mmol/L glycine-HCl (pH 1.5) was used. All measurements were carried out at 25°C.

Mouse FcγR binding activity can be represented by relative mouse FcγR binding activity. The antibody was captured with protein L, and the amount of change in the sensorogram before and after capture of the antibody was determined as X1. Next, the mouse FcγR was allowed to interact with the antibody, and the value obtained by subtracting the binding activity of the mouse FcγR corresponding to the amount of change on the sensogram before and after allowing the running buffer to interact with the antibody captured by protein L (ΔA2) from the value obtained by multiplying by The 1500 value obtained by dividing the mouse FcγR binding activity corresponding to the amount of change in the sensorogram before and after this interaction (ΔA1) by the amount (X) of each antibody captured was divided by the amount of each antibody captured (X) and then multiplied by 1500 to give mouse FcγR binding activity (Y) (Equation 1).

[Equation 1]

Mouse FcγR binding activity (Y)=(ΔA1-ΔA2)/X×1500

The results are presented in table 7 below.

As shown in Tables 2 and 3, Fv4-IgG1-F140 and Fv4-IgG1-F424 showed reduced binding to mouse FcγR without affecting human FcRn binding activity compared to Fv4-IgG1-F21 and Fv4-IgG1- F157.

(4-5) In vivo PK study using transgenic mice with human FcRn

The study of PK when the obtained antibodies Fv4-IgG1-F140, Fv4-IgG1-F424, Fv4-IgG1-F21 and Fv4-IgG1-F157 were administered to transgenic mice with human FcRn was performed by the method presented below.

Anti-human IL-6 receptor antibody was administered at 1 mg/kg in a single tail vein injection of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32+/+ mouse, Jackson Laboratories, Methods Mol. Biol. (2010) 602, 93-104). Blood sampling was carried out 15 minutes, 7 hours and 1, 2, 3, 4, 7, 14, 21 and 28 days after administration of antibodies against human IL-6 receptor. Plasma was obtained by centrifuging immediately collected blood for 15 minutes at 4°C and 15,000 rpm. The separated plasma was stored in a freezer set at -20° C. or lower until the time of measurement.

(4-6) Measurement of anti-human IL-6 receptor antibody concentration by ELISA

The concentration of anti-human IL-6 receptor antibody in mouse plasma was determined by ELISA. First, anti-human IgG (γ-chain specific) F(ab') 2 fragment (SIGMA) was dispensed into Nunc-Immuno Plate, MaxiSoup (Nalge nunc International) and allowed to stand overnight at 4°C to give tablets with anti-human IgG antibody in solid phase. Samples for a calibration curve were prepared having plasma concentrations of anti-human IgG antibody of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μg/mL, and mouse plasma samples for measurement, diluted 100 times or more. Then mixtures prepared by adding 200 µl of soluble human IL-6 receptor at 20 ng/ml to 100 µl of standard curve samples and measurement plasma samples were allowed to stand intact for 1 hour at room temperature. The anti-human IgG solid phase plate to each well of which the mixtures were dispensed was then allowed to stand intact for 1 hour at room temperature. Then, the reaction liquid obtained by reacting for one hour with a biotinylated anti-human IL-6 R antibody (R&D) at room temperature and further reacting for one hour with Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) at room temperature was carried out. TMB One Component HRP Microwell Substrate (BioFX Laboratories) as substrate. After stopping the reaction with 1 N sulfuric acid (Showa Chemical), the absorbance of the reaction liquids in each well was measured at 4-50 nm on a microplate reader. Plasma antibody concentrations in mice were calculated from absorbance values on the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

The concentrations of antibodies with pH-dependent binding of human IL-6 receptor after intravenous administration of antibodies with pH-dependent binding of human IL-6 receptor to human FcRn transgenic mice are shown in FIG. 14.

Based on the results presented in Fig. 14, it was found that Fv4-IgG1-F140, whose binding to mouse FcγR was lower compared to Fv4-IgG1-F21, showed an increase in plasma retention time compared to Fv4-IgG1-F21. Similarly, Fv4-IgG1-F424, whose binding to mouse FcγR was lower compared to Fv4-IgG1-F157, was found to exhibit prolonged plasma retention compared to Fv4-IgG1-F157.

Based on this, it was shown that an antibody that has binding activity to human FcRn under neutral pH conditions and has an FcγR binding domain whose FcγR binding activity is lower than that of the normal FcγR binding domain has more higher plasma retention than an antibody having a normal FcγR binding domain.

Although the present invention is not bound by a particular theory, it is believed that the reason for observing such an increase in plasma retention of antigen-binding molecules is that since antigen-binding molecules have binding activity to human FcRn under conditions of the neutral pH region, and have an FcγR domain whose binding activity with FcγR being lower than that of the naturally occurring FcγR binding domain, the formation of the quaternary complex described in Example 3 was inhibited. In other words, Fv4-IgG1-F21 and Fv4-IgG1-F157, which form a four-component complex on the cell membrane of antigen-presenting cells, are believed to be more readily incorporated into antigen-presenting cells. On the other hand, it is believed that in the case of Fv4-IgG1-F140 and Fv4-IgG1-F424, which are classified as Embodiment 1 of Example 3, and which do not form a quaternary complex on the cell membrane of antigen-presenting cells, incorporation into antigen-presenting cells is inhibited. . In this case, the incorporation of antigen-binding molecules into cells such as vascular endothelial cells that do not express FcγR is believed to mainly involve non-specific incorporation or incorporation mediated by FcRn on the cell membrane, and is believed not to be affected by reduced binding activity. with FcγR. In other words, the increase in plasma retention that was observed as described above is believed to result from selective inhibition of incorporation into immune cells, including antigen presenting cells.

[Example 5] Evaluation of plasma retention of human antibodies that have human FcRn binding activity in the neutral pH range but no mouse FcγR binding activity

(5-1) Production of human antibodies that do not have human and mouse FcγR binding activity and bind to the human IL-6 receptor in a pH-dependent manner

Received antibodies in the same way as shown below, to obtain human antibodies that do not have the activity of binding to human and mouse FcγR and bind to the human IL-6 receptor in a pH-dependent manner. VH3-IgG1-F760 (SEQ ID NO: 53), which has no activity of binding to human and mouse FcγR, was obtained by amino acid substitution obtained by substitution of Arg for Leu at position 235 (EU numbering) and amino acid substitution obtained by substitution of Lys instead of Ser at position 239 of the VH3-IgG1 amino acid sequence.

Similarly, VH3-IgG1-F821 (SEQ ID NO: 57), VH3-IgG1-F939 (SEQ ID NO: 58), and VH3-IgG1-F1009 (SEQ ID NO: 59) were obtained, which do not have human FcγR binding activity. and mouse, by an amino acid substitution obtained by substituting Arg for Leu at position 235 (EU numbering) and an amino acid substitution obtained by substituting Lys for Ser at position 239 of the respective amino acid sequences VH3-IgG1-F11 (SEQ ID NO: 54), VH3 -IgG1-F890 (SEQ ID NO: 55) and VH3-IgG1-F947 (SEQ ID NO: 56).

Fv4-IgG1, Fv4-IgG1-F11, Fv4-IgG1-F890, Fv4-IgG1-F947, Fv4-IgG1-F760, Fv4-IgG1-F821, Fv4-IgG1-F939 and Fv4-IgG1-F1009 containing these antibodies in as the heavy chain and VL3-CK as the light chain were obtained using the method of Reference Example 2.

(5-2) Confirmation of binding activity to human FcRn and mouse FcγR

Binding activity (dissociation constant KD) to human FcRn at pH 7.0 of antibodies containing VH3-IgG1, VH3-IgG1-F11, VH3-IgG1-F890, VH3-IgG1-F947, VH3-IgG1-F760, VH3-IgG1- F821, VH3-IgG1-F939 or VH3-IgG1-F1009 as a heavy chain and L(WT)-CK as a light chain obtained using the method of Reference Example 2 were measured using the method of Example 4. The measurement results are shown in the table below. 8 below.

Mouse FcγR binding activity at pH 7.4 of antibodies containing VH3-IgG1, VH3-IgG1-F11, VH3-IgG1-F890, VH3-IgG1-F947, VH3-IgG1-F760, VH3-IgG1-F821, VH3-IgG1 -F939 or VH3-IgG1-F1009 as a heavy chain and L(WT)-CK as a light chain were measured in the same way as in the method of example 4. The measurement results are presented in table 9 below.

As shown in Tables 4 and 5, Fv4-IgG1-F760, Fv4-IgG1-F821, Fv4-IgG1-F939, and Fv4-IgG1-F1009 showed reduced binding to mouse FcγR without affecting human FcRn binding activity by compared to Fv4-IgG1, Fv4-IgG1-F11, Fv4-IgG1-F890 and Fv4-IgG1-F947.

(5-3) In vivo PK study using transgenic mice with human FcRn

The study of PK when the obtained antibodies Fv4-IgG1 and Fv4-IgG1-F760 were administered to transgenic mice with human FcRn was performed in accordance with the method presented below.

Anti-human IL-6 receptor antibody was administered at 1 mg/kg in a single tail vein injection of transgenic human FcRn mice (B6.mFcRn-/-.hFcRn Tg line 32+/+ mouse, Jackson Laboratories, Methods Mol. Biol. (2010) 602, 93-104). Blood sampling was carried out 15 minutes, 7 hours and 1, 2, 3, 4, 7, 14, 21 and 28 days after administration of the antibody against the human IL-6 receptor. Plasma was obtained by centrifuging immediately collected blood for 15 minutes at 4°C and 15,000 rpm. The separated plasma was stored in a freezer set at -20° C. or lower until the time of measurement.

The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA in a manner similar to the method of Example 4. The results are shown in FIG. 15. Fv4-IgG1-F760, which reduced Fv4-IgG1 binding activity to mouse FcγR, showed a plasma retention nearly equal to that of Fv4-IgG1-F11; however, the effect of improving plasma retention by reducing FcγR binding activity was not observed.

(5-4) In vivo PK study using transgenic mice with human FcRn

The study of PK when the obtained antibodies Fv4-IgG1-F11, Fv4-IgG1-F890, Fv4-IgG1-F947, Fv4-IgG1-F821, Fv4-IgG1-F939 and Fv4-IgG1-F1009 were administered to transgenic mice with human FcRn was carried out in accordance with in the way shown below.

Anti-human IL-6 receptor antibody was administered at 1 mg/kg as a single injection under the dorsal skin of human FcRn transgenic mice (mouse B6.mFcRn-/-.hFcRn Tg line 32+/+, Jackson Laboratories, Methods Mol. Biol. (2010) 602, 93-104). Blood sampling was carried out 15 minutes, 7 hours and 1, 2, 3, 4, 7, 14, 21 and 28 days after administration of antibodies against human IL-6 receptor. Plasma was obtained by centrifuging immediately collected blood for 15 minutes at 4°C and 15,000 rpm. The separated plasma was stored in a freezer set at -20° C. or lower until the time of measurement.

The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA in a manner similar to the method of Example 4. The results are shown in FIG. 16. Fv4-IgG1-F821, which reduced Fv4-IgG1-F11 binding activity to mouse FcγR, exhibited a plasma retention time substantially equal to that of Fv4-IgG1-F11. On the other hand, Fv4-IgG1-F939, which reduced Fv4-IgG1-F890 binding activity to mouse FcγR, was found to show increased plasma retention compared to Fv4-IgG1-F890. Similarly, Fv4-IgG1-F1009, which reduced Fv4-IgG1-F947 binding activity to mouse FcγR, was found to exhibit increased plasma retention compared to Fv4-IgG1-F947.

On the other hand, since there was no difference in plasma retention for both Fv4-IgG1 and IgG1-F760, and Fv4-IgG1, which does not have FcRn binding activity in the neutral pH region, is able to form a binary complex with FcγR at immune cells, but unable to form a four-component complex, it was believed that there would be no improvement in plasma retention inherent in the decrease in FcγR binding activity. That is, it can be argued that an increase in plasma retention time was observed only as a result of a decrease in the FcγR binding activity of antigen-binding molecules with FcRn binding activity in the neutral pH region and inhibition of the formation of a four-component complex. Based also on these data, it is believed that the formation of a four-component complex plays an important role in increasing the retention time in plasma.

(5-5) Production of human antibodies that do not have human and mouse FcγR binding activity and bind to the human IL-6 receptor in a pH-dependent manner

VH3-IgG1-F1326 (SEQ ID NO: 155), in which the activity of binding to human and mouse FcγR is reduced, was obtained by an amino acid substitution obtained by substituting Ala for Leu at position 234 (EU numbering) and an amino acid substitution obtained by substituting Ala instead of Leu at position 235 of the amino acid sequence VH3-IgG1-F947 (SEQ ID NO: 56).

Fv4-IgG1-F1326 containing VH3-IgG1-F1326 as a heavy chain and VL3-CK as a light chain was prepared using the method of Reference Example 2.

(5-6) Confirmation of binding activity to human FcRn and mouse FcγR

The binding activity (dissociation constant KD) to human FcRn at pH 7.0 of an antibody containing VH3-IgG1-F1326 as a heavy chain and L(WT)-CK as a light chain obtained using the method of Reference Example 2 was measured using the method of example 4. In addition, the activity of binding to mouse FcγR at pH 7.4 was measured in the same way as in the method of example 4. The measurement results are presented in table 10 below.

As shown in Table 10, Fv4-IgG1-F1326 showed reduced binding to mouse FcγR without affecting human FcRn binding activity compared to Fv4-IgG1-F947.

(5-7) In vivo PK study using transgenic mice with human FcRn

The study of PK when the obtained antibody Fv4-IgG1-F1326 was administered to transgenic mice with human FcRn was carried out similarly to the method of example 5-4. The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA in the same manner as in the method of Example 4. The results are shown in FIG. 54 along with the results for Fv4-IgG1-F947 prepared according to Example 5-4. It was found that Fv4-IgG1-F1326, which reduced the binding activity of Fv4-IgG1-F947 to mouse FcγR, showed an increase in plasma retention time compared to Fv4-IgG1-F947.

Based on the above, in the case of a human antibody having increased binding to human FcRn under neutral conditions, it has been shown that it is possible to increase the plasma retention time in transgenic mice with human FcRn by reducing binding activity to mouse FcγR and inhibiting the formation of a four-component complex. In this case, in order to demonstrate the effect of increasing plasma retention time by reducing mouse FcγR binding activity, the affinity (KD) for human FcRn at pH 7.0 should preferably exceed 310 nM and more preferably 110 nM or less.

As a result, it was confirmed that the plasma retention time was increased by imparting the properties of Embodiment 1 to the antigen-binding molecules analogously to Example 4. In this case, the observed increase in plasma retention time is believed to be due to selective inhibition of incorporation into immune cells, including antigen-presenting cells, and as a result, it is expected that it is possible to inhibit the induction of an immune response.

[Example b] Evaluation of plasma retention of mouse antibodies that have mouse FcRn binding activity in the neutral pH region but no mouse FcγR binding activity

(6-1) Generation of mouse antibodies that bind to the human IL-6 receptor but lack mouse FcγR binding activity

In Examples 4 and 5, antigen-binding molecules were shown to have human FcRn binding activity under neutral pH conditions and to contain an FcγR binding domain whose binding activity to mouse FcγR is lower than that of the native FcγR binding domain. , demonstrate improved plasma retention in transgenic mice with human FcRn. Similarly, for antigen-binding molecules that have mouse FcRn binding activity under neutral pH conditions and contain an FcγR binding domain whose binding activity to mouse FcγR is lower than the binding activity of the native FcγR binding domain, whether they increased plasma retention in normal mice.

mPM1H-mIgG1-mF40 (SEQ ID NO: 60) was obtained by an amino acid substitution obtained by substituting Lys for Pro at position 235 (EU numbering) and an amino acid substitution obtained by substituting Lys for Ser at position 239 in the amino acid sequence mPM1H-mIgG1- mF38 obtained according to Example 2, while mPM1H-mIgG1-mF39 (SEQ ID NO: 61) was obtained by amino acid substitution obtained by substituting Lys for Pro at position 235 (EU numbering) and amino acid substitution obtained by substituting Lys instead of Ser at position 239 of the mPM1H-mIgG1-mF14 amino acid sequence.

(6-2) Confirmation of binding activity to mouse FcRn and mouse FcγR

Binding activity (dissociation constant KD) to mouse FcRn at pH 7.0 was measured using the method of Example 2. The results are shown in Table 11 below.

FcγR binding activity at pH 7.4 was measured using the method of Example 4. The results are shown in Table 12 below.

(6-3) In vivo PK study using normal mice

The study of PK with the introduction of the obtained mPM1-mIgG1-mF14, mPM1-mIgG1-mF38, mPM1-mIgG1-mF39 and mPM1-mIgG1-mF40 to normal mice was performed in accordance with the method described below.

Anti-human IL-6 receptor antibody was administered at 1 mg/kg in a single injection under the dorsal skin of normal mice (C57BL/6J mouse, Charles River Japan). Blood sampling was carried out 5 minutes, 7 hours and 1, 2, 4, 7, 14, 21 and 28 days after administration of antibodies against human IL-6 receptor. Plasma was obtained by immediate centrifugation of the taken blood for 15 minutes at 4°C and 15,000 rpm. The separated plasma was stored in a freezer set at -20° C. or lower until the time of measurement.

(6-4) Measurement of mouse anti-human IL-6 receptor antibody concentration by ELISA

The concentration of mouse anti-human IL-6 receptor antibody in mouse plasma was determined by ELISA. Soluble human IL-6 receptor was first dispensed into a Nunc-Immuno Plate, MaxiSoup (Nalge Nunc International) and then allowed to stand intact overnight at 4°C to prepare a solid phase human soluble IL-6 receptor plate. Samples for a calibration curve were obtained having a concentration of mouse anti-human IL-6 receptor antibody in plasma of 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, and 0.020 μg/mL, and mouse plasma samples for measurement diluted 100-fold or more . Aliquots of these standard curve samples and 100 μl plasma samples for measurement were dispensed into each well of a soluble human IL-6 receptor solid phase plate and then allowed to stand intact for 2 hours at room temperature. Then, the reaction liquid obtained by reaction with anti-mouse IgG antibody with peroxidase (SIGMA) was chromogenically reacted for 1 hour at room temperature and further reacted with Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 1 hour at room temperature using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as substrate. After stopping the reaction by adding 1 N sulfuric acid (Showa Chemical), the absorbance of the reaction liquids in each well at 450 nm was measured on a microplate reader. Plasma antibody concentrations in mice were calculated from absorbance values on the calibration curve using SOFTmax PRO analytical software (Molecular Devices). Changes in plasma antibody concentration in normal mice after intravenous administration as measured by this method are shown in FIG. 17.

Based on the results shown in Fig. 17, it was found that mPM1-mIgG1-mF40, which has no mouse FcγR binding activity, exhibits an increase in plasma retention time compared to mPM1-mIgG1-mF38. In addition, mPM1-mIgG1-mF39, which lacks mouse FcγR binding activity, was found to exhibit an increase in plasma retention time compared to mPM1-mIgG1-mF14.

Based on the above, it has been shown that an antibody having mouse FcRn binding activity under neutral pH conditions and having an FcγR binding domain that does not have mouse FcγR binding activity has a higher plasma retention time in normal mice. than an antibody having a normal FcγR binding domain.

As a result, similarly to Examples 4 and 5, it was confirmed that the plasma retention time is high for the antigen-binding molecules having the properties of the antigen-binding molecules of Embodiment 1. Although the present invention is not bound to a particular theory, it is believed that the increase in retention in plasma observed in this case is the result of selective inhibition of incorporation into immune cells, including antigen presenting cells, and as a result, inhibition of the induction of an immune response is expected to be possible.

[Example 7] Evaluation of the in vitro immunogenicity of a humanized antibody (anti-human IL-6 receptor antibody) having a human FcRn binding activity in the neutral pH region and containing an FcγR binding domain whose binding activity to a human FcγR is lower than that of binding at the native FcγR binding domain

To evaluate the immunogenicity in humans of an antigen-binding molecule according to Option 1, namely, an antigen-binding molecule having FcRn binding activity under neutral pH conditions and containing an antigen-binding domain whose binding activity to active FcγR is lower than that of native FcγR- binding domain, the T cell response to the antigen-binding molecule in vitro was evaluated according to the method shown below.

(7-1) Confirmation of human FcRn binding activity

Association constants (KD) of VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F21 and VH3/L(WT)-IgG1-F140 with human FcRn under neutral pH conditions (pH 7.0) measured according to example 4, are presented in table 13 below.

(7-2) Evaluation of human FcγR binding activity

The binding activity of VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F21 and VH3/L(WT)-IgG1-F140 to human FcγR at pH 7.4 was measured using the method below.

The binding activity between antibodies and human FcγRIa, FcγRIIa (H), FcγRIIa (R), FcγRIIb and FcγRIIIa (F) (hereinafter referred to as human FcγR) was evaluated using Biacore T100 or T200 (GE Healthcare). Antibodies of interest were captured with protein L (ACTIGEN) which was immobilized in appropriate amounts on a CM4 sensor chip (GE Healthcare) by the amine coupling method. Next, diluted human FcγRs and running buffer were injected and used as a blank to ensure interaction with antibodies captured on the sensor chip. A buffer consisting of 20 mmol/L ACES, 150 mmol/L NaCl and 0.05% (w/v) Tween 20 (pH 7.4) was used as running buffer and this buffer was also used to dilute human FcγR. To regenerate the sensor chip, 10 mmol/L glycine-HCl (pH 1.5) was used. All measurements were carried out at 25°C.

FcγR binding activity can be reflected by relative human FcγR binding activity. The antibody was captured with protein L, and the amount of change in the sensogram before and after capture of the antibody was determined as X1. Next, the human FcγR was allowed to interact with the antibody, and the value obtained by subtracting the binding activity of the human FcγR corresponding to the amount of change in the sensorgram before and after allowing the running buffer to interact with the antibody captured by protein L (ΔA2) from the value obtained by multiplying by 1500 value obtained by dividing the binding activity of human FcγR corresponding to the amount of change in the sensogram before and after interaction (ΔA1) by the amount (X) of each antibody captured, divided by the amount of each antibody captured (X), and then multiplied by 1500 to obtain the binding activity Human FcγR (Y) (Equation 2).

[Equation 2] Human FcγR binding activity (Y)=(ΔA1-ΔA2)/X×1500

The results are presented in table 14 below.

According to the results shown in Table 14, Fv4-IgG1-F140 showed reduced binding to each human FcγR without affecting human FcRn binding activity compared to Fv4-IgG1-F21.

(7-3) In vitro immunogenicity study using human PBMC

An in vitro immunogenicity study was performed as shown below using Fv4-IgG1-F21 and Fv4-IgG1-F140 prepared according to Example 1.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood taken from healthy volunteers. After separation of PBMCs from blood by Ficoll density gradient centrifugation (GE Healthcare), CD8 ⁺ T cells were magnetically isolated from PBMCs using Dynabeads CD8 (Invitrogen) according to the standard protocol provided. Next, CD25 ^hi T cells were isolated magnetically using Dynabeads CD25 (Invitrogen) according to the standard protocol provided.

Proliferation assay was performed in the same manner as described below. That is, PBMCs from each donor from which CD8 ⁺ T cells and CD25 ^hi T cells were isolated and resuspended in AIMV medium (Invitrogen) containing 3% deactivated human serum to a concentration of 2 x 10 ⁶ /ml were added in a 24-well flat bottom plate at 2 x 10 ⁶ cells per well. After culturing for 2 hours at 37°C and 5% CO ₂ , cells to which the test substance was added to a final concentration of 10, 30, 100 and 300 μg/ml were cultured for 8 days. During cultivation, after transfer to a round-bottom 9b-well plate, BrdU (bromodeoxyuridine) was added to 150 μl of the suspension on days 6, 7, and 8 of cultivation, after which the cells were further cultured for 24 hours. BrdU that incorporated into the nuclei of these cells cultured with BrdU was stained using the BrdU Flow Kit (BD Bioscience) according to the standard protocol provided, while surface antigens (CD3, CD4 and CD19) were stained with anti-CD3 antibodies. , anti-CD4 and anti-CD19 (BD Bioscience). Further, the percentage of BrdU-positive CD4 ⁺ T cells was detected using BD FACS Calibur or BD FACS CantII (BD). The percentage of BrdU-positive CD4 ⁺ T cells was calculated at each concentration of the test substance of 10, 30, 100 and 300 μg/ml on days 6, 7 and 8 of cultivation, and then its average values were calculated.

The results are shown in FIG. 18. In FIG. 18 shows the proliferative responses of CD4 ⁺ T cells to Fv4-IgG1-F21 and Fv4-IgG1-F140 in PBMCs of five human donors from which CD8 ⁺ T cells and CD25 ^hi T cells were isolated. At first, no increase in the CD4 ⁺ T cell proliferative response attributable to the addition of the test substance was observed in PBMCs of donors A, B and D compared to the negative control. It is believed that these donors are not inherently immune to the test substances. On the other hand, a proliferative CD4 ⁺ T cell response inherent in the addition of test substance was observed in PBMCs of donors C and E compared to the negative control. One point to be noted is that the CD4 ⁺ T cell proliferative response of Fv4-IgG1-F140 tended to decrease compared to Fv4-IgG1-F21 for both C and E donors. As described above, Fv4-IgG1 -F140 has a lower human FcγR binding activity than Fv4-IgG1-F21 and has the properties of Embodiment 1. Based on the above results, it is believed that immunogenicity can be suppressed against antigen-binding molecules having FcRn binding activity under conditions of the region neutral pH values and containing an antigen-binding domain, the binding activity of which to human FcγR is lower than the binding activity of the native FcγR-binding domain.

[Example 8] In vitro evaluation of the immunogenicity of a humanized antibody (anti-human A33 antibody) having a human FcRn binding activity in the neutral pH region and containing an antigen-binding domain whose binding activity to a human FcγR is lower than the binding activity of a native FcγR- binding domain

(8-1) Production of hA33-IgG1

Since human PBMCs inherently have a low immune response to Fv4-IgG1-F21, as shown in Example 7, it was suggested that they are not suitable for assessing the suppression of the immune response to Fv4-IgG1-F140 containing an antigen-binding domain whose binding activity with FcγR is lower than the binding activity of the native FcγR binding domain. Thus, a humanized anti-A33 antibody (ha33-IgG1), which is a humanized IgG1 antibody against A33 antigen, was obtained to enhance the ability to detect immunogenicity reduction effects in an in vitro immunogenicity assessment system.

In the case of ha33-IgG1, it was confirmed in a clinical trial that anti-antibody is produced in 33% to 73% (Hwang, et al. (Methods (2005) 36, 3-10) and Walle, et al. (Expert Opin. Bio Ther (2007) 7(3), 405-418)). Since the high immunogenicity of hA33-IgG1 originates in the variable region sequence, for molecules in which FcRn binding activity in the neutral pH region is enhanced for ha33-IgG1, it would be easy to detect immunogenicity-reducing effects that result from inhibition of the formation of a four-component complex by reducing the activity of binding to FcγR.

The amino acid sequences of ha33H (SEQ ID NO: 62) used as the heavy chain variable region of the humanized antibody against A33 and hA33L (SEQ ID NO: 63) used as the light chain variable region were obtained from known information (British Journal of Cancer (1995) 72, 1364-1372). In addition, a naturally occurring human IgG1 (SEQ ID NO: 11, hereinafter referred to as IgG1) was used as the heavy chain constant region, and a naturally occurring human kappa sequence (SEQ ID NO: 64, hereinafter referred to as k0) was used as light chain constant region.

An expression vector containing the ha33H-IgG1 heavy chain and hA33L-k0 light chain base sequences was prepared according to the method of Reference Example 1. In addition, a humanized anti-A33 antibody in the form of ha33-IgG1 containing the ha33H-IgG1 heavy chain and light chain hA33L-k0, according to the method of Reference Example 2.

(8-2) Preparation of A33 binding antibody having human FcRn binding activity under neutral pH conditions

Because the resulting ha33-IgG1 is a human antibody having a naturally occurring human Fc region, it does not have binding activity to human FcRn under neutral pH conditions. Thus, an amino acid modification was made to the ha33-IgG1 heavy chain constant region to confer the ability to bind to human FcRn under neutral pH conditions.

More specifically, hA33H-IgG1-F21 (SEQ ID NO: 65) was generated by substituting Tyr for Met at position 252 (EU numbering), substituting Pro for Val at position 308 (EU numbering), and substituting Tyr for Asn at position 434 (EU numbering). EU) in the ha33-IgG1 heavy chain constant region in the form of ha33H-IgG1. Using the method of Reference Example 2, an A33 binding antibody having human FcRn binding activity under neutral pH conditions was obtained in the form of hA33-IgG1-F21 containing hA33H-IgG1-F21 as a heavy chain and hA33L-k0 as a light chain. chains.

(8-3) Preparation of an A33 binding antibody containing an FcγR binding domain whose binding activity to human FcγR under neutral pH conditions is lower than the binding activity of the native FcγR binding domain

Received hA33H-IgG1-F140 (SEQ ID NO: 66), in which Lys replaced Ser at position 239 (EU numbering) in the amino acid sequence hA33H-IgG1-F21, to reduce the binding activity of hA33-IgG1-F21 to human FcγR.

(8-4) Evaluation of the immunogenicity of various types of A33-binding antibodies by in vitro T-cell assay

The immunogenicity of the resulting ha33-IgG1-F21 and hA33-IgG1-F140 was evaluated using the same method as in Example 7. Moreover, the healthy volunteers serving as donors were not the same individuals as the healthy volunteers from whom the PBMCs used in example 7 were obtained. In other words, donor A in example 7 and donor A in this trial were different healthy volunteers.

The results of the study are presented in Fig. 19. In FIG. 19 compares the results for hA33-IgG1-F21, which has human FcRn binding activity in the neutral pH region, and hA33-IgG1-F140, which contains an FcγR binding domain whose binding activity to human FcγR is lower than binding activity at the native FcγR binding domain. Because no response to ha33-IgG1-F21 was detected in PBMCs from donors C, D, and F compared to the negative control, it is believed that donors C, D, and F are donors that have an immune response to hA33-IgG1-F21 does not occur. A strong immune response to hA33-IgG1-F21 was observed in PBMCs from the other seven donors (donors A, B, E, G, H, I and J) compared to the negative control; and hA33-IgG1-F21 showed a high level of immunogenicity in vitro, as expected. On the other hand, an effect was observed in which the immune response of isolated PBMCs from all of these seven donors (donors A, B, E, G, H, I and J) to hA33-IgG1-F140, which contains an FcγR-binding domain, activity binding of which to human FcγR is lower than the binding activity of the native FcγR-binding domain was reduced compared to the immune response to hA33-IgG1-F21. In addition, since the immune response of PBMCs from E and J donors to hA33-IgG1-F140 was also approximately the same as that of the negative control, it was hypothesized that immunogenicity may be reduced in antigen-binding molecules with binding activity to FcRn human in the region of neutral pH values, by reducing the activity of binding to human FcγR to a level lower than the activity of binding at the native FcγR-binding domain and inhibition of the formation of a four-component complex.

[Example 9] Evaluation of in vitro immunogenicity of a humanized antibody (anti-human A33 antibody) that has binding activity to human FcRn under neutral pH conditions but no binding activity to human FcγR

(9-1) Obtaining an A33 binding antibody having potent human FcRn binding activity under neutral pH conditions

hA33H-IgG1-F698 (SEQ ID NO: 67) was prepared according to the method of Reference Example 1 by substituting Tyr for Met at position 252 (EU numbering), substituting Glu for Asn at position 286 (EU numbering), substituting Gln for Thr at position 307 (EU numbering), an Ala substitution for Gln at position 311 (EU numbering), and a Tyr substitution for Asn at position 434 (EU numbering) in the ha33H-IgG1 amino acid sequence. A human A33 binding antibody having strong binding activity to human FcRn under neutral pH conditions was obtained in the form of hA33-IgG1-F698 containing hA33H-IgG1-F698 as a heavy chain and hA33L-k0 as a light chain.

(9-2) Preparation of an A33-binding antibody containing an antigen-binding domain whose binding activity to human FcγR under neutral pH conditions is lower than the binding activity of a native FcγR-binding domain

Received hA33H-IgG1-F699 (SEQ ID NO: 68), in which Lys replaced Ser at position 239 (EU numbering) of hA33H-F698 and which contains an antigen-binding domain, the binding activity of which to human FcγR is lower than the binding activity of native FcγR binding domain.

The binding activity of VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F698 and VH3/L(WT)-IgG1-F699 to human FcRn at pH 7.0 was measured using the method of Example 4. Moreover , binding activity of VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F698 and VH3/L(WT)-IgG1-F699 to human FcγR at pH 7.4 was measured using the method of Example 7. Results for both measurements are presented in Table 15 below.

As shown in Table 15, VH3/L(WT)-IgG1-F699 in which Lys replaces Ser at position 239 (EU numbering) and which contains an antigen-binding domain whose binding activity for each type of human FcγR is lower than the binding activity at the native FcγR binding domain showed binding activity to hFcgRI even though binding to hFcgRIIa (R), hFcgRIIa (H), hFcgRIIb and hFcgRIIIa (F) was reduced.

(9-3) Evaluation of the immunogenicity of various types of A33-binding antibodies by in vitro T-cell assay

Immunogenicity against the obtained hA33-IgG1-F698 and ha33-IgG1-F699 was evaluated according to the same method as in Example 7. Moreover, healthy volunteers serving as donors were not the same individuals as healthy volunteers. from which the PBMCs used according to examples 7 and 8 were obtained. In other words, donor A in examples 7 and 8 and donor A in this study were different healthy volunteers.

The results of the study are presented in Fig. 20. In FIG. 20, a comparison is made between the results for hA33-IgG1-F698, which has a potent binding activity to human FcRn under neutral pH conditions, and hA33-IgG1-F699, which contains an FcγR binding domain whose binding activity to human FcγR is more lower than the binding activity of the naturally occurring FcγR domain. Since no response to hA33-IgG1-F698 was observed in PBMCs derived from G and I donors compared to the negative control, G and I donors are believed to be donors that do not mount an immune response to hA33-IgG1-F698. A high immune response to hA33-IgG1-F698 was observed in PBMCs from the other seven donors (donors A, B, C, D, E, F and H) compared to the negative control, and a high level of immunogenicity was demonstrated in vitro similar to the above hA33-IgG1-F21. On the other hand, an effect was observed in which the immune response of PBMCs obtained from five donors (donors A, B, C, D, and F) to hA33-IgG1-F699, which contains an FcγR-binding domain whose binding activity to human FcγR is lower than the binding activity of the native FcγR-binding domain was reduced compared to the immune response to hA33-IgG1-F698. In particular, the immune response of PBMCs isolated from donors C and F to hA33-IgG1-F699 was confirmed to be approximately the same as that of the negative control. The fact that the effect of reducing immunogenicity was confirmed not only for hA33-IgG1-F21, but also for hA33-IgG1-F698, which has a high binding activity to human FcRn, showed that immunogenicity can be reduced in antigen-binding molecules having binding activity with human FcRn in the neutral pH region, by ensuring that the binding activity to human FcγR is lower than the binding activity at the native FcγR binding domain, and inhibiting the formation of a four-component complex.

(9-4) Preparation of an A33 binding antibody lacking human FcγRIa binding activity under neutral pH conditions

As described in (9-3) above, hA33-IgG1-F699 showed reduced binding activity to various human FcγR types by replacing Lys instead of Ser at position 239 (EU numbering) in hA33-IgG1-F698, and hFcgRI binding was maintained, although binding with hFcgRIIa (R), hFcgRIIa (H), hFcgRIIb and hFcgRIIIa (F) decreased significantly.

Thus, in order to obtain an A33 binding antibody that does not contain an FcγR binding domain lacking binding activity for all human FcγRs, including hFcgRIa, hA33H-IgG1-F763 (SEQ ID NO: 69) was prepared in which Arg replaced Leu at position 235 (EU numbering) and Lys replaced Ser at position 239 (EU numbering) in hA33H-IgG1-F698 (SEQ ID NO: 67).

Association constants (KD) for human FcRn under neutral pH conditions (pH 7.0) were measured for VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F698 and VH3/L(WT)-IgG1 -F763 using the method of Example 4. In addition, human FcγR binding activity was evaluated for VH3/L(WT)-IgG1, VH3/L(WT)-IgG1-F698 and VH3/L(WT)-IgG1-F763 in in accordance with the method described in example 7. These results are also presented in table 16 below.

As shown in Table 16, IgG1-F763, in which Arg replaced Leu at position 235 (EU numbering) and Lys replaced Ser at position 239 (EU numbering), was shown to exhibit reduced binding activity to all human FcγRs, including hFcγRIa.

(9-5) Evaluation of the immunogenicity of various types of A33 binding antibodies by in vitro T cell assay

The immunogenicity of the obtained hA33-IgG1-F698 and hA33-IgG1-F763 was evaluated using the same method as in Example 7. Moreover, similarly as described above, healthy volunteers serving as donors were not the same individuals as well as healthy volunteers from which the PBMCs used in the examples mentioned above were obtained. In other words, donor A in the above examples and donor A in this study were different healthy volunteers.

The results of the study are presented in Fig. 21. In FIG. 21, a comparison is made between the results for hA33-IgG1-F698, which has a potent binding activity to human FcRn under neutral pH conditions, and hA33-IgG1-F763, which contains an FcγR binding domain whose binding activity to human FcγR is more lower than the binding activity of the native FcγR binding domain. Since no response to hA33-IgG1-F698 was observed in PBMCs from donors B, E, F, and K compared to the negative control, it is believed that donors B, E, F, and K are donors that have an immune response to hA33 -IgG1-F698 does not occur. A strong immune response to ha33-IgG1-F698 was observed in PBMCs from the other seven donors (donors A, C, D, G, H, I and J) compared to the negative control. On the other hand, an effect was observed in which the immune response of PBMCs derived from four donors (donors A, C, D, and H) to hA33-IgG1-F763, which contains an FcγR-binding domain whose binding activity to human FcγR is lower than the binding activity of the naturally occurring FcγR domain was reduced compared to the immune response to ha33-IgG1-F698. Among these four donors, the immune response of PBMCs derived from donors C, D, and H, in particular to hA33-IgG1-F763, was approximately the same as in the negative control, and among the four donors, in which the immune response of PBMCs was reduced as a result of reduced FcγR binding, it was in fact possible to completely inhibit the immune response of three-donor PBMCs. Also, based on these data, antigen-binding molecules containing an FcγR binding domain, the binding activity of which to human FcγR is low, are considered to be extremely effective molecules with reduced immunogenicity.

Based on the results of examples 7, 8 and 9, it was confirmed that the immune response to antigen-binding molecules in which the formation of a four-component complex was inhibited by reducing binding to active FcγR (embodiment 1) was inhibited in multiple donors compared to antigen-binding molecules that are able to form four-component complex on antigen-presenting cells. The results described above indicated that the formation of a quaternary complex on antigen-presenting cells is important for the immune response of antigen-binding molecules, and that antigen-binding molecules that do not form this quaternary complex offer the opportunity to reduce immunogenicity in multiple donors.

[Example 10] Evaluation of the in vivo immunogenicity of a humanized antibody which has binding activity to human FcRn in the neutral pH region but no binding activity to mouse FcγR

Using an in vitro experiment, it was demonstrated in Examples 7, 8 and 9 that immunogenicity is reduced in antigen-binding molecules having human FcRn binding activity in the neutral pH region and containing an FcγR binding domain whose FcγR binding activity is lower than the binding activity of the native FcγR binding domain compared to antigen-binding molecules in which the FcγR binding activity is not reduced. The following study was carried out to confirm whether this effect is also shown in vivo.

(10-1) In vivo immunogenicity study in transgenic mice with human FcRn

The production of antibodies to Fv4-IgG1-F11, Fv4-IgG1-F890, Fv4-IgG1-F947, Fv4-IgG1-F821, Fv4-IgG1-F939 and Fv4-IgG1-F1009 was evaluated using mouse plasma obtained according to example 5, in according to the method below.

(10-2) Detection of anti-injection antibody in plasma by electrochemical luminescence

Antibody against the injected antibody sample present in the mouse was quantified by electrochemical luminescence. First, injected antibody was dispensed into an uncoated Uncoated Multi-Array Plate (Meso Scale Discovery) and then allowed to stand overnight at 4° C. to form a solid phase antibody injected plate. Samples for alteration in mouse plasma were prepared by diluting 50-fold, followed by dispensing into a solid phase plate and reacting overnight at 4°C. Then, anti-mouse IgG (whole molecule) (Sigma) ruthenated with Sulfo-Tag NHS Ester (Meso Scale Discovery) was allowed to react for 1 hour at room temperature and T (×4) reading buffer (Meso Scale Discovery) was added. ), and then immediately measured using the Sector PR 400 (Meso Scale Discovery). Measurement in the plasma of five animals that did not receive the antibody was performed as a negative control sample for each measurement system, and the value (X) obtained by adding the result of multiplying the standard deviation (SD) of the values measured using the plasma of these five animals by 1.645, and the mean value (MEAN) of the values measured using five animals was used as a criterion for determining a positive reaction (Equation 3). Animals that showed a reaction exceeding the positive criterion even once on any of the days of blood sampling were considered to have a positive response in the form of antibody production to the test substance.

[Equation 3]

Positive criterion for antibody production (X) = MEAN + 1.645 × SD

(10-3) Inhibitory effect on in vivo immunogenicity by reducing FcγR binding activity

The results are shown in FIG. 22-27. In FIG. 22 shows mouse antibody titers produced in response to Fv4-IgG1-F11 3, 7, 14, 21 and 28 days after administration of Fv4-IgG1-F11 to human FcRn transgenic mice. Mouse anti-Fv4-IgG1-F11 antibody production was shown to be positive in one out of three mice (#3) each day that blood was drawn after administration (positive: 1/3). On the other hand, in FIG. 23 shows mouse antibody titers produced in response to Fv4-IgG1-F821 3, 7, 14, 21, and 28 days after administration of Fv4-IgG1-F821 to human FcRn transgenic mice. Mouse anti-Fv4-IgG1-F821 antibody production was shown to be negative in all three mice each day of blood sampling after administration (positive score: 0/3).

In FIG. 24A and its enlarged view in the form of FIG. 24B shows mouse antibody titers produced in response to Fv4-IgG1-F890 3, 7, 14, 21, and 28 days after administration of Fv4-IgG1-F890 to human FcRn transgenic mice. The production of mouse antibodies to Fv4-IgG1-F890 was shown to be positive in two of three mice (#1 and #3) 21 and 28 days after injection (positive: 2/3). On the other hand, in FIG. 25 shows titers of mouse antibody produced in response to Fv4-IgG1-F939 3, 7, 14, 21 and 28 days after administration of Fv4-IgG1-F939 to human FcRn transgenic mice. The production of mouse anti-Fv4-IgG1-F939 antibody was shown to be negative in all three mice each day of blood sampling after administration (positive score: 0/3).

In FIG. 26 shows mouse antibody titers produced in response to Fv4-IgG1-F947 3, 7, 14, 21, and 28 days after administration of Fv4-IgG1-F947 to human FcRn transgenic mice. Mouse anti-Fv4-IgG1-F947 antibody production was shown to be positive in two out of three mice (#1 and #3) 14 days after injection (positive: 2/3). On the other hand, in FIG. 27 shows mouse antibody titers produced in response to Fv4-IgG1-F1009 3, 7, 14, 21, and 28 days after administration of Fv4-IgG1-F1009 to human FcRn transgenic mice. Mouse anti-Fv4-IgG1-F1009 antibody production was shown to be positive in two of three mice (#4 and #5) starting 7 days after injection (positive score: 2/3).

As shown in Example 5, Fv4-IgG1-821 has reduced binding to various mouse FcγR types relative to Fv4-IgG1-F11, Fv4-IgG1-F939 similarly has reduced binding to various mouse FcγR types relative to Fv4-IgG1-F890, and Fv4- IgG1-F1009 similarly has reduced binding to various mouse FcγR types for Fv4-IgG1-F947.

It has been shown that in vivo immunogenicity can be significantly reduced by reducing the binding of Fv4-IgG1-F11 and Fv4-IgG1-F890 to various mouse FcγR types. On the other hand, the effect of reducing immunogenicity in vivo by reducing the binding of Fv4-IgG1-F947 to various mouse FcγR types has not been demonstrated.

Without being bound by a particular theory, it is believed that the reason for the observation of this inhibitory effect on immunogenicity can be explained as described below.

As described in Example 3, inhibition of the formation of a quaternary complex on the cell membrane of antigen-presenting cells is believed to be possible by reducing the FcγR binding activity of antigen-binding molecules having FcRn binding activity under neutral pH conditions. It is believed that by inhibiting the formation of the quaternary complex, the incorporation of antigen-binding molecules into antigen-presenting cells is inhibited, and it is believed that as a result, the induction of immunogenicity against antigen-binding molecules is suppressed. Thus, Fv4-IgG1-F11 and Fv4-IgG1-F890 are believed to have reduced induction of immunogenicity by reducing FcγR binding activity.

On the other hand, Fv4-IgG1-F947 did not show an immunogenicity-suppressing effect by reducing FcγR binding activity. Without being bound by theory, the reason for this can be commented as follows.

As shown in FIG. 16, elimination of Fv4-IgG1-F947 and Fv4-IgG1-F1009 from plasma is extremely fast. In this regard, it is believed that Fv4-IgG1-F1009 has undergone a decrease in binding activity to mouse FcγR, and it is believed that the formation of a four-component complex on antigen-presenting cells is inhibited. Therefore, Fv4-IgG1-F1009 is believed to be incorporated into cells by binding only to FcRn expressed on the cell membrane of cells such as vascular endothelial cells or hematopoietic cells. In this regard, since FcRn is also expressed on the cell membranes of some antigen-presenting cells, Fv4-IgG1-F1009 can also be incorporated into antigen-presenting cells by binding to FcRn only. In other words, in addition to rapid elimination of Fv4-IgG1-F1009 from plasma, a portion may be incorporated into antigen-presenting cells.

Moreover, Fv4-IgG1-F1009 is a human antibody, and it is a completely foreign protein in mice. In other words, mice are believed to have numerous populations of T cells that specifically respond to Fv4-IgG1-F1009. Only a small amount of Fv4-IgG1-F1009 incorporated into antigen-presenting cells is presented to T cells after cell processing, and since mice have numerous populations of T cells that specifically respond to Fv4-IgG1-F1009, it is believed that the immune response to Fv4 -IgG1-F1009 is easily induced. In fact, when a foreign protein in the form of a soluble human IL-6 receptor is administered to mice as described in Reference Example 4, the soluble human IL-6 receptor is eliminated in a short period of time, and an immune response to the soluble human IL-6 receptor is induced. It is believed that the fact that immunogenicity was induced even though the soluble human IL-6 receptor does not have FcγR and FcRn binding activity in the neutral pH region is due to the rapid elimination of the soluble human IL-6 receptor and the fact that a large amount was incorporated into antigen-presenting cells.

In other words, in the case where the antigen-binding molecule is a foreign protein (such as when a human protein is administered to mice), it is believed that it will be more difficult to suppress the immune response by inhibiting the formation of a four-component complex on antigen-presenting cells compared to the case when the antigen-binding molecule is a homologous protein (eg, as in the case of administering a mouse protein to mice).

In fact, in the case where the antigen-binding molecule is an antibody, since the antibody administered to a human is a humanized antibody or a human antibody, an immune response to the homologous protein occurs. Thus, in Example 11, an assessment was made as to whether inhibition of the formation of a quaternary complex resulted in a decrease in immunogenicity by administration of a mouse antibody to mice.

[Example 11] Evaluation of in vivo immunogenicity of mouse antibodies that have mouse FcRn binding activity under neutral pH conditions but no mouse FcγR binding activity

(11-1) In vivo immunogenicity study in normal mice

The following study was performed to test the inhibitory effect on immunogenicity obtained by inhibiting the formation of a quaternary complex on antigen-presenting cells in the case where the antigen-binding molecule is a homologous protein (as in the case of administering a mouse antibody to mice).

The production of antibodies to mPM1-mIgG1-mF38, mPM1-mIgG1-mF40, mPM1-mIgG1-mF14 and mPM1-mIgG1-mF39 was evaluated using mouse plasma, according to example 6, according to the method below.

(11-2) Plasma anti-injection antibody measurement by electrochemical luminescence

Antibody against the injected antibody sample present in the mouse was measured by electrochemical luminescence. First, the injected antibody was dispensed into a 96-well Multi-Array plate and then allowed to react for 1 hour at room temperature. After washing the tablet, samples were obtained for measurement in mouse plasma diluted 50 times; and after reacting for 2 hours at room temperature and washing the plate, the injected sample was dispensed, ruthenated with Sulfo-Tag NHS Ester (Meso Scale Discovery), and then the reaction was allowed to proceed overnight at 4°C. The next day, the plate was washed and then read buffer T (×4) (Meso Scale Discovery) was dispensed and then measured immediately with a Sector PR 2400 Reader (Meso Scale Discovery). Measurement in the plasma of five animals that did not receive the antibody was performed as a negative control sample for each measurement system, and the value (X) obtained by adding the result of multiplying the standard deviation (SD) of the values measured using the plasma of these five animals by 1.645, and the mean value (MEAN) of the values measured using five animals was used as a criterion for determining a positive reaction (Equation 3). Animals that showed a reaction exceeding the positive criterion even once on any of the days of blood sampling were considered to have a positive response in the form of antibody production to the test substance.

[Equation 3]

Positive criterion for antibody production (X) = MEAN + 1.645 × SD

(11-3) Inhibitory effect on in vivo immunogenicity by reducing FcγR binding activity

The results are shown in FIG. 28-31. In FIG. 28 shows the mouse antibody titers produced in response to mPM1-mIgG1-mF14 at 14, 21 and 28 days after administration of mPM1-mIgG1-mF14 to normal mice. It was shown that the production of mouse antibodies to mPM1-mIgG1-mF14 is positive in all three mice 21 days after injection (positive rate: 3/3). On the other hand, in FIG. 29 shows mouse antibody titers produced in response to mPM1-mIgG1-mF39 14, 21 and 28 days after mPM1-mIgG1-mF39 administration to normal mice. The production of mouse anti-mPM1-mIgG1-mF39 antibody was shown to be negative in all three of the mice on each day of blood sampling after administration (positive score: 0/3).

In FIG. 30 shows mouse antibody titers produced in response to mPM1-mIgG1-mF38 at 14, 21, and 28 days after administration of mPM1-mIgG1-mF38 to normal mice. Mouse anti-mPM1-mIgG1-mF38 antibody production was shown to be positive in two out of three mice (#1 and #2) 28 days after injection (positive: 2/3). On the other hand, in FIG. 31 shows mouse antibody titers produced in response to mPM1-mIgG1-mF40 at 14, 21 and 28 days after administration of mPM1-mIgG1-mF40 to normal mice. Mouse anti-mPM1-mIgG1-mF40 antibody production was shown to be negative in all three mice each day of blood sampling after administration (positive score: 0/3).

As shown in Example 6, relative to mPM1-mIgG1-mF38, mPM1-mIgG1-mF40 has reduced binding to various mouse FcγR types, and similarly to mPM1-mIgG1-mF14, mPM1-mIgG1-mF39 has reduced binding to various mouse FcγR types.

These results confirmed that even when mouse antibodies mPM1-mIgG1-mF38 and mPM1-mIgG1-mF14, which are homologous proteins, were administered to normal mice, antibody production was confirmed in response to the administered antibody and immune response was confirmed. As indicated in examples 1 and 2, this is believed to be due to the acceleration of incorporation into antigen-presenting cells through the formation of a four-component complex on antigen-presenting cells by increasing the activity of binding to FcRn in the region of neutral pH values.

It has been shown that it is possible to reduce in vivo immunogenicity by inhibiting the formation of a quaternary complex by reducing the binding of such antigen-binding molecules having human FcRn binding activity in the neutral pH region to various mouse FcγR types.

These results indicate that by reducing the FcγR binding activity of an antigen binding molecule having FcRn binding activity under neutral pH conditions, the immunogenicity of the antigen binding molecule can be reduced extremely effectively both in vitro and in vivo. In other words, it has been shown that the immunogenicity of an antigen-binding molecule which has FcRn-binding activity under neutral pH conditions and whose binding activity to active FcγR is lower than the binding activity of a native FcγR-binding domain (namely, an antigen-binding molecule according to embodiment 1 described in Example 3) is significantly reduced compared to an antigen-binding molecule having a binding activity approximately comparable to the binding activity of a native FcγR-binding domain (i.e., an antigen-binding molecule capable of forming a quaternary complex as described in Example 3) .

[Example 12] Production and evaluation of human antibodies having human FcRn binding activity in the neutral pH region and whose binding activity to human FcγR is lower than the binding activity of the native FcγR binding domain

(12-1) Production and evaluation of human IgG1 antibodies having human FcRn binding activity in the neutral pH region and whose binding activity to human FcγR is lower than the binding activity of the native FcγR binding domain

In a non-limiting aspect of the present invention, although preferred examples of an Fc region whose binding activity to an active FcγR is lower than the binding activity to an active FcγR of a naturally occurring Fc region include Fc regions in which one or more amino acids in any of positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325 and 329 (EU numbering) among the amino acids of the above Fc region are modified to an amino acid that is different from the naturally occurring Fc region, modification of the Fc- area is not limited to the modification described above, but instead may be, for example, deglycosylation (N297A, N297Q) described in Current Opinion in Biotechnology (2009) 20(6), 685-691, modifications such as IgG1-L234A/ L235A, IgG1-A325A/A330S/P331S, IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S, IgG1-S267E/L328F, I gG2-V234A/G237A, IgG2- H268Q/V309L/A330S/A331S, IgG4-L235A/G237A/E318A or IgG4-L236E, as well as modifications such as G236R/L328R, L235G/G236R, N325A/L328R or N325LL328R described in WO 2008 /092117, insertion of amino acids into provisions 233, 234, 235 and 237 (EU numbering), and modifications of the areas described in WO 2000/042072.

Fv4-IgG1-F890 and Fv4-IgG1-F947 prepared according to Example 5 are antibodies that have binding activity to human FcRn under neutral pH conditions and bind to the human IL-6 receptor in a pH dependent manner. Various variants were generated in which binding to human FcγRs was reduced by amino acid substitutions in their amino acid sequences (Table 17). More specifically, variants were generated including VH3-IgG1-F938 (SEQ ID NO: 156) in which Lys replaced Leu at position 235 (EU numbering) and Lys replaced Ser at position 239 of the amino acid sequence of VH3-IgG1-F890, VH3- IgG1-F1315 (SEQ ID NO: 157), in which Lys replaced Gly at position 237 (EU numbering) and Lys replaced Ser at position 239 of the amino acid sequence of VH3-IgG1-F890, VH3-IgG1-F1316 (SEQ ID NO: 158) , in which Arg replaced Gly at position 237 (EU numbering) and Lys replaced Ser at position 239 in the amino acid sequence VH3-IgG1-F890, VH3-IgG1-F1317 (SEQ ID NO: 159), in which Lys replaced Ser at position 239 (EU numbering) and Lys replaced Pro at position 329 in the amino acid sequence VH3-IgG1-F890, VH3-IgG1-F1318 (SEQ ID NO: 160), in which Lys replaced Ser at position 239 (EU numbering) and Arg replaced Pro at position 329 of the amino acid sequence VH3-IgG1-F890, VH3-IgG1-F1324 (SEQ ID NO: 161), in which Ala replaced Leu at position 234 (EU numbering) and Ala replaced Leu at position 235 of the amino acid sequence VH3-IgG1-F890, VH3-IgG1-F1325 (SEQ ID NO: 162), in which Ala replaced Leu at position 234 (EU numbering), Ala replaced Leu at position 235, and Ala replaced Asn at position 297 of the amino acid sequence of VH3-IgG1-F890, VH3-IgG1 -F1333 (SEQ ID NO: 163), in which Arg replaced Leu at position 235 (EU numbering), Arg replaced Gly at position 236, and Lys replaced Ser at position 239 of the amino acid sequence VH3-IgG1-F890, VH3-IgG1-F1356 ( SEQ ID NO: 164), in which Arg replaced Gly at position 236 (EU numbering) and Arg replaced Leu at position 328 of the amino acid sequence VH3-IgG1-F890, VH3-IgG1-F1326 (SEQ ID NO: 155), in which Ala replaced Leu at position 234 (EU numbering) and Ala replaced Leu at position 235 of the amino acid sequence VH3-IgG1-F947, and VH3-IgG1-F1327 (SEQ ID NO: 165) where Ala replaced Leu at position 234 (EU numbering) , Ala replaced Leu at position 235, and Ala replaced Asn at position 297 of the VH3-IgG1-F947 amino acid sequence.

(12-2) Confirmation of binding activity to human FcRn and human FcγR

The binding activity (dissociation constant KD) to human FcRn at pH 7.0 of antibodies containing each of the amino acid sequences obtained according to (12-1) as heavy chains and containing L(WT)-CK as light chain was measured with using the method of Example 4. In addition, human FcγR binding activity was measured at pH 7.4 using the method of Example 7. The measurement results are shown in Table 18 below.

According to the results presented in Table 18, there are no specific restrictions on the amino acid modifications made to reduce the binding activity to different types of human FcγR compared to the binding activity of the native FcγR binding domain, and this can be done using various amino acid modifications.

(12-3) Obtaining an antibody that binds to glypican-3

Conducted a detailed analysis of the binding to each FcγR variants of amino acid residues, putative binding sites for FcγR in the Fc region of IgG1, to identify modifications in which binding to FcgR is reduced compared to naturally occurring IgG1. The variable region of an anti-glypican 3 antibody having improved plasma dynamics described in WO 2009/041062, in the form of an anti-glypican-3 antibody containing a GpH7 CDR (SEQ ID NO: 74), was used as the H chain of the antibody. Similarly, anti-glypican-3 antibody GpL16-k0, which has improved plasma dynamics as described in WO 2009/041062 (SEQ ID NO: 75), has been commonly used as the L chain of the antibody. In addition, B3, obtained by introducing the K439E mutation in G1d, in which Gly and Lys are removed from the C-terminus of IgG1 (SEQ ID NO: 76), was used as the constant region of the H chain of the antibody. This H chain was subsequently designated as GpH7-B3 (SEQ ID NO: 77), while the L chain was subsequently designated as GpL16-k0 (SEQ ID NO: 75).

(12-4) Kinetic analysis of binding to different types of FcγR

First, to confirm the robustness of the detailed assay using GpH7-B3/GpL16-k0 as control, binding capacity was compared with each FcgR between GpH7-B3/GpL16-k0 and GpH7-G1d/GpL16-k0 (Table 19). Binding to each human FcγR (FcγRIa, FcγRIIa (H), FcγRIIa (R), FcγRIIb and FcγRIIa (F)) of both antibodies after expression and purification according to the method of Reference Example 2 was evaluated according to the method below.

The interaction between each modified antibody and the Fcγ receptor prepared as described above was analyzed using Biacore T100 (GE Healthcare), Biacore T200 (GE Healthcare), Biacore A100 or Biacore 4000. HBS-EP + (GE Healthcare) was used in as running buffer and measurements were carried out at 25°C. The chips used were those on which antigenic peptides, protein A (Thermo Scientific), protein A/G (Thermo Scientific), or protein L (ACTIGEN or BioVision) were immobilized on a Series S Sensor Chip CM5 (GE Healthcare) or Series S Sensor Chip CM4 (GE Healthcare) by the amine attachment method, or chips on which pre-biotinylated antigenic peptides were allowed to interact and then immobilized on a Series S Sensor Chip SA (certified) (GE Healthcare). Target antibodies were immobilized on these sensor chips, the Fcγ receptor diluted with running buffer was allowed to interact, and then the amount of bound antibody was measured. The amounts of bound antibody were compared between antibodies. However, since the amount of Fcγ receptor bound depends on the amount of antibody captured, the values used for comparison were first adjusted by dividing the amount of Fcγ receptor bound by the amount of each antibody captured. By reaction with 10 mM glycine-HCl at pH 1.5, the sensor chips were regenerated by washing away the antibody captured on the sensor chips and reused.

The binding strength was analyzed according to the method based on the results of the interaction analysis with each FcγR. The value obtained by dividing the amount of GpH7-B3/GpL16-k0 bound to FcγR by the amount of GpH7-G1d/GpL16-k0 bound to FcγR and multiplying this value by 100 was used as an indicator of the relative binding activity with each FcγR. Based on the results presented in Table 19, since the binding of GpH7-B3/GpL16-k0 to each FcgR was approximately equal to the binding of GpH7-G1d/GpL16-k0, it was decided that GpH7-B3/GpL16-k0 could be used as a control. in subsequent studies.

(12-5) Preparation and evaluation of Fc mutants

Further amino acids putatively involved in FcγR binding and their surrounding amino acids in the GpH7-B3 amino acid sequence (position 234 to position 239, position 265 to position 271, position 295, position 296, position 298, position 300, and position 324 to position 327 (EU numbering)), respectively, were replaced by 18 types of amino acids, excluding the original amino acids and Cys. These Fc mutants are referred to as B3 variants. The binding of B3 variants expressed and purified by the method of Reference Example 2 to each of the FcγRs (FcγRIa, FcγRIIa (H), FcγRIIa (R), FcγRIIb and FcγRIIIa (F)) was evaluated in detail according to method (12-4).

Binding strength was evaluated according to the following method based on the results of the interaction analysis with each FcγR. The amount of antibody derived from each FcγR-bound B3 variant was divided by the amount of the reference antibody in which B3 was not mutated (antibody having the sequence of naturally occurring human IgG1 at positions 234 to position 239, from position 265 to position 271, position 295, position 296, position 298, position 300, and from position 324 to position 337, according to the EU numbering) associated with FcγR. This value was then further multiplied by 100 and the resulting value was used as an indication of the relative binding activity with each FcγR.

Modifications that reduced binding to all FcgRs among the variants analyzed are shown in Table 21. The 236 types of modifications shown in Table 20 are modifications that reduced binding to at least one FcgR type compared to the antibody before the modification (GpH7- B3/GpL16-k0) and are believed to be modifications similarly having the effect of binding to at least one type of FcgR even when introduced into naturally occurring IgG1.

Therefore, there are no specific limitations on the amino acid modifications made to reduce the binding activity to each type of human FcγR compared to the binding activity of the native FcγR binding domain, and it has been shown that this can be achieved by making the amino acid modifications shown in Table 20, by at least one area. In addition, the amino acid modifications made in this case may be at a single position or a combination of several positions.

(12-6) Production and evaluation of a human IgG2 antibody and a human IgG4 antibody that have a human FcRn binding activity in the neutral pH region and whose binding activity to a human FcγR is lower than the binding activity of the native FcγR binding domain

An Fc region that has binding activity to human FcRn in the neutral pH region and whose binding activity to human FcγR is lower than the binding activity of the native FcγR binding domain was prepared in the same manner as described below using human IgG2 or human IgG4.

An antibody containing VH3-IgG2 (SEQ ID NO: 166) as a heavy chain and L(WT)-CK (SEQ ID NO: 41) as a light chain was obtained according to the method of Reference Example 2, for use in as an antibody that binds the human IL-6 receptor having IgG2 as a constant region. Similarly, an antibody containing VH3-IgG4 (SEQ ID NO: 167) as a heavy chain and L(WT)-CK (SEQ ID NO: 41) as a light chain was prepared according to the method of Reference Example 2, for use as a human IL-6 receptor-binding antibody having human IgG4 as a constant region.

Amino acid substitutions were made in each constant region of VH3-IgG2 and VH3-IgG4 to report human FcRn binding activity under neutral pH conditions. More specifically, VH3-IgG2-F890 (SEQ ID NO: 168) and VH3-IgG4-F890 (SEQ ID NO: 169) were generated by amino acid substitution of VH3-IgG2 and VH3-IgG4 in which Tyr replaced Met at position 252 (numbering EU), Tyr replaced Asn at position 434 and Val replaced Tyr at position 436.

Amino acid substitutions were made in each constant region of VH3-IgG2-F890 and VH3-IgG4-F890 to reduce binding to human FcγR. More specifically, VH3-IgG2-F939 (SEQ ID NO: 170) was generated by amino acid substitution VH3-IgG2-F890 in which Arg replaced Ala at position 235 (EU numbering) and Lys replaced Ser at position 239. In addition, VH3- IgG4-F939 (SEQ ID NO: 171) was generated by the VHE-IgG4-F890 amino acid substitution in which Arg replaced Leu at position 235 (EU numbering) and Lys replaced Ser at position 239.

An antibody containing obtained VH3-IgG2-F890, VH3-IgG4-F890, VH3-IgG2-F939 or VH3-IgG4-F939 as a heavy chain and L(WT)-CK (SEQ ID NO: 41) as a light chain, was obtained in accordance with the method presented in Reference Example 2.

(12-7) Evaluation of a human IgG2 antibody and a human IgG4 antibody that have a human FcRn binding activity in the neutral pH region and whose binding activity to a human FcγR is lower than the binding activity of the native FcγR binding domain

The binding activity (dissociation constant KD) of the human FcRn at pH 7.0 of the antibody (Table 21) obtained according to (12-6) was measured using the method of Example 4. In addition, the binding activity of the human FcγR at pH 7.4 was measured using the method of Example 7. The measurement results are shown in Table 22 below.

The results shown in Table 22 show that it is possible to obtain an Fc region which has a human FcRn binding activity in the neutral pH region and whose binding activity to a human FcγR is lower than the binding activity of the native FcγR binding domain using Human IgG1 is not particularly limited, and human IgG2 or IgG4 can also be used.

[Example 13] Preparation and evaluation of an antigen-binding molecule in which only one of the two polypeptides that constitute the FcRn-binding domain has FcRn-binding activity under neutral pH conditions

An antigen-binding molecule in which only one of the two polypeptides that make up the FcRn binding domain has FcRn binding activity under neutral pH conditions, while the other has no FcRn binding activity under neutral pH conditions, as shown for option implementation 3 in example 3, received as indicated below.

(13-1) Obtaining an antigen-binding molecule in which only one of the two polypeptides that make up the FcRn-binding domain has FcRn-binding activity under the conditions of the neutral pH range, while the other has no FcRn-binding activity under the conditions of the neutral pH values

First, VH3-IgG1-F947 (SEQ ID NO: 70) was prepared according to the method of Reference Example 1 as a heavy chain of an anti-human IL-6R antibody having FcRn binding activity under neutral pH conditions. In addition, VH3-IgG1-F46 (SEQ ID NO: 71) was prepared by introducing an amino acid substitution obtained by substituting Ala for Ile at position 253 (EU numbering) of VH3-IgG1 for use as an antigen-binding molecule that does not have binding activity for FcRn both in the region of acidic pH values and in the region of neutral pH values.

The use of Fc regions in which one Fc region of the antibody contains substitutions in which Lys replaces Asp at position 356 (EU numbering) and Lys replaces Glu at position 357 (EU numbering) and the other Fc region contains substitutions in which Glu replaces Lys at position 370 (EU numbering), Arg replaces His at position 435 (EU numbering), and Glu replaces Lys at position 439 (EU numbering) is known as a method for producing a high purity antibody heterodimer (WO 2006/106905).

Received VH3-IgG1-FA6a (SEQ ID NO: 72), in which Lys replaces Asp at position 356 (EU numbering) and Lys replaces Glu at position 357 (EU numbering) VH3-IgG1-F947 (hereinafter referred to as heavy chain A) . In addition, VH3-IgG1-FB4a (SEQ ID NO: 73) was prepared in which Glu replaces Lys at position 370 (EU numbering), Arg replaces His at position 435 (EU numbering), and Glu replaces Lys at position 439 (EU numbering). ) VH3-IgG1-F46 (hereinafter referred to as heavy chain B) (Table 23).

Fv4-IgG1-FA6a/FB4a was prepared according to the method of Reference Example 2, which has VH3-IgG1-FA6a and VH3-IgG1-FB4a as heavy chains and VL3-CK as light chain by adding equal amounts of heavy chain plasmids in the form of VH3-IgG1-FA6a and VH3-IgG1-FB4a.

(13-2) PK study of an antigen-binding molecule in which only one of the two polypeptides that constitute the FcRn-binding domain has FcRn-binding activity under neutral pH conditions, while the other has no FcRn-binding activity under conditions neutral pH ranges

A PK assay was performed according to the method described below when Fv4-IgG1-F947 and Fv4-IgG1-FA6a/FB4a were administered to human FcRn transgenic mice.

Anti-human IL-6 receptor antibody was administered at 1 mg/kg as a single injection under the dorsal skin of transgenic mice with human FcRn (B6.mFcRn-/-.hFcRn Tg mouse line 32+/+, Jackson Laboratories, Methods Mol. Biol. (2010)602, 93-104). Blood sampling was carried out 15 minutes, 7 hours and 1, 2, 3, 4 and 7 days after administration of antibodies against the human IL-6 receptor. Plasma was obtained by centrifuging immediately collected blood for 15 minutes at 4°C and 15,000 rpm. The separated plasma was stored in a freezer set at -20° C. or lower until the time of measurement.

The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA in the same manner as in the method of Example 4. The results are shown in FIG. 32. Fv4-IgG1-FA6a/FB4a, which is able to bind to only one human FcRn molecule through one binding region, has been shown to show a shift towards higher plasma concentrations compared to Fv4-IgG1-F947, which is able to bind to two human FcRn molecules through two binding regions.

As described previously, although there are two FcRn-binding regions in the Fc region of IgG, it has been described that molecules having an Fc from which one of the two FcRn-binding regions has been removed are eliminated from plasma more rapidly compared to molecules having an Fc-occurring nature Fc region (Scand. J. Immunol., 1994; 40:457-465). In other words, IgG having two binding regions that bind to FcRn under acidic pH conditions is known to exhibit increased plasma retention compared to IgG having a single FcRn binding region. It has been shown that IgG incorporated into cells is recycled back into plasma by binding to FcRn in endosomes and, since naturally occurring IgG is able to bind to two FcRn molecules via two FcRn binding regions, it binds to FcRn with high binding capacity, and, thus, it is believed that a large amount of IgG is recycled. On the other hand, IgG having only one FcRn-binding region has low binding capacity for FcRn in endosomes, and because it cannot be sufficiently recycled, it is believed to be eliminated from plasma faster.

Therefore, in Fv4-IgG1-FA6a/FB4a having only one FcRn binding site under neutral pH conditions as shown in FIG. 32, the phenomenon whereby an increase in plasma retention is observed was completely unexpected as it is the opposite of what occurs in the case of naturally occurring IgG.

Although the present invention is not bound by a particular theory, it is believed that a possible reason for the observed change in these high plasma retention levels is an increase in the rate of subcutaneous absorption when the antibody is administered subcutaneously to mice.

It is generally believed that an antibody that has been injected subcutaneously migrates into the plasma after being absorbed through the lymphatic system (J. Pharm. Sci. (2000)89(3), 297-310). Since there are a large number of immune cells in the lymphatic system, it is believed that the antibody, which was injected subcutaneously, migrates into the plasma after being exposed to a large number of immune cells. For the most part, immunogenicity is known to be enhanced when antibody-based pharmaceuticals are administered subcutaneously as compared to intravenous administration, and one possible reason for this is that subcutaneously administered antibodies are exposed to a large number of immune cells in the lymphatic system. In fact, as shown in Example 1, Fv4-IgG1-F1 was confirmed to be rapidly eliminated from plasma when administered subcutaneously, indicating the production of anti-Fv4-IgG1-F1 antibody. On the other hand, in the case of intravenous administration, rapid elimination of Fv4-IgG1-F1 from plasma was not confirmed, indicating that no mouse anti-Fv4-IgG1-F1 antibody was produced.

Therefore, during absorption of a subcutaneously administered antibody, when the antibody is incorporated into immune cells present in the lymphatic system, it causes a decrease in bioavailability and at the same time becomes a cause of immunogenicity.

However, in the case of subcutaneous administration of the antigen-binding molecule shown as an example in Embodiment 3 of Example 3, in which only one of the two polypeptides that constitute the FcRn-binding domain has FcRn-binding activity under the conditions of the neutral pH region, while the other polypeptide does not have FcRn binding activity under the conditions of the neutral pH region, even when exposed to immune cells present in the lymphatic system during absorption, it is believed that the four-component complex is not formed on the cell membrane of immune cells. Therefore, the increase in bioavailability is due to the inhibition of incorporation into immune cells present in the lymphatic system, and as a result, it is considered possible that an increase in plasma concentration may occur.

Methods for providing an increase in plasma concentration or a decrease in immunogenicity by increasing the bioavailability of a subcutaneously administered antibody are not limited to the antigen-binding molecules presented as Embodiment 3 of Example 3. Instead, any antigen-binding molecule can be used, provided that it is an antigen-binding molecule that does not form four-component complex on the cell membrane of immune cells. Therefore, when administered subcutaneously, it is believed that any of the antigen-binding molecules of embodiments 1, 2 and 3 are able to increase plasma retention and at the same time increase bioavailability, and further cause a decrease in immunogenicity compared to antigen-binding molecules capable of forming a four-component complex.

It is believed that some of the antigen-binding molecules that remain in the plasma always migrate to the lymphatic system. In addition, immune cells are also present in the blood. Therefore, although the adaptation of the present invention is in no way limited to a specific route of administration, it is believed that an example that will demonstrate the effects particularly easily is the route of administration that is mediated by the lymphatic system during absorption of the antigen-binding molecule, and one of these examples is subcutaneous administration.

[Example 14] Preparation of an antibody having human FcRn binding activity under neutral pH conditions and selective binding activity for inhibitory FcγR

In addition, the antigen-binding molecule according to Embodiment 2 shown in Example 3 can be prepared using a modification that results in increased selective binding activity with inhibitory FcγRIIb for an antigen-binding molecule having increased binding to FcRn under neutral conditions. In other words, an antigen-binding molecule that has FcRn binding activity under neutral conditions and that has been modified to increase selective binding activity for inhibitory FcγRIIb is capable of forming a four-component complex mediated by two FcRn molecules and one FcγR molecule. However, since the selective binding to the inhibitory FcγR is due to the effect of this modification, the binding activity to the active FcγR is reduced. It is believed that as a result, a four-component complex containing an inhibitory FcγR is preferably formed on antigen-presenting cells. As described previously, it is believed that immunogenicity is caused by the formation of a four-component complex containing an active FcγR, and thus, as a result of the formation of a four-component complex containing an inhibitory FcγR, the immune response can be inhibited.

Thus, the following study was carried out to discover amino acid mutations that lead to increased selective binding activity with inhibitory FcγRIIb.

(14-1) Detailed analysis of Fc variant in relation to FcγR binding

Conducted a detailed analysis of the activity of binding to each FcγR of many variants of IgG1 antibodies, which introduced a mutation that reduces Fc-mediated binding to active FcγR, in particular, with any of the FcγRIIa H-type and R-type polymorphisms, compared with naturally occurring IgG1, and enhances binding to FcγRIIb.

The variable region of an anti-glypican-3 antibody with improved plasma dynamics described in WO 2009/041062, in the form of an anti-glypican-3 antibody containing a CDR from GpH7 (SEQ ID NO: 74), was used as the H chain of the antibody. Similarly, GpL16-k0 of the anti-glypican-3 antibody with enhanced plasma dynamics described in WO 2009/041062 (SEQ ID NO: 75) has been commonly used as the antibody L chain in combination with another H chain. In addition, B3, obtained by introducing the K439E mutation in G1d, in which Gly and Lys are removed from the C-terminus of IgG1 (SEQ ID NO: 76), was used as the constant region of the H chain of the antibody. This H chain was subsequently designated as GpH7-B3 (SEQ ID NO: 77), while the L chain was subsequently designated as GpL16-k0 (SEQ ID NO: 75).

Amino acids putatively involved in FcγR binding and their surrounding amino acids in the GpH7-B3 amino acid sequence (position 234 to position 239, position 265 to position 271, position 295, position 296, position 298, position 300, and position 324 to position 327 (EU numbering)), respectively, were replaced by 18 types of amino acids, excluding the original amino acids and Cys. These Fc mutants are referred to as B3 variants. The binding of B3 variants expressed and purified by the method of Reference Example 2 to each of the FcγRs (FcγRIa, FcγRIIa(H), FcγRIIa(R), FcγRIIb and FcγRIIIa) was evaluated in detail according to the method described in Example 9.

For each FcγR built charts in accordance with the method described below. In other words, the amount of antibody obtained for the B3 variant bound to each FcγR was divided by the amount of control antibody that does not have mutations introduced in B3 (the antibody having the sequence of naturally occurring human IgG1 at position 234 to position 239 , from position 265 to position 271, at position 295, at position 296, at position 298, at position 300 and from position 324 to position 337 (EU numbering)). Then, this value was further multiplied by 100, and the resulting value was expressed as the amount of binding to each FcγR. The binding of each variant to FcγRIIb was displayed on the horizontal axis, and the values of each active FcγR in the form of FcγRIa, FcγRIIa(H), FcγRIIa(R) and FcγRIIIa, respectively, were displayed on the vertical axis (Fig. 33, 34, 35 and 36).

As a result, as indicated by the symbols in FIG. 33-36, among all the modifications, mutation A (modification obtained by substituting Asp for Pro at position 238 (EU numbering)) and mutation B (modification obtained by substituting Glu for Leu at position 328 (EU numbering)) showed significant increased binding with FcγRIIb compared to naturally occurring IgG1, and were found to exhibit a significant inhibition of binding to both types of FcγRIIa.

(14-2) SPR analysis of FcγRIIb selective binding variants

Conducted a more detailed analysis of the binding to each FcγR variant obtained by replacing Asp instead of Pro at position 238 (EU numbering) identified in (14-1).

For the H chain of IgG1, the variable region of IL6R-H described in WO 2009/125825 (SEQ ID NO: 78), which is the variable region of an anti-human interleukin-6 receptor antibody, is used as the variable region of the H chain of the antibody, and IL6R-G1d (SEQ ID NO: 79) containing the constant region of Gld, from which Gly and Lys are removed from the C-terminus of human IgG1, is used as the constant region of the H chain of the antibody. Received IL6R-G1d_v1 (SEQ ID NO: 80), in which Asp replaced Pro at position 238 (EU numbering) IL6R-G1d. Next, IL6R-G1d_v2 (SEQ ID NO: 81) was obtained in which Glu replaced Leu at position 328 (EU numbering) of IL6R-G1d. For comparison, IL6R-G1d_v3 (SEQ ID NO: 82) was prepared, which is a variant of IL6R-G1d that has been introduced with a known mutation (Mol. Immunol. (2008)45, 3926-3933) by substituting Glu for Ser at position 267 (EU numbering) and replacing Phe with Leu at position 328 (EU numbering). IL6R-L (SEQ ID NO: 83), which is the L chain of tocilizumab, has been commonly used as an antibody L chain in combination with these heavy chains. The antibodies were expressed and purified according to the method of Reference Example 2. Antibodies containing IL6R-G1d, IL6R-G1d_v1, IL6R-Gld_v2, and IL6R-G1d_v3 antibodies as the H chain were hereinafter respectively designated as IgG1, IgG1-v1, IgG1- v2 and IgG1-v3.

Next, the kinetics of interactions between these antibodies and FcγR was analyzed using Biacore T100 (GE Healthcare). Interaction measurements were performed at 25° C. using HBS-EP+ (GE Healthcare) as running buffer. A Series S Sensor Chip CM5 (GE Healthcare) was used after protein A was immobilized by amine addition. The binding of each FcγR to the antibody was measured by allowing each FcγR diluted with running buffer to act on a chip that captured the target antibody. The antibody captured on the chip was washed off, allowing 10 mM glycine-HCl (pH 1.5) to react after measurement. The chip thus regenerated was reused. The dissociation constant KD (mol/l) was calculated from the association rate constant ka (l/mol/s) and the dissociation rate constant kd (1/s) calculated by fitting the measurement results globally to a 1:1 Langmuir binding model using Biacore software evaluation software.

Because the binding of IgG1-v1 and IgG1-v2 to FcγRIIa(H) or FcγRIIIa was extremely weak, KD could not be calculated by global approximation of the measurement results to the above 1:1 Langmuir binding model using the Biacore Evaluation Software. For IgG1-v1 and IgG1-v2 interaction with FcγRIIa(H) or FcγRIIIa, KD could be calculated using the following 1:1 binding model described in Biacore T100 Software Handbook BR1006-48, Edition AE.

The behavior of the interacting molecules in the Biacore system using the 1:1 binding model can be mapped using Equation 4 below.

[Equation 4]

Req=C×Rmax/(KD+C)+RI

The meaning of each parameter in Equation 4 above is as follows:

Req (RU): Steady State Binding Level

C (M): Concentration of the analyzed compound

C: Concentration

Rmax (RU): Surface binding capacity of the analyte

RI (RU): Cumulative contribution of the refractive index in the sample

KD(M): Equilibrium dissociation constant

KD can be expressed according to Equation 5 below by transforming Equation 4.

[Equation 5]

KD=C × Rmax/(Req-RI) - C

KD can be calculated by substituting Rmax, RI and C into this equation. Under the measurement conditions used in this example, the values plugged into this equation were RI = 0 and C = 2 µcol/L. Rmax was the value obtained by dividing the Rmax value obtained from the global approximation of the results of the analysis of the interaction of IgG1 with each FcγR using the Langmuir 1:1 binding model by the amount of IgG1 captured and multiplied by the amounts of IgG1-v1 and IgG1-v2 captured.

Under the measurement conditions used in this example, the binding of IgG1-v1 and IgG1-v2 to FcγRIIa(H) was approximately 2.5 IU and 10 IU, respectively, and the binding of IgG1-v1 and IgG1-v2 to FcγRIIIa was approximately 2.5 OE and 5 OE, respectively. The amounts of captured IgG1-v1 and IgG1-v2 antibodies on the sensor chip during the analysis of the interaction of IgG1 with FcγRIIa(H) were 469.2 FU and 444.2 FU, and the amounts of captured IgG1-v1 and IgG1-v2 antibodies in the sensor analysis during analysis of the interaction of IgG1 with FcγRIIIa were 470.8 OU and 447.1 OU. In addition, the Rmax values obtained by globally fitting the results of the analysis of the interaction of IgG1 with FcγRIIa(H) and FcγRIIIa using the Langmuir 1:1 binding model were 69.8 FU and 63.8 FU, respectively, and the amount of antibody captured on the sensor chip , were 452 OU and 454.5 OU, respectively. The Rmax values calculated using these values for IgG1-v1 and IgG1-v2 with FcγRIIa(H) were 72.5 U and 68.6 U, respectively, while the calculated Rmax values for IgG1-v1 and IgG1-v2 with FcγRIIIa were 66.0 OU and 62.7 OU, respectively. KD values for IgG1-v1 and IgG1-v2 with FcγRIIa(H) and FcγRIIIa were calculated by substituting these values into Equation 5.

[Equation 5]

KD=C × Rmax/(Req-RI) - C

The KD values for IgG1, IgG1-v1, IgG1-v2 and IgG1-v3 for each FcγR (KD values for each antibody against each FcγR) are shown in Table 24, while the relative KD values for IgG1-v1, IgG1-v2 and IgG1-v3 obtained by dividing IgG1 KD values for each FcγR by IgG1-v1, IgG1-v2 and IgG1-v3 KD values for each FcγR (relative KD values for each antibody against each FcγR) are shown in Table 25.

In Table 24 above, asterisks indicate KD values that were calculated using Equation 5 when FcγR binding to IgG was not sufficiently observed.

[Equation 5]

KD=C × Rmax/(Req-RI) - C

As shown in Table 25, the affinity of IgG1-v1 for FcγRIa decreased to 0.047-fold compared to IgG1, the affinity for FcγRIIa(R) decreased to 0.10-fold, the affinity for FcγRIIa(H) decreased to 0.014-fold, and the affinity for in relation to FcγRIIIa decreased to 0.061 times. On the other hand, the affinity for FcγRIIb increased 4.8-fold.

In addition, as also shown in Table 25, the affinity of IgG1-v2 for FcγRIa decreased to 0.74 times compared to IgG1, the affinity for FcγRIIa(R) decreased to 0.41 times, the affinity for FcγRIIa(H) decreased to 0.064 times, and the affinity for FcγRIIIa decreased to 0.14 times. On the other hand, the affinity for FcγRIIb increased 2.3-fold.

In other words, based on these results, IgG1-v1, in which Asp replaced Pro at position 238 (EU numbering), and IgG1-v2, in which Glu replaced Leu at position 328 (EU numbering), showed reduced binding to all active forms. FcγR, including both FcγRIIa polymorphisms; and binding to FcγRIIb, which is an inhibitory FcγR, clearly increased. To date, no changes having such properties have been described and they are very rare, as shown in FIG. 33-36. Changes obtained by replacing Pro at position 238 (EU numbering) with Asp or replacing Leu at position 328 (EU numbering) with Glu are highly useful for developing drugs for immunological inflammatory diseases and the like.

Moreover, as shown in Table 25, IgG1-v3 definitely shows a 408-fold increased binding to FcγRIIb, while binding to FcγRIIa (H) is reduced to 0.51-fold, and binding to FcγRIIa (R) is increased by 522-fold. . Thus, since IgG1-v1 and IgG1-v2 suppress their binding to both FcγRIIa (R) and FcγRIIa (H), and increase their binding to FcγRIIb, they are considered variants that bind with greater selectivity for FcγRIIb compared to IgG1-v3. In particular, changes obtained by replacing Pro at position 238 (EU numbering) with Asp or replacing Leu at position 328 (EU numbering) with Glu are highly suitable for developing drugs for immunological inflammatory diseases and the like.

(14-3) Effects of combining FcγRIIb selective binding modification and other amino acid substitutions in the Fc region

In (14-2), it was found that the variant obtained by substituting Asp for Pro at position 238 (EU numbering) in the amino acid sequence of naturally occurring human IgG1, or the variant obtained by substituting Glu for Leu at position 328 (EU numbering) , shows reduced Fc-mediated binding to FcγRIa, FcγRIIIa and any of the FcγRIIa polymorphisms, as well as increased binding to FcγRIIb. Thus, Fc variants were designed to have further reduced binding to any of FcγRI, FcγRIIa(H), FcγRIIa(R), and FcγRIIIa, and further increased binding to FcγRIIb by making additional amino acid substitutions in the variant obtained by substituting Asp instead of Pro at position 238 (EU numbering), or a variant obtained by substituting Glu for Leu at position 328 (EU numbering).

(14-4) Production of antibodies having human FcRn binding activity under neutral pH conditions and whose binding activity to human FcγRIIb is selectively enhanced

Received antibodies in accordance with the method below, to selectively enhance the binding activity of VH3-IgG1 and VH3-IgG1-F11 with human FcγRIIb. VH3-IgG1-F648 (SEQ ID NO: 84) was obtained by introducing an amino acid substitution obtained by substituting Asp for Pro at position 238 (EU numbering) in VH3-IgG1 according to the method of Reference Example 1. Similarly, VH3-IgG1-F652 (SEQ ID NO: 85) was obtained by introducing an amino acid substitution obtained by substituting Asp for Pro at position 238 (EU numbering) in VH3-IgG1-F11 according to the method of Reference Example 1.

(14-5) Evaluation of antibodies having human FcRn binding activity under neutral pH conditions and whose binding activity to human FcγRIIb is selectively enhanced

Antibodies containing VH3-IgG1, VH3-IgG1-F648, VH3-IgG1-F11 or VH3-IgG1-F652 as heavy chain and L(WT)-CK as light chain were prepared according to the method of Reference Example 2.

The interaction of these antibodies with FcγRIIa(R) and FcγRIIb was analyzed using Biacore T100 (GE Healthcare). The measurement was carried out at 25°C using a buffer consisting of 20 mm ACES, 150 mm NaCl and 0.05% Tween 20 (pH 7.4) as running buffer. A Series S Sensor Chip CM4 (GE Healthcare) was used after protein L was immobilized by amine addition. The interaction of each FcγR with the antibody was measured by allowing each FcγR diluted with running buffer to act on the chip on which the target antibody was captured. The antibody captured on the chip was washed by reaction with 10 mM glycine-HCl (pH 1.5) after measurement, and the chip thus regenerated was reused.

The measurement results were analyzed using the Biacore Evaluation Software. The antibody was captured with protein L, and the amount of change in the sensogram before and after capture of the antibody was determined as X1. Further, the human FcγR was allowed to interact with the antibody, and the value obtained by subtracting the binding activity of the human FcγR corresponding to the amount of change in the sensogram before and after allowing the running buffer to interact with the antibody captured by protein L (ΔA2) from the value obtained by multiplying by 1500 the value obtained by dividing the human FcγR binding activity corresponding to the amount of change in the sensorogram before and after this interaction (ΔA1) by the amount (X) of each antibody captured, divided by the amount of each antibody captured (X), and then multiplied by 1500 s obtaining the binding activity of human FcγR (Y) (Equation 1).

[Equation 1]

Mouse FcγR binding activity (Y)=(ΔA1-ΔA2)/X×1500

The results are presented in table 26 below. It was confirmed that the effect of selectively enhancing binding activity to human FcγRIIb by introducing a mutation obtained by replacing Asp instead of Pro at position 238 (EU numbering) was equally observed even when introduced into an antibody having activity with human FcRn under neutral range conditions. pH.

IgG1-F652 obtained in this example is an antibody that has FcRn binding activity under neutral pH conditions and that results in increased selective binding activity for inhibitory FcγRIIb. In other words, this antibody corresponds to the antigen-binding molecule according to embodiment 2 shown in Example 3. In other words, IgG1-F652 is capable of forming a four-component complex mediated by two FcRn molecules and one FcγR molecule; however, since this leads to an increase in the selective binding activity of the inhibitory FcγR, the binding activity of the active FcγR is reduced. As a result, it is believed that a four-component complex containing an inhibitory FcγR is preferably formed on antigen-presenting cells. As described previously, it is believed that immunogenicity is caused by the formation of a quaternary complex containing an active FcγR, and that thus the immune response is inhibited by the formation of a quaternary complex containing an inhibitory FcγR.

[Reference Example 1] Construction of expression vectors for IgG antibodies with amino acid substitution

Mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as described in the accompanying instructions. Plasmid fragments containing the mutants were inserted into animal cell expression vectors to construct the desired H chain and L chain expression vectors. The nucleotide sequences of the resulting expression vectors were determined by methods known to those skilled in the art.

[Reference Example 2] Expression and purification of IgG antibodies

Antibodies were expressed using the following method. Cells of a cell line derived from human embryonic kidney cancer HEK293H (Invitrogen) were suspended in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). Cells were plated at 10 ml per dish in non-adherent cell dishes (10 cm diameter; CORNING) at a cell density of 5-6 x 10 ⁵ cells/ml and cultured in a CO ₂ incubator (37° C., 5% CO ₂ ) throughout the day and night. The medium was then removed by aspiration and 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added. The resulting plasmid was introduced into cells by lipofection. The resulting culture supernatants were collected, centrifuged (approximately 2000×g, 5 min, room temperature) to remove cells, and sterilized by filtration through a 0.22 µm MILLEX (registered trademark)-GV (Millipore) filter to obtain supernatants. Antibodies were purified from purified culture supernatants in a manner known to those skilled in the art using rProtein A Sepharose™ Fast Flow (Amersham Biosciences). To determine the concentration of purified antibody, absorbance was measured at 280 nm using a spectrophotometer. From the determined values, antibody concentrations were calculated using the absorbance calculated by the method described in Protein Science (1995) 4: 2411-2423.

[Reference Example 3] Preparation of Soluble Human IL-6 Receptor (hsIL-6R)

The recombinant human IL-6 receptor, which is an antigen, was prepared as follows. A CHO cell line was produced constitutively expressing the soluble human IL-6 receptor (hereinafter referred to as hsIL-6R) having an amino acid sequence consisting of positions 1 to 357 from the N-terminus as described in J. Immunol. 152: 4958-4968 (1994), in a manner known to those skilled in the art. Soluble human IL-6 receptor was expressed by culturing this CHO line. Soluble human IL-6 receptor was purified from the culture supernatant of the resulting CHO line using two steps: Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. The fraction eluted as the main peak in the last step was obtained as the final purification product.

[Reference Example 4] PK study of human soluble IL-6 receptor and human antibody in normal mice

To study the plasma retention and immunogenicity of soluble human IL-6 receptor and human antibodies in normal mice, the following test was performed.

(4-1) Plasma retention time and immunogenicity study of soluble human IL-6 receptor in normal mice

To study the plasma retention and immunogenicity of the soluble human IL-6 receptor in normal mice, the following test was performed.

A single dose (50 μg/kg) of soluble human IL-6 receptor (prepared according to Reference Example 3) was injected into the tail vein of a normal mouse (C57BL/6J mouse, Charles River Japan). Blood sampling was performed 15 minutes, 7 hours and 1, 2, 3, 4, 7, 14 and 21 days after administration of the soluble human IL-6 receptor. The blood samples were immediately centrifuged for 15 minutes at 4°C and 15,000 rpm. for plasma separation. The separated plasma was stored in a freezer at -20° C. or below until the time of measurement. The plasma concentration of soluble human IL-6 receptor and the titer of soluble mouse anti-human IL-6 antibody were determined as described below.

The concentration of soluble human IL-6 receptor in mouse plasma was determined by the electrochemiluminescence method. Soluble IL-β receptor calibration curve sample prepared at 2000, 1000, 500, 250, 125, 62.5, or 31.25 pg/mL and mouse plasma measurement sample diluted 50 times or more were mixed with anti-human IL-6R monoclonal antibody (R&D) ruthenated with SULFO-TAG NHS Ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D), and tocilizumab, followed by overnight reaction at 37°C . Tocilizumab was received at a final concentration of 333 μg/ml. The reaction liquid was then dispensed into a MA400 PR Streptavidin Plate (Meso Scale Discovery). In addition, after washing off the reaction liquid, which was allowed to react for 1 hour at room temperature, reading buffer T (×4) (Meso Scale Discovery) was distributed. Then, measurement was immediately carried out in the reaction liquid using a SECTOR PR 400 (Meso Scale Discovery) reader. Soluble human IL-6 receptor concentration was calculated from the response of the standard curve using SOFTmax PRO analysis software (Molecular Devices).

The titer of mouse anti-human IL-6 receptor antibody in mouse plasma was determined by the electrochemiluminescence method. First, the human IL-6 receptor was distributed into an uncoated MA100 PR Uncoated Plate (Meso Scale Discovery). The plate was allowed to stand intact overnight at 4° C. to obtain a solid phase human IL-6 receptor plate. The human IL-6 receptor solid phase plate, into which a sample of 50 times diluted mouse plasma was dispensed for measurement, was allowed to stand undisturbed overnight at 4°C. This plate, which was then allowed to react with anti-mouse IgG (whole molecule) (Sigma-Aldrich) routineized with SULFO-TAG NHS Ester (Meso Scale Discovery), was washed for 1 hour at room temperature. Reading Buffer T (x4) (Meso Scale Discovery) was dispensed into said plate and immediately followed by measurement using a SECTOR PR 400 (Meso Scale Discovery) reader.

The results are shown in FIG. 37. The results demonstrate that the soluble human IL-6 receptor in mouse plasma rapidly disappeared. Of the three mice treated with soluble human IL-6 receptor, two mice (#1 and 3) showed an increased titer of soluble mouse anti-human IL-6 antibody in plasma. This indicates that these two mice developed an immune response to the soluble human IL-6 receptor leading to the production of mouse antibodies.

(4-2) Evaluation of the immunogenicity of the soluble human IL-6 receptor in a stationary model

To investigate the effects of the production of mouse antibodies against soluble IL-6 receptor on the plasma concentration of human soluble IL-6 receptor, the following test was performed.

The following study model was created as a model for maintaining the plasma concentration of the soluble human IL-6 receptor at steady state (approximately 20 ng/ml). An infusion pump (MODEL2004, alzet MINI-OSMOTIC PUMP) filled with human soluble IL-6 receptor was implanted subcutaneously in the back of a healthy mouse (C57BL/6J mouse, Charles River Japan) to generate an animal model with plasma concentrations of soluble IL-6 receptor person maintained in a stationary state.

The study was carried out in two groups (n=4 per group). In a group of mice that mimic immune tolerance, a single dose (20 mg/kg) of anti-mouse CD4 monoclonal antibody (R&D) was injected into the tail vein to inhibit the production of mouse antibodies against human soluble IL-6 receptor. Then, the antibody was similarly administered once every 10 days (hereinafter, this group is referred to as the anti-mouse CD4 antibody administration group). The other group was used as a control group, ie. a group without administration of anti-mouse CD4 antibody, in which no anti-mouse CD4 monoclonal antibody was administered. An infusion pump filled with 92.8 μg/ml soluble human IL-6 receptor was then implanted subcutaneously into the back of a mouse. After implantation of the infusion pump, blood samples were taken over time and then immediately centrifuged for 15 minutes at 4° C. and 15,000 rpm. to obtain plasma. The separated plasma was stored in a freezer at -20° C. or below until the time of measurement. Plasma concentration of soluble human IL-6 receptor (hsIL-6R) was determined in the same manner as in Reference Example 4-1.

Changes in plasma concentration of soluble human IL-6 receptor in selected normal mice, determined as described above, are shown in FIG. 38.

As a result, on day 14 after the infusion pump was subcutaneously implanted in the dorsal region of the mouse, it was observed that the concentrations of soluble human IL-6 receptor were reduced in all three mice in the group without administration of anti-mouse CD4 antibody. On the other hand, no decrease in plasma concentrations of soluble human IL-6 receptor was observed in all mice administered anti-CD4 antibody to inhibit the production of mouse antibodies against soluble human IL-6 receptor.

The results (4-1) and (4-2) indicate the following three points:

(1) Soluble human IL-6 receptor rapidly disappears from plasma after administration to mice;

(2) the soluble human IL-6 receptor is a foreign protein to mice that is immunogenic when administered to a mouse, inducing the production of mouse antibodies against the soluble human IL-6 receptor; And

(3) if the production of mouse antibodies against human soluble IL-6 receptor occurs, the disappearance of soluble human IL-6 receptor is further accelerated, even in a model with plasma concentration of human soluble IL-6 receptor maintained at a certain level, there is a decrease in plasma concentration .

(4-3) Study of plasma retention time and immunogenicity of a human antibody in a normal mouse

To study the plasma retention time and immunogenicity of a human antibody in a normal mouse, the following test was performed.

A single dose (1 mg/kg) of the anti-human IL-6 receptor antibody, Fv4-IgG1, was injected into the tail vein of a normal mouse (C57BL/6J mouse, Charles River Japan). Blood samples were taken at 15 minutes, 7 hours and 1, 2, 3, 4, 7, 14 and 21 days after administration of the anti-human IL-6 receptor antibody. The blood samples taken were immediately centrifuged at 15,000 rpm. for 15 minutes at 4°C to separate the plasma. The separated plasma was stored in a freezer at -20° C. or below until the time of measurement.

Plasma concentration of anti-human IL-6 receptor antibody in mice was determined by antibody ELISA. First, anti-human IgG (γ-chain specific) F(ab')2 fragment (SIGMA) was dispensed into Nunc-Immuno Plate, MaxiSoup (Nalge nunc International) and allowed to stand overnight at 4°C to give tablets with anti-human IgG antibody in solid phase. Samples for a calibration curve were prepared having plasma concentrations of anti-human IL-6 receptor antibody of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μg/mL, and samples for analysis mouse plasma diluted 100-fold or more. Mixtures of 100 μl of standard curve sample and measurement plasma sample and 200 μl of 20 ng/ml soluble human IL-6 receptor were allowed to stand intact for 1 hour at room temperature. The anti-human IgG solid phase plate to each well of which the mixtures were dispensed was then allowed to stand intact for 1 hour at room temperature. The plate was then allowed to react with a biotinylated anti-human IL-6R (R&D) antibody for 1 hour at room temperature. The reaction liquid obtained by reaction with Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was subjected to a chromogenic reaction for 1 hour at room temperature using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. After stopping the reaction with 1 H sulfuric acid (Showa Chemical), the absorbance of the reaction liquids in each well at 450 nm was measured on a microplate reader. Plasma antibody concentrations in mice were calculated from absorbance values on the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

The results are shown in FIG. 39. The plasma retention time of human antibody when a single dose of human antibody was administered to mice was significantly higher than that of soluble human IL-6 receptor when a single dose of soluble human IL-6 receptor was administered (Fig. 37) and was demonstrated that a high plasma concentration persists even 21 days after administration. This is likely a consequence of the fact that human antibodies that are incorporated into cells bind to mouse FcRn in the endosome for recycling into plasma. On the other hand, the soluble human IL-6 receptor, which is incorporated into cells, is thought to rapidly disappear from plasma because it has no pathway for recycling from the endosome.

Moreover, none of the three mice injected with the human antibody exhibited the reduced plasma concentration observed in the stationary model for the soluble human IL-6 receptor (FIG. 38). In other words, it was hypothesized that, unlike the human IL-6 receptor, no mouse anti-human antibody was produced.

The results (4-1), (4-2) and (4-3) may indicate the following. First of all, both human soluble IL-6 receptor and human antibody are foreign proteins in mice. Thus, mice are believed to have a large population of T cells that specifically respond to them.

When the soluble human IL-6 receptor, i.e. foreign protein was injected into mice, it disappeared from the plasma in a short period of time, and the immune response to the human soluble IL-6 receptor was confirmed. In this case, the rapid disappearance of the soluble human IL-6 receptor from plasma indicates that a plurality of soluble human IL-6 receptors are incorporated into antigen-presenting cells in a short period of time and processed in the cells, and then activate T cells that specifically respond on the soluble human IL-6 receptor. The result is believed to be an immune response to the soluble human IL-6 receptor (ie, production of a mouse antibody against the soluble human IL-6 receptor).

On the other hand, when a human antibody, i.e. foreign protein was injected into mice, its plasma retention was significantly lower than that of the soluble human IL-6 receptor, and no immune response to the human antibody occurred. Longer plasma retention indicates the presence of only a small amount of human antibodies that are incorporated into antigen-presenting cells and processed. Thus, it is believed that even if a mouse has a T cell population that specifically responds to a human antibody, the T cells are not activated by presenting the antigen and resulting in an immune response to the human antibody (i.e. production of mouse anti-human antibody) does not occur.

[Reference Example 5] Generation and evaluation of various Fc variants of antibodies with increased binding affinity to human FcRn at neutral pH

(5-1) Generation and evaluation of binding activity of various Fc variants of antibodies with increased binding affinity to human FcRn at neutral pH

To increase binding affinity to human FcRn in the neutral pH range, various mutations were introduced into VH3-IgG1 (SEQ ID NO: 35) for evaluation. Variants (IgG1-F1 to IgG1-F1052) containing the resulting heavy and light chains of L (WT)-CK (SEQ ID NO: 41) were expressed and purified according to the method described in Reference Example 2.

The binding of the antibody to human FcRn was analyzed according to the method described in Example 4. In other words, the binding activity of human FcRn variants under neutral conditions (pH 7.0) determined using Biacore is shown in Tables 27-1 to 27-32 .

Table 27-2 is a continuation of Table 27-1.

Table 27-3 is a continuation of Table 27-2.

Table 27-4 is a continuation of Table 27-3.

Table 27-5 is a continuation of Table 27-4.

Table 27-6 is a continuation of Table 27-5.

Table 27-7 is a continuation of Table 27-6.

Table 27-8 is a continuation of Table 27-7.

Table 27-9 is a continuation of Table 27-8.

Table 27-10 is a continuation of Table 27-9.

Table 27-11 is a continuation of Table 27-10.

Table 27-12 is a continuation of Table 27-11.

Table 27-13 is a continuation of Table 27-12.

Table 27-14 is a continuation of Table 27-13.

Table 27-15 is a continuation of Table 27-14.

Table 27-16 is a continuation of Table 27-15.

Table 27-17 is a continuation of Table 27-16.

Table 27-18 is a continuation of table 27-17.

Table 27-19 is a continuation of Table 27-18.

Table 27-20 is a continuation of table 27-19.

Table 27-21 is a continuation of Table 27-20.

Table 27-22 is a continuation of Table 27-21.

Table 27-23 is a continuation of Table 27-22.

Table 27-24 is a continuation of Table 27-23.

Table 27-25 is a continuation of Table 27-24.

Table 27-26 is a continuation of table 27-25.

Table 27-27 is a continuation of Table 27-26.

Table 27-28 is a continuation of table 27-27.

Table 27-29 is a continuation of table 27-28.

Table 27-30 is a continuation of Table 27-29.

Table 27-31 is a continuation of table 27-30.

Table 27-32 is a continuation of Table 27-31.

(5-2) In vivo study of human IL-6 receptor pH dependent antibodies with enhanced human FcRn binding under neutral pH conditions

Antibodies with pH-dependent binding to the human IL-6 receptor having the ability to bind human FcRn under neutral conditions were prepared using the heavy chains prepared as described in Example 5-1 to have the ability to bind human FcRn under neutral conditions. The antibodies were evaluated for their in vivo antigen-clearing effect. In particular, the antibodies below were expressed and purified by methods known to those skilled in the art, as described in Reference Example 2:

Fv4-IgG1 containing VH3-IgG1 (SEQ ID NO: 35) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-v2 containing VH3-IgG1-v2 (SEQ ID NO: 37) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F14 containing VH3-IgG1-F14 (SEQ ID NO: 86) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F20 containing VH3-IgG1-F20 (SEQ ID NO: 39) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F21 containing VH3-IgG1-F21 (SEQ ID NO: 40) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F25 containing VH3-IgG1-F25 (SEQ ID NO: 87) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F29 containing VH3-IgG1-F29 (SEQ ID NO: 88) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F35 containing VH3-IgG1-F35 (SEQ ID NO: 89) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F48 containing VH3-IgG1-F48 (SEQ ID NO: 90) and VL3-CK (SEQ ID NO: 36);

Fv4-IgG1-F93 containing VH3-IgG1-F93 (SEQ ID NO: 91) and VL3-CK (SEQ ID NO: 36); And

Fv4-IgG1-F94 containing VH3-IgG1-F94 (SEQ ID NO: 92) and VL3-CK (SEQ ID NO: 36).

The resulting antibodies with pH-dependent binding to the human IL-6 receptor were examined in vivo by the method described below using human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 276 +/+ mouse, Jackson Laboratories; Mol Biol Methods (2010) 602: 93-104).

Transgenic mice with human FcRn (mice B6.mFcRn-/-.hFcRn Tg line 276 +/+, Jackson Laboratories, Methods Mol Biol. 2010; 602: 93-104) and normal mice (C57BL/6J mouse, Charles River Japan) , administered hsIL-6R (soluble human IL-6 receptor prepared according to Reference Example 3) alone, or administered soluble human IL-6 receptor and anti-human IL-6 receptor antibody simultaneously to study the pharmacokinetics of soluble human IL-6 receptor and anti-human IL-6 receptor antibody Human IL-6 in vivo. Single dose solution (10 ml/kg) of soluble human IL-6 receptor (5 µg/ml) or mixture of soluble human IL-6 receptor and anti-human IL-6 receptor antibody (5 µg/ml and 0.1 mg/ml, respectively) was injected into the tail vein. At this time, the antibody against the soluble human IL-6 receptor existed in sufficient or excessive amounts. Thus, most of the soluble human IL-6 receptors are believed to bind to the antibody. Blood samples were collected at 15 minutes, 7 hours and 1, 2, 3, 4, 7, 14, 21 and 28 days after administration. The obtained blood samples were immediately centrifuged for 15 minutes at 4°C and 15000 rpm. for plasma separation. The separated plasma was stored in a freezer at -20° C. or below until the time of measurement.

(5-3) Determination of Plasma Concentration of Soluble Human IL-6 Receptor by Electrochemiluminescence Method

Human soluble IL-6 receptor calibration curve sample prepared at 2000, 1000, 500, 250, 125, 62.5, or 31.25 pg/mL, and mouse plasma measurement sample diluted 50-fold or higher, mixed with anti-human IL-6R monoclonal antibody (R&D) rutened with SULFO-TAG NHS Ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D), and tocilizumab, followed by reaction overnight at 37° WITH. Tocilizumab was received at a final concentration of 333 μg/ml. The reaction liquid was then dispensed into a MA400 PR Streptavidin Plate (Meso Scale Discovery). In addition, after washing off the reaction liquid, which was allowed to react for 1 hour at room temperature, reading buffer T (×4) (Meso Scale Discovery) was distributed. Then, measurement was immediately carried out in the reaction liquid using a SECTOR PR 400 (Meso Scale Discovery) reader. Soluble human IL-6 receptor concentration was calculated from the response of the standard curve using SOFTmax PRO analysis software (Molecular Devices).

Plasma concentration over time of soluble human IL-6 receptor following intravenous administration to human FcRn transgenic mice is shown in FIG. 40. The result of the test showed that the plasma concentration of soluble human IL-6 receptor remained low over time in the presence of any of the antibodies with pH-dependent binding of human IL-6 receptor with increased binding to human FcRn under neutral conditions, compared with the concentration in the presence of Fv4-IgG1, which has almost no ability to bind to human FcRn under neutral conditions. Among others, antibodies that produced a noticeable effect include, for example, Fv4-IgG1-F14. It has been demonstrated that the plasma concentration of human soluble IL-6 receptor co-administered with Fv4-IgG1-F14 decreased approximately 54-fold one day after administration compared to the concentration of human soluble IL-6 receptor co-administered with Fv4-IgG1 . Moreover, it was demonstrated that the plasma concentration of soluble human IL-6 receptor co-administered with Fv4-IgG1-F21 was reduced approximately 24-fold seven hours after administration compared to the concentration of soluble human IL-6 receptor co-administered with Fv4-IgG1. In addition, the plasma concentration of soluble human IL-6 receptor co-administered with Fv4-IgG1-F25 seven hours after administration was below the detection limit (1.56 ng/mL). Thus, Fv4-IgG1-F25 was expected to provide a significant 200-fold or greater reduction in the concentration of soluble human IL-6 receptor relative to the concentration of soluble human IL-6 receptor co-administered with Fv4-IgG1.

The data described above demonstrate that enhancing antibody binding with pH-dependent antigen binding to human FcRn under neutral conditions is highly effective in enhancing the effect of antigen elimination. Meanwhile, the type of amino acid change to enhance the binding of human FcRn under neutral conditions, which is made to enhance the antigen elimination effect, is not particularly limited; and such changes include those shown in Table 16. It can be predicted that the antigen elimination effect will be enhanced in vivo by any change introduced.

[Reference Example 6] Obtaining antibodies that bind to the IL-6 receptor in a Ca-dependent manner from an antibody library using phage display technology

(6-1) Obtaining a phage display library for naive human antibodies

A phage display library for human antibodies, consisting of a plurality of phages representing the Fab domains of mutually distinct human antibody sequences, was constructed according to a method known to those skilled in the art using poly-A RNA derived from human PBMC and a commercial poly- And human RNA as a template.

(6-2) Obtaining antibody fragments that bind to an antigen in a Ca-dependent manner from a library by bead panning

The engineered phage display library for naïve human antibodies was subjected to initial selection by concentrating only those antibody fragments that have antigen-binding ability (IL-6 receptor) or concentrating antibody fragments using Ca concentration-dependent antigen-binding ability (IL-6 receptor) in as an indicator. Concentration of antibody fragments using Ca concentration-dependent antigen-binding capacity (IL-6 receptor) as an indicator was performed by eluting phage library phages bound to the IL-6 receptor in the presence of Ca ions with EDTA, which chelates Ca ions. The biotinylated IL-6 receptor was used as an antigen.

Phages were obtained from Escherichia coli containing the engineered phage display phagemid. The phage library solution was prepared by diluting with TBS a population of phages precipitated by adding 2.5 M NaCl/10% PEG to the E. coli culture solution in which the phages were produced. Then BSA and CaCl ₂ were added to the phage library solution at a final concentration of 4% BSA and a calcium ion concentration of 1.2 mM. The conventional panning method using an antigen immobilized on magnetic beads has been called the panning method (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1- 2), 191-203; Biotechnol Prog. (2002) 18(2) 212-20; Mol Cell Proteomics (2003) 2(2), 61-9). Neutravidin-coated beads (Sera-Mag SpeedBeads Neutravidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.

Specifically, 250 pmol of biotin-labeled antigen was added to the resulting phage library solution to ensure that said phage library solution was contacted with the antigen for 60 minutes at room temperature. BSA-blocked magnetic beads were added to bind to antigen-phage complexes for 15 minutes at room temperature. The beads were washed once with 1 ml 1.2 mM CaCl ₂ /TBS (TBS containing 1.2 mM CaCl ₂ ). The phage solution was then isolated using the general elution method to concentrate the antibody fragment having the ability to bind the IL-6 receptor, or by elution from beads suspended in 2 mM EDTA/TBS (TBS containing 2 mM EDTA) to concentrate the antibody fragment with using the ability to bind the IL-6 receptor in a Ca concentration dependent manner as an index. The isolated phage solution was added to 10 ml of E. coli TG1 strain in logarithmic growth phase (OD600 0.4-0.7). E. coli were cultured with gentle agitation at 37° C. for 1 hour to allow the phages to infect E. coli. Infected E. coli were inoculated into a 225 mm x 225 mm dish. The phages were then isolated from the E. coli culture medium after inoculation to obtain a phage library solution.

In the second and subsequent panning, phages were concentrated using Ca-dependent binding capacity as an indicator. Specifically, 40 pmol of biotin labeled antibody was added to the resulting phage library solution to allow the phage library to contact the antigen for 60 minutes at room temperature. BSA-blocked magnetic beads were added to bind to antigen-phage complexes for 15 minutes at room temperature. The beads were washed once with 1 ml of 1.2 mM CaCl ₂ /TBST and 1.2 mM CaCl ₂ /TBS. Then, the beads, to which 0.1 ml of 2 mM EDTA/TBS was added, were suspended at room temperature. Immediately afterwards, the beads were separated using a magnetic stand to collect the phage solution. The isolated phage solution was added to 10 ml of E. coli TG1 strain in logarithmic growth phase (OD600 0.4-0.7). E. coli were cultured with gentle agitation at 37° C. for 1 hour to allow the phages to infect E. coli. Infected E. coli were inoculated into a 225 mm x 225 mm dish. The phages were then isolated from the E. coli culture medium after inoculation to obtain a phage library solution. Panning using Ca-dependent binding capacity as an index was repeated several times.

(6-3) ELISA study of phages

The phage containing the culture supernatant was harvested in a conventional manner (Methods Mol. Biol. (2002) 178, 133-145) from a single E. coli colony prepared as described above.

Culture supernatant containing phages to which BSA and CaCl ₂ were added to a final concentration of 4% BSA and a calcium ion concentration of 1.2 mM was subjected to ELISA as described below. A 96-well StreptaWell microtiter plate (Roche) was loaded overnight with 100 μl of PBS containing biotin labeled antigen. Each well of said plate was washed with PBST to remove antigen, and then the wells were blocked with 250 μl of 4% BSA-TBS for 1 hour or longer. This plate, with the resulting culture supernatant added to each well from which 4% BSA-TBS had been removed, was allowed to stand intact at 37° C. for 1 hour, allowing the phage-presenting antibody to bind to the antigen present in each well. To each well washed with 1.2 mM CaCl ₂ /TBST was added 1.2 mM CaCl ₂ /TBS or 1 mM EDTA/TBS. The tablet was allowed to stand intact for 30 minutes at 37°C for incubation. After washing with 1.2 mM CaCl ₂ /TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS at a final concentration of 4% BSA and 1.2 mM ionized calcium was added to each well, and the plate was incubated for 1 hours. After washing with 1.2 mM CaCl ₂ /TBST, the chromogenic reaction of the solution in each well, to which only TMB solution (ZYMED) was added, was stopped by adding sulfuric acid. Then the specified color was determined by measuring the absorbance at 450 nm.

As a result of the above phage ELISA, the base sequence of the gene amplified with the specific primers and the antibody fragment identified as having the ability of Ca-dependent antigen binding as a template was analyzed.

(6-4) Expression and purification of antibodies

As a result of the phage ELISA described above, a clone identified as having a Ca-dependent antigen-binding ability was introduced into an expression plasmid for animal cells. Antibodies were expressed as described below. Freestyle 293-F (Invitrogen), derived from human embryonic kidney cells, was suspended in Freestyle 293 Expression Medium (Invitrogen) and then inoculated at 3 ml into each well of a 6-well plate at a cell density of 1.33×10 ⁶ cells /ml The resulting plasmid was introduced into cells by lipofection. Cells were cultured for 4 days in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm). Antibodies were purified from the culture supernatant prepared as described above by a method known in the art using rProtein A Sepharose (trade mark) Fast Flow (Amersham Biosciences). The absorbance of the purified antibody solution was measured at 280 nm using a spectrophotometer. The antibody concentration was calculated from the measurement data obtained using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[Reference Example 7] Investigation of Ca-Dependent Binding Ability of Obtained Human IL-6 Receptor Antibodies

To investigate whether the antibody binding activity of 6RL#9-IgG1 [heavy chain (constant region sequence derived from IgG1 linked to SEQ ID NO: 9) and light chain (SEQ ID NO: 93)] and FH4-IgG1 [ heavy chain (SEQ ID NO: 94) and light chain (SEQ ID NO: 95)] obtained according to Reference Example 6, with Ca-dependent human IL-6 receptor, a kinetic analysis of antigen-antibody reactions of these antibodies with IL receptor was performed. -6 people using Biacore T100 (GE Healthcare). H54/L28-IgG1 [heavy chain variable region (SEQ ID NO: 96) and light chain variable region (SEQ ID NO: 97)] described in WO 2009/125825 was used as a control antibody which has Ca-dependent activity binding to the human IL-6 receptor. Kinetic analysis of antigen-antibody reactions was performed in solutions with calcium ion concentrations of 2 mM and 3 μM as high and low calcium ion conditions, respectively. The antibody of interest was immobilized on a CM4 sensor chip (GE Healthcare) on which an appropriate amount of Protein A (Invitrogen) was immobilized by the amine coupling method. Two buffers were used as running buffers [10 mM ACES, 150 mM NaCl, 0.05% (w/v) Tween 20, and 2 mM CaCl ₂ (pH 7.4) or 10 mM ACES, 150 mM NaCl, 0. 05% (w/v) Tween 20, and 3 µl/l CaCl ₂ (pH 7.4)]. These buffers were used to dilute the human IL-6 receptor. All measurements were carried out at 37°C.

In an antigen-antibody kinetic assay using the H54L28-IgG1 antibody, the H54L28-IgG1 antibody captured on the sensor chip was allowed to interact with the IL-6 receptor by injecting IL-6 receptor diluent and running buffer (blank sample) at a flow rate of 20 µl /min for 3 minutes. Then, after detecting dissociation of the IL-6 receptor using running buffer at a flow rate of 20 μl/min for 10 minutes, the sensor chip was regenerated by injecting 10 mM glycine-HCl (pH 1.5) at a flow rate of 30 μl/min for 30 seconds. From the sensorgrams obtained during the measurement, the kinetic parameters were calculated: the binding constant (ka) (1/Ms) and the dissociation rate constant (kd) (1/s). These values were used to calculate the dissociation constant (KD) (M) of the H54L28-IgG1 antibody against the human IL-6 receptor. Each parameter was calculated using Biacore T100 Evaluation Software (GE Healthcare).

In an antigen-antibody kinetic assay using FH4-IgG1 and 6RL#9-IgG1 antibodies, an FH4-IgG1 or 6RL#9-IgG1 antibody captured on a sensor chip was allowed to interact with the IL-6 receptor by injection of an IL-6 receptor diluent and running buffer (blank) at a flow rate of 5 µl/min for 15 minutes. The sensor chip was then regenerated by injecting 10 mM glycine-HCl (pH 1.5) at a flow rate of 30 μl/min for 30 seconds. Dissociation constants (KD) (M) were calculated from sensograms when measured using a stationary affinity model. Each parameter was calculated using Biacore T100 Evaluation Software (GE Healthcare). The dissociation constants (KD) between each antibody and the IL-6 receptor in the presence of 2 mM CaCl ₂ , determined as described above, are shown in Table 28.

The KD value for the H54/L28-IgG1 antibody under conditions of 3 μM Ca concentration can be calculated in the same way as in the presence of 2 mM Ca concentration. Under conditions of Ca concentration of 3 μM, it was observed that the FH4-IgG1 and 6RL#9-IgG1 antibodies hardly bind to the IL-6 receptor, thus calculating KD values by the method described above is difficult. However, the KD values for these antibodies under 3 μM Ca concentration conditions can be estimated using Formula 5 (Biacore T100 Software Handbook, BR-1006-48, AE 01/2007) described in Example 13.

Approximate results for the dissociation constant KD values for antibodies and IL-6 receptor at Ca 3 µl/l concentration, estimated using formula 3 described in example 13, are presented in table 29. In table 29, the values of _Req , R _max , RI and C are estimated based on the result of the analysis.

Based on the data described above, it was predicted that the K _D between the IL-6 receptor and the FH4-IgG1 antibody or the 6RL#9-IgG1 antibody increased by approximately 60 or 120-fold (affinity decreased by 60 or 120-fold or more) when the concentration of CaCl ₂ in the buffer was reduced from 2 mm to 3 μm. Table 30 summarizes the K _D values at CaCl ₂ concentrations of 2 mM and 3 μM and the dependence on Ca for the three types of antibodies H54/L28-IgG1, FH4-IgG1 and 6RL#9-IgG1.

There were no differences in the binding of the H54/L28-IgG1 antibody to the IL-6 receptor due to differences in Ca concentration. On the other hand, it was found that the binding of antibodies FH4-IgG1 and 6RL#9-IgG1 to the IL-6 receptor was significantly attenuated under conditions of low Ca concentration (table 30).

[Reference Example 8] Calcium ion binding study of the resulting antibody

Then, the intermediate temperature of thermal denaturation (Tm value) was measured using differential scanning calorimetry (DSC) as an indicator for the study of calcium ion binding to the antibody (MicroCal VP-Capillary DSC, MicroCal). The thermal denaturation intermediate temperature (Tm value) is an indicator of stability. The thermal denaturation intermediate temperature (Tm value) becomes higher when the protein is stabilized by calcium ion binding compared to no calcium ion binding (J. Biol. Chem. (2008) 283, 37, 25140-25149). The activity of calcium ion binding to the antibody was investigated by examining changes in the Tm value of the antibody, depending on changes in the concentration of calcium ions in the antibody solution. The purified antibody was dialyzed (EasySEP, TOMY) using an external solution of 20 mM Tris-HCl, 150 mM NaCl, and 2 mM CaCl ₂ (pH 7.4), or 20 mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl ₂ (pH 7.4). DSC measurement was performed at a heating rate of 240°C/h from 20 to 115°C using an antibody solution prepared at a concentration of approximately 0.1 mg/ml with dialysate as the test substance. The intermediate thermal denaturation temperatures (Tm values) of the Fab domains of each antibody, calculated from the DSC denaturation curve, are shown in Table 31.

The results presented in Table 31 show that the Tm values for FH4-IgG1 and 6RL#9-IgG1 Fab antibodies, which exhibit calcium-dependent binding ability, varied with calcium ion concentration, while the Tm values for H54 antibody Fab /L28-IgG1, which does not exhibit calcium-dependent binding ability, does not change with changes in the concentration of calcium ions. The variation in Tm values for Fab antibodies FH4-IgG1 and 6RL#9-IgG1 demonstrates that the calcium ions associated with these antibodies stabilize the Fab portions. The results described above show that calcium ions bound to the FH4-IgG1 and 6RL#9-IgG1 antibodies, while calcium ions did not bind to the H54/L28-IgG1 antibody.

[Reference Example 9] Identification of the calcium ion binding site in the 6RL#9 antibody by X-ray crystallography

(9-1) X-ray crystallography

As described in Reference Example 8, measurement of the thermal denaturation temperature Tm showed that the 6RL#9 antibody binds to calcium ions. However, it was unpredictable how much of the 6RL#9 antibody binds to the calcium ion. Therefore, residues of the 6RL#9 antibody that interact with calcium ion were identified using X-ray crystallography.

(9-2) Expression and purification of 6RL#9 antibody

The 6RL#9 antibody was expressed and purified for x-ray crystallography. In particular, animal expression plasmids designed to be capable of expressing the heavy chain (constant region sequence derived from IgG1 linked to SEQ ID NO: 9) and light chain (SEQ ID NO: 93) of the antibody 6RL# 9 was introduced temporarily into animal cells. The engineered plasmids were introduced by lipofection into FreeStyle 293-F human embryonic kidney cell line (Invitrogen) suspended in 800 ml of FreeStyle 293 Expression Medium (Invitrogen) (final cell density: 1×10 ⁶ cells/ml) . Cells with the introduced plasmid were cultured in a CO ₂ incubator (37°C, 8% CO ₂ , 90 rpm) for five days. From the culture supernatant prepared as described above, antibodies were purified in a manner known to those skilled in the art using rProtein A Sepharose™ Fast Flow (Amersham Biosciences). The absorbance at 280 nm of the purified antibody solutions was measured using a spectrophotometer. From the measured values, antibody concentrations were calculated using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

(9-3) Purification of the Fab fragment of the 6RL#9 antibody

The 6RL#9 antibody was concentrated to 21 mg/ml using a 10,000 MWCO ultrafilter. An antibody sample at a concentration of 5 mg/ml (2.5 ml) was prepared by diluting the antibody solution using 4 mM L-cysteine/5 mM EDTA/20 mM sodium phosphate (pH 6.5) buffer. 0.125 mg papain (Roche Applied Science) was added to the sample. After mixing, the sample was incubated at 35°C for two hours. After incubation, an EDTA-free Protease Inhibitor Cocktail Mini tablet (Roche Applied Science) was dissolved in 10 ml of 25 mM MES buffer (pH 6) and added to the sample. The sample was incubated on ice to stop proteolytic cleavage with papain. The sample was then applied to a 1 ml HiTrap SP HP cation exchange column (GE Healthcare) equilibrated with 25 mM MES buffer (pH 6), below which a 1 ml HiTrap MabSelect Sure Protein A column (GE Healthcare) was connected in series. A purified fraction of the Fab fragment of the 6RL#9 antibody was obtained by eluting with a linear concentration gradient of NaCl up to 300 mm in the buffer described above. The resulting purified fraction was then concentrated to approximately 0.8 ml using a 5000 MWCO ultrafilter. The concentrate was applied to a Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated with 100 mM HEPES buffer (pH 8) containing 50 mM NaCl. The purified Fab fragment of the 6RL#9 antibody for crystallization was eluted from the column using the same buffer. All column treatments described above were performed at a low temperature of 6 to 7.5°C.

(9-4) Crystallization of the Fab fragment of the antibody 6RL#9 in the presence of Ca

Seed crystals of the 6RL#9 Fab fragment were prepared in advance according to general conditions. The purified Fab fragment of the 6RL#9 antibody in 5 mM CaCl ₂ was then concentrated to 12 mg/ml using a 5000 MWCO ultrafilter. Next, the sample concentrated as described above was crystallized by hanging drop method by vapor diffusion using 100 mM HEPES buffer (pH 7.5) containing 20%-29% PEG4000 as a reservoir solution. The seed crystals described above were crushed in 100 mM HEPES buffer (pH 7.5) containing 29% PEG4000 and 5 mM CaCl ₂ and serially diluted from 100 to 10,000 times. Then 0.2 µl of the diluted solutions were combined with a mixture of 0.8 µl of the reservoir solution and 0.8 µl of the concentrated sample to obtain crystallization droplets on a coverslip. The crystal droplets were allowed to stand at 20° C. for two to three days until flat crystals were obtained. Using crystals, X-ray diffraction data were obtained.

(9-5) Crystallization of the Fab fragment of the 6RL#9 antibody in the absence of Ca

The purified Fab fragment of the 6RL#9 antibody was concentrated to 15 mg/ml using a 5000 MWCO ultrafilter. Next, the sample concentrated as described above was crystallized by hanging drop method by vapor diffusion using 100 mM HEPES buffer (pH 7.5) containing 18%-25% PEG4000 as a reservoir solution. The seed crystals described above were crushed in 100 mM HEPES buffer (pH 7.5) containing 25% PEG4000 and serially diluted from 100 to 10,000 times. Then 0.2 µl of the diluted solutions were combined with a mixture of 0.8 µl of the reservoir solution and 0.8 µl of the concentrated sample to obtain crystallization droplets on a coverslip. The crystal droplets were allowed to stand at 20° C. for two to three days until flat crystals were obtained. Using crystals, X-ray diffraction data were obtained.

(9-6) X-ray crystallographic measurement of a crystal of the Fab fragment from the antibody 6RL#9 in the presence of Ca

Crystals of the Fab fragment of the antibody 6RL#9 obtained in the presence of Ca were moistened with 100 mM HEPES buffer (pH 7.5) containing 35% PEG4000 and 5 mM CaCl ₂ . After removing the external solution from the surface of the single crystal with a micropin in the form of a nylon loop, the single crystal was frozen in liquid nitrogen. Frozen crystal X-ray diffraction data were obtained from a BL-17A Photon Factory beam line at the High Energy Accelerator Research Organization. The frozen crystal was kept frozen during the measurement by constantly placing it in a flow of nitrogen gas at -178°C. A total of 180 diffraction images were obtained using a Quantum315r CCD detector (ADSC) attached to the beamline while rotating the crystal at 1° intervals. Lattice constant determination, diffraction spot indexing, and diffraction data analysis were performed using the Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch) and Scala (CCP4 Software Suite) programs. Finally, diffraction intensity data were obtained with a resolution of up to 2.2 angstroms. The crystal belongs to the P212121 symmetry group with a lattice constant a=45.47 angstroms, b=79.86 angstroms, c=116.25 angstroms, α=90°, β=90° and γ=90°.

(9-7) X-ray crystallographic measurement of a crystal of the Fab fragment from the antibody 6RL#9 in the absence of Ca

Crystals of the Fab fragment of the 6RL#9 antibody obtained in the absence of Ca were wetted with 100 mM HEPES buffer (pH 7.5) containing 35% PEG4000. After removing the external solution from the surface of the single crystal with a micropin in the form of a nylon loop, the single crystal was frozen in liquid nitrogen. Frozen crystal X-ray diffraction data were obtained from a BL-5A Photon Factory beamline at the High Energy Accelerator Research Organization. The frozen crystal was kept frozen during the measurement by constantly placing it in a flow of nitrogen gas at -178°C. A total of 180 diffraction images were obtained using a Quantum210r CCD detector (ADSC) attached to the beamline while rotating the crystal at 1° intervals. Lattice constant determination, diffraction spot indexing, and diffraction data analysis were performed using Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch), and Scala (CCP4 Software Suite). Finally, diffraction intensity data were obtained with a resolution of up to 2.3 angstroms. The crystal belongs to the symmetry group P212121 with lattice constant a=45.40 angstroms, b=79.63 angstroms, c=116.07 angstroms, α=90°, β=90° and γ=90°, and thus it is structurally identical to the crystal obtained in the presence of Ca.

(9-8) Measurement by X-ray crystallography of a crystal of Fab fragment from antibody 6RL#9 in the presence of Ca

The crystal structure of the Fab fragment of the 6RL#9 antibody in the presence of Ca was determined by the molecular displacement method using the Phaser program (CCP4 Software Suite). The number of molecules in the asymmetric element was estimated based on the size of the crystal lattice and the molecular weight of the Fab fragment of the antibody 6RL#9. Based on primary sequence homology, part of amino acid positions 112-220 of the A chain and part of amino acid positions 116-218 of the B chain in the conformational coordinate of the PDB code 1ZA6 were used as model molecules for the analysis of the CL and CH1 regions. Then, a portion of amino acid positions 1-115 of the B chain in the conformational coordinate of the PDB code 1ZA6 was used as a model molecule for analysis of the VH region. Finally, a portion of amino acid positions 3-147 of the light chain in the conformational coordinate of the PDB code 2A9M was used as a model molecule for analysis of the VL region. From this order, an initial structural model of the 6RL#9 antibody Fab fragment was generated by determining positions and orientations of the model molecules for lattice analysis from the shear and rotation functions. Crystallographic confidence factor R for reflectance data at 25 to 3.0 resolution was 46.9% and free R was 48.6% after rigid framework detailing where each of the VH, VL, CH1 and CL domains was allowed to deviate. from the original structural model. Model refinement was then achieved by repeating the structural refinement using Refmac5 (CCP4 Software Suite) followed by model validation using Coot (Paul Emsley) based on electron density maps Fo-Fc and 2Fo-F, where the coefficients Fo-Fc and 2Fo-Fc was calculated using the experimentally determined structure factor Fo, the structure factor Fc calculated from this model, and the phases. The final detailing was performed using the Refmac5 program (CCP4 Software Suite) based on Fo-Fc and 2Fo-F electron density maps by adding a water molecule and a Ca ion to the model. Using 21020 reflectance data at a resolution of 25 to 2.2 angstroms, the final crystallographic confidence factor R was 20.0% and free R was 27.9% for a model consisting of 3440 atoms.

(9-9) Determination of X-ray diffraction data of the 6RL#9 Fab fragment crystal in the absence of Ca

The crystal structure of the Fab fragment of the 6RL#9 antibody in the absence of Ca was determined based on the structure of the crystal obtained in the presence of Ca. Water molecules and Ca ions were neglected in the conformational coordinates of the crystal of the Fab fragment of the 6RL#9 antibody obtained in the presence of Ca. The crystallographic confidence factor R for reflection data at a resolution of 25 to 3.0 angstroms was 30.3% and free R was 31.7% after rigid framework detailing where each of the VH-, VL-, CH1- and CL domains were allowed to deviate. Model refinement was then achieved by repeating the structural refinement using Refmac5 (CCP4 Software Suite) followed by model validation using Coot (Paul Emsley) based on electron density maps Fo-Fc and 2Fo-F, where the coefficients Fo-Fc and 2Fo-Fc was calculated using the experimentally determined structure factor Fo, the structure factor Fc calculated from this model, and the phases. The final detailing was performed using the Refmac5 program (CCP4 Software Suite) based on Fo-Fc and 2Fo-F electron density maps by adding a water molecule and a Ca ion to the model. Using 18357 reflection data at a resolution of 25 to 2.3 angstroms, the final crystallographic confidence factor R was 20.9% and the free R was 27.7% for a model consisting of 3351 atoms.

(9-10) Comparison of X-ray crystallographic diffraction data of Fab fragments of the 6RL#9 antibody in the presence and absence of Ca

When comparing the crystallographic structures of the Fab fragments of the 6RL#9 antibody in the presence and absence of Ca, significant changes in the CDR3 of the heavy chain were revealed. The heavy chain CDR3 structure of the Fab fragment of the 6RL#9 antibody, determined by X-ray crystallography, is shown in FIG. 41. In particular, the calcium ion was located in the center of the CDR3 loop region of the heavy chain of the Fab fragment of the 6RL#9 antibody obtained in the presence of Ca. It has been suggested that the calcium ion interacts with positions 95, 96 and 100a (Kabat numbering) of the heavy chain CDR3. It was proposed that the heavy chain CDR3 loop, which is important for antigen binding, was stabilized by calcium binding in the presence of Ca, and became the optimal structure for antigen binding. There are no reports demonstrating that calcium binds to the heavy chain CDR3 of the antibody. Thus, the calcium-linked structure of the antibody heavy chain CDR3 is a novel structure.

The calcium-binding motif present in the heavy chain CDR3, identified in the structure of the Fab fragment of the 6RL#9 antibody, may also become a new design element for the Ca library. For example, a library containing the heavy chain CDR3 of the 6RL#9 antibody and non-strict residues in other CDRs, including the light chain, is thought to be possible.

[Reference Example 10] Obtaining antibodies that bind to IL-6 in a Ca-dependent manner from a human antibody library using phage display methods

(10-1) Construction of a phage display library of naive human antibodies

A human antibody phage display library containing a plurality of phages that display different human antibody Fab domain sequences was constructed in a manner known to those skilled in the art using, as a template, a poly-A-RNA derived from human PBMC, a commercially available poly- human A-RNA, and the like.

(10-2) Obtaining antibody fragments that bind to an antigen in a Ca-dependent manner from a library using bead panning

Primary selection from engineered phage display libraries of naïve human antibodies was performed by enrichment for antibody fragments that have antigen binding activity (IL-6). The antigen used was biotin labeled IL-6.

Phages were generated using E. coli bearing phage display phagemids. 2.5 M NaCl/10% PEG was added to the sediment of phages obtained with E. coli in the culture medium for E. coli. The phage fraction was diluted with TBS to obtain a solution with a phage library. BSA and CaCl ₂ were then added to the phage library solution to final concentrations of 4% and 1.2 mM calcium ions, respectively. The panning method used was the conventional panning method using antigen-immobilized magnetic beads (J Immunol Methods. 2008 Mar 20, 332(1-2): 2-9; J Immunol Methods. 2001 Jan 1, 247(1-2 ): 191-203; Biotechnol Prog. 2002 Mar-Apr, 18(2): 212-20; Mol Cell Proteomics 2003 Feb, 2(2): 61-9). The magnetic beads used were neutravidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) and streptavidin-coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotinylated antigen was added to the resulting phage library solution. Thus, the solution was contacted with the antigen at room temperature for 60 minutes. The BSA-blocked magnetic beads were added and the antigen-phage complex was allowed to bind to the magnetic beads at room temperature for 15 minutes. The beads were washed three times with 1 ml 1.2 mM CaCl ₂ /TBS (TBS containing 1.2 mM CaCl ₂ ). After that, 0.5 ml of trypsin at a concentration of 1 mg/ml was added to the granules. After dispersing for 15 minutes at room temperature, the beads were immediately separated using a magnetic stand to collect the phage suspension. The resulting phage suspension was added to 10 ml of E. coli strain TG1 in the logarithmic growth phase (OD600 = 0.4-0.5). E. coli were incubated with gentle agitation at 37° C. for one hour for infection with phages. Infected E. coli were plated (225 mm x 225 mm). Then, phages were collected from the culture medium of the seeded E. coli to obtain a solution with a phage library.

At the second and subsequent pannings, phages were enriched using Ca-dependent binding capacity as an indicator. Specifically, 40 pmol of biotin labeled antigen was added to the resulting phage library solution. Thus, the phage library was contacted with the antigen at room temperature for 60 minutes. The BSA-blocked magnetic beads were added and the antigen-phage complex was allowed to bind to the magnetic beads at room temperature for 15 minutes. The beads were washed once with 1 ml 1.2 mM CaCl ₂ /TBST (TBS containing 1.2 mM CaCl ₂ and 0.1% Tween-20) and 1.2 mM CaCl ₂ /TBS. Then 0.1 ml of 2 mM EDTA/TBS was added to the beads. After dispersion at room temperature, the beads were immediately separated using a magnetic stand to collect the phage suspension. The pIII protein (pIII protein derived from helper phage) was cleaved from phages that did not exhibit Fab by adding 5 μl of trypsin at a concentration of 100 mg/ml to the harvested phage suspension to eliminate the ability of phages not exhibiting Fab to infect E. coli. Phages collected from the stock solution with phage after trypsin treatment were added to 10 ml of E. coli strain TG1 cells in the logarithmic growth phase (OD600=0.4-0.7). E. coli were incubated with gentle agitation at 37° C. for one hour for infection with phages. Infected E. coli were plated (225 mm x 225 mm). Phages were then collected from the culture medium of the seeded E. coli to obtain a liquid stock solution with a phage library. Panning was performed three times using Ca-dependent binding activity as an indicator.

(10-3) Evaluation by ELISA of phages

Phage-containing culture supernatants were collected from single colonies of E. coli obtained by the method described above, according to the conventional method (Methods Mol. Biol. (2002) 178, 133-145). BSA and CaCl ₂ were added to phage-containing culture supernatants to final concentrations of 4% and 1.2 mM calcium ions, respectively. Supernatants were subjected to ELISA according to the following method. StreptaWell 96-well microtiter plates (Roche) were coated overnight with 100 µl of PBS containing biotin labeled antigen. The antigen was removed from the well by washing each well of the PBST plate. The wells were then blocked with 250 μl 4% BSA-TBS for one hour or more. After removal of 4% BSA-TBS, the resulting culture supernatants were added to each well. The plate was incubated at 37° C. for one hour to allow antibody-expressing phages to bind to the antigen on each well. After washing each well with 1.2 mM CaCl ₂ /TBST, 1.2 mM CaCl ₂ /TBS or 1 mM EDTA/TBS was added. The tablet was left to incubate at 37°C for 30 minutes. After washing with 1.2 mM CaCl ₂ /TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS containing BSA and calcium ions at final concentrations of 4% and 1.2 mM calcium ions was added to each well, and a plate incubated for one hour. After washing with 1.2 mM CaCl ₂ /TBST, only TMB solution (ZYMED) was added to each well. The chromogenic reaction in the solution in each well was stopped by adding sulfuric acid. The developed color was then evaluated by measuring the absorbance at 450 nm.

From 96 isolated clones, the 6KC4-1#85 antibody was obtained by phage ELISA, having a Ca-dependent IL-6 binding activity. Using antibody fragments predicted to have Ca-dependent antigen-binding activity based on the phage ELISA result described above as a template, the genes were amplified with specific primers and their sequences analyzed. The sequences of the heavy chain and light chain variable regions of the 6KC4-1#85 antibody are shown in SEQ ID NOS: 10 and 98, respectively. A polynucleotide encoding the heavy chain variable region of the antibody 6KC4-1#85 (SEQ ID NO: 10) was linked to a polynucleotide encoding a sequence derived from IgG1 by PCR. The obtained DNA fragment was inserted into an expression vector of animal cells to construct an expression vector for the heavy chain of SEQ ID NO: 99. The polynucleotide encoding the light chain variable region of the antibody 6KC4-1#85 (SEQ ID NO: 98) was linked to the polynucleotide encoding the constant region natural kappa chain (SEQ ID NO: 100) by PCR. A DNA fragment containing the linked sequence shown in SEQ ID NO: 101 was inserted into an animal cell expression vector. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art.

(10-4) Expression and purification of antibodies

Clone 6KC4-1#85, which was evaluated as having the ability to Ca-dependent binding using phage ELISA, was introduced into the expression plasmids of animal cells. Antibodies were expressed using the following method. FreeStyle 293-F embryonic kidney-derived cells (Invitrogen) were suspended in FreeStyle 293 Expression Medium (Invitrogen) and 3 ml aliquots plated in each well of 6-well plates at a cell density of 1.33 x 10 ⁶ cells/ml. The resulting plasmids were introduced into cells by lipofection. Cells were cultured in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm) for four days. From the resulting culture supernatants, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. Purified antibody concentrations were determined by measuring absorbance at 280 nm using a spectrophotometer. Antibody concentrations were calculated from established values based on the extinction coefficient determined by the PACE method (Protein Science 1995; 4: 2411-2423).

[Reference Example 11] Evaluation of 6KC4-1#85 Antibody Calcium Ion Binding

(11-1) Evaluation of 6KC4-1#85 antibody binding to calcium ion

The calcium-dependent binding antibody 6KC4-1#85, which was isolated from a human antibody library, was evaluated for its calcium binding. Whether the measured Tm value varies depending on the concentration of ionized calcium was judged by the method described in Reference Example 6.

The Tm values for the Fab domain of the 6KC4-1#85 antibody are shown in Table 32. As shown in Table 32, the Tm value for the Fab domain of the 6KC4-1#85 antibody varied depending on the concentration of calcium ions. This demonstrates that the 6KC4-1#85 antibody binds to calcium.

(11-2) Identification of the calcium ion binding site in the 6KC4-1#85 antibody

As shown in (11-1) of Reference Example 11, the 6KC4-1#85 antibody binds to calcium ion. However, 6KC4-1#85 does not have a calcium binding motif, such as the hVk5-2 sequence, which has been evaluated to have a calcium binding motif. Thus, to identify the residues responsible for calcium ion binding by the 6KC4-1#85 antibody, altered heavy chains (6_H1-11 (SEQ ID NO: 102), 6_H1-12 (SEQ ID NO: 103), 6_H1-13 ( SEQ ID NO: 104), 6_H1-14 (SEQ ID NO: 105), 6_H1-15 (SEQ ID NO: 106)) and altered light chains (6_L1-5 (SEQ ID NO: 107) and 6_L1-6 (SEQ ID NO: 108)) by replacing the Asp (D) residue in the CDR of the 6KC4-1#85 antibody with an Ala (A) residue that does not participate in calcium ion binding or chelation. By the method described in Reference Example 6, the altered antibodies were purified from the culture supernatants of animal cells into which expression vectors carrying genes of the altered antibodies were introduced. Purified modified antibodies were evaluated for their calcium binding in the manner described in Example 6. The result of the measurement is shown in Table 33. As shown in Table 33, substitution of an Ala residue at position 95 or 101 (Kabat numbering) in the CDR3 of the heavy chain of the antibody 6KC4-1 #85 resulted in loss of activity of the 6KC4-1#85 antibody in relation to calcium binding. This indicates that these residues are responsible for calcium binding. The calcium binding motif present at the base of the heavy chain CDR3 loop of the 6KC4-1#85 antibody, as revealed from the calcium binding properties of the modified 6KC4-1#85 antibody, could also be a new design element for the Ca library, as described in the reference Example 9 In other words, in addition to a library with a calcium binding motif introduced into the light chain variable region provided as a specific example in Reference Example 20, etc., a library containing a calcium binding motif present, for example, in the CDR3 of the heavy chain of the antibody 6KC4-1#85 and containing non-strict residues in other amino acid residues.

[Reference Example 12] Investigation of the Effects of Ca-Dependent Binding Antibody on Antigen Plasma Retention Using Normal Mice

(12-1) In vivo study using normal mice

Normal mouse (C57BL/6J mouse, Charles River Japan), administered hsIL-6R (soluble human IL-6 receptor prepared according to Reference Example 3) alone, or administered soluble human IL-6 receptor and anti-human IL-6 receptor antibody simultaneously to study the kinetics of soluble human IL-6 receptor and antibodies against human IL-6 receptor in vivo. A single dose (10 ml/kg) of a solution of soluble human IL-6 receptor (5 μg/ml) or a mixture of soluble human IL-6 receptor and anti-human IL-6 receptor antibody was injected into the tail vein. The above H54/L28-IgG1, 6RL#9-IgG1 and FH4-IgG1 were used as antibodies against human IL-6 receptor.

The concentration of soluble human IL-6 receptor in all mixtures is 5 μg/ml. Anti-human IL-6 receptor antibody concentrations vary for antibodies: 0.1 mg/ml for H54/L28-IgG1 and 10 mg/ml for 6RL#9-IgG1 and FH4-IgG1. At this time, it was believed that most of the soluble human IL-6 receptors bind to the antibody, since the antibody against the soluble human IL-6 receptor is present in sufficient or excessive amounts. Blood samples were taken 15 minutes, 7 hours and 1, 2, 4, 7, 14, 21 and 28 days after administration. The obtained blood samples were immediately centrifuged for 15 minutes at 4°C and 12000 rpm. for plasma separation. The separated plasma was stored in a freezer at -20° C. or below until the time of measurement.

(12-2) Plasma concentration determination of anti-human IL-6 receptor in normal mice by ELISA

The concentration of anti-human IL-6 receptor antibody in mouse plasma was determined by ELISA. First, anti-human IgG (γ chain specific) F(ab')2 fragment (SIGMA) was dispensed into Nunc-Immuno Plate, MaxiSoup (Nalge nunc International) and allowed to stand overnight at 4°C to give tablets with anti-human IgG antibody in solid phase. Calibration curve samples having plasma concentrations of 0.64, 0.32, 0.16, 0.08, 0.04, 0.02, or 0.01 µg/mL and mouse plasma samples for measurement diluted 100 times or more, dispensed into a solid phase anti-human IgG antibody plate, and then incubated for 1 hour at 25°C. The plate was then allowed to react with a biotinylated anti-human IL-6 R antibody (R&D) for 1 hour at 25°C and then reacted with streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 0.5 hour at 25°C. The chromogenic reaction was performed using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as substrate. After stopping the chromogenic reaction with 1 N sulfuric acid (Showa Chemical), the absorbance of the color solution at 450 nm was measured on a microplate reader. Plasma antibody concentrations in mice were calculated from absorbance values on the calibration curve using SOFTmax PRO analytical software (Molecular Devices). Changes in plasma concentrations of H54/L28-IgG1, 6RL#9-IgG1, and FH4-IgG1 antibodies in normal mice after intravenous administration, measured as described above, are shown in FIG. 42.

(12-3) Plasma concentration determination of human soluble IL-6 receptor by electrochemiluminescence method

The concentration of soluble human IL-6 receptor in mouse plasma was determined by the electrochemiluminescence method. Soluble IL-6 receptor calibration curve sample prepared at a concentration of 2000, 1000, 500, 250, 125, 62.5, or 31.25 pg/mL and a mouse plasma sample for measurement diluted 50 times or more were mixed with anti-human IL-6R monoclonal antibody (R&D) rutenized with SULFO-TAG NHS Ester (Meso Scale Discovery), biotinylated anti-human IL-6R antibody (R&D), and tocilizumab (heavy chain SEQ ID NO: 109, light chain SEQ ID NO: 83), followed by overnight reaction at 4°C. By this time, the assay buffer contained 10 mM EDTA to reduce the concentration of free Ca in the sample and dissociate virtually all soluble human IL-6 receptors in the sample from 6RL#9-IgG1 or FH4-IgG1 to bind to the added tocilizumab. This reaction liquid was then dispensed into a MA400 PR Streptavidin Plate (Meso Scale Discovery). In addition, after washing each well of the plate, which was allowed to react for 1 hour at 25°C, reading buffer T (×4) (Meso Scale Discovery) was dispensed into each well. Immediately thereafter, the reaction liquid was measured using a SECTOR PR 400 (Meso Scale Discovery) reader. Soluble human IL-6 receptor concentration was calculated from the response of the standard curve using SOFTmax PRO analysis software (Molecular Devices). Changes in plasma concentration of soluble human IL-6 receptor in normal mice following intravenous administration, determined as described above, are shown in FIG. 43.

As a result, the disappearance of the soluble human IL-6 receptor was very rapid when the soluble human IL-6 receptor was administered alone, while the disappearance of the soluble human IL-6 receptor was significantly delayed when the soluble human IL-6 receptor was administered simultaneously with H54. /L28-IgG1, a conventional antibody lacking Ca-binding activity for the soluble human IL-6 receptor. In contrast, the disappearance of the soluble human IL-6 receptor was significantly accelerated when the soluble human IL-6 receptor was administered concomitantly with 6RL#9-IgG1 or FH4-IgG1, which has a 100-fold or higher Ca-dependent binding capacity to the soluble IL-6 receptor. 6 people. Plasma concentrations of the soluble human IL-6 receptor one day after administration of the soluble human IL-6 receptor simultaneously with 6RL#9-IgG1 and FH4-IgG1 were reduced by 39 times and 2 times, respectively, compared with simultaneous administration with H54/ L28-IgG1. Thus, it was confirmed that antibodies with calcium-dependent binding are able to accelerate the disappearance of antigen from plasma.

[Reference Example 13] Trial for improving the effect of accelerating antigen elimination by Ca-dependent antigen binding (antibody production)

(13-1) IgG antibody binding to FcRn

IgG antibodies have a longer plasma retention time as a result of FcRn binding. Binding between IgG and FcRn is observed only under acidic conditions (pH 6.0). In contrast, binding is practically undetectable under neutral conditions (pH 7.4). The IgG antibody is taken up into cells in a non-specific manner. The antibody returns to the cell surface by binding to endosomal FcRn under the acidic conditions of endosomes and then dissociates from the FcRn under neutral plasma conditions. When FcRn binding under acidic conditions is lost due to the introduction of mutations in the IgG Fc domain, the plasma retention time of the antibody is significantly reduced because the antibody is no longer recycled into plasma from the endosome.

A disclosed method for increasing plasma retention of an IgG antibody is to enhance FcRn binding under acidic conditions. Amino acid mutations are introduced into the Fc domain of an IgG antibody to increase its binding to FcRn under acidic conditions. This increases the efficiency of recycling to plasma from the endosome, resulting in increased plasma retention of IgG antibodies. When making a substitution, it is considered important not to increase binding to FcRn under neutral conditions. IgG antibodies that bind to FcRn under neural conditions can return to the cell surface by binding to FcRn under acidic conditions of the endosome, however, IgG antibodies do not dissociate from FcRn in plasma under neutral conditions and are not recycled to plasma, and thus that retention in plasma of IgG antibodies, on the contrary, will worsen.

For example, as described in Dall' Acqua et al. (J. Immunol. (2002) 169 (9), 5171-5180), plasma retention of an IgG1 antibody that was allowed to bind to mouse FcRn under neutral conditions (pH 7.4) was enhanced by amino acid substitution in the mouse. In addition, as described by Yeung et al. (J. Immunol. (2009) 182 (12), 7663-7671), Datta-Mannan et al. (J. Biol. Chem. (2007) 282 (3), 1709-1717), and Dall' Acqua et al. (J. Immunol. (2002) 169 (9), 5171-5180), it was found that IgG1 antibody variants whose binding to human FcRn under acidic conditions (pH 6.0) is improved by introducing an amino acid substitution also bind to FcRn human under neutral conditions (pH 7.4). Plasma retention time of said antibody administered to cynomolgus monkey was reportedly not increased, showing no change in plasma retention. Thus, in antibody engineering technology to enhance antibody functions, attempts have been made to increase plasma retention of an antibody by increasing its binding to human FcRn under acidic conditions without increasing its binding to human FcRn under neutral conditions (pH 7.4). In other words, no reports have been published regarding the benefits of IgG1 antibodies whose binding activity to human FcRn under neutral conditions (pH 7.4) is increased by introducing amino acid substitutions in the Fc region.

Antibodies that bind to an antigen in a Ca-dependent manner are extremely useful because they have the effect of hastening the disappearance of the soluble antigen and rebinding a single antibody molecule to the soluble antigen. A method for enhancing binding to FcRn under neutral conditions (pH 7.4) was investigated as a method for further improving the effect of accelerating the disappearance of the antigen.

(13-2) Obtaining antibodies with Ca-dependent binding of human IL-6 receptor having the ability to bind FcRn under neutral conditions

An amino acid mutation was introduced into the Fc regions of FH4-IgG1 and 6RL#9-IgG1 having the ability of calcium-dependent antigen binding, and H54/L28-IgG1, not having the ability of calcium-dependent antigen binding (used as control) to obtain variants, having the ability to bind to FcRn under neutral conditions (pH 7.4). The amino acid mutation was introduced in a manner known in the art using PCR. Specifically, FH4-N434W (heavy chain SEQ ID NO: 110, light chain SEQ ID NO: 95), 6RL#9-N434W (heavy chain SEQ ID NO: 111, light chain SEQ ID NO: 93), and H54 /L28-N434W (heavy chain SEQ ID NO: 112, light chain SEQ ID NO: 97) with Asn (amino acid at position 434 according to EU numbering) replaced by Trp in the IgG1 heavy chain constant region. An animal cell expression vector into which a polynucleotide encoding an amino acid substitution variant was inserted was generated using the QuikChange Site-Directed Mutahenesis Kit (Stratagene) in the manner described in the accompanying instructions. Expression and purification of the antibody and concentration measurement were performed according to the method described in Reference Example 6.

[Reference 14] Investigation of the effect of accelerating the disappearance of antibodies with Ca-dependent binding using normal mice

(14-1) In vivo study using normal mice

Normal mice (C57BL/6J mouse; Charles River Japan) were treated with hsIL-6R (soluble human IL-6 receptor prepared as described in Reference Example 3) alone or treated with soluble human IL-6 receptor and anti-human IL-6 antibody simultaneously to study the kinetics of soluble human IL-6 receptor and antibodies against human IL-6 receptor in vivo. A single dose (10 ml/kg) of a solution of soluble human IL-6 receptor (5 μg/ml) or a mixture of soluble human IL-6 receptor and anti-human IL-6 antibody was injected into the tail vein. H54/L28-N434W, 6RL#9-N434W and FH4-N434W described above were used as antibodies against the human IL-6 receptor.

The concentration of soluble human IL-6 receptor in all mixtures is 5 μg/ml. Anti-human IL-6 receptor antibody concentrations vary for antibodies: prepared at 0.042 mg/mL for H54/L28-N434W, at 0.55 mg/mL for 6RL#9-N434W, and at 1 mg/mL for FH4 -N434W. At this time, it was believed that most of the soluble human IL-6 receptors bound to the antibody, since the antibody against the soluble human IL-6 receptor exists in sufficient or excessive amounts. Blood samples were taken 15 minutes, 7 hours and 1, 2, 4, 7, 14, 21 and 28 days after administration. The blood samples were immediately centrifuged for 15 minutes at 4°C and 12,000 rpm. for plasma separation. The separated plasma was stored in a freezer at -20° C. or below until the time of measurement.

(14-2) Determination of anti-human IL-6 receptor antibody concentration in normal mice by ELISA

Plasma concentration of anti-human IL-6 receptor antibody in mice was determined by ELISA as described in Reference Example 12. injections measured as described above are shown in FIG. 44.

(14-3) Determination of the concentration of soluble human IL-6 receptor by electrochemiluminescence method

The concentration of soluble human IL-6 receptor in mouse plasma was determined by the electrochemiluminescence method. Soluble IL-6 receptor calibration curve sample prepared at 2000, 1000, 500, 250, 125, 62.5, or 31.25 pg/mL and mouse plasma measurement sample diluted 50 times or more were mixed with anti-human IL-6R monoclonal antibody (R&D) rutened with SULFO-TAG NHS Ester (Meso Scale Discovery) and biotinylated anti-human IL-6R antibody (R&D), followed by overnight reaction at 4°C. By this time, the assay buffer contained 10 mM EDTA to reduce the concentration of free Ca in the sample and dissociate virtually all soluble human IL-6 receptors in the sample from 6RL#9-N434W or FH4-N434W to exist in the free state. This reaction liquid was then dispensed into a MA400 PR Streptavidin Plate (Meso Scale Discovery). In addition, after washing each well of the plate, which was allowed to react for 1 hour at 25°C, reading buffer T (×4) (Meso Scale Discovery) was dispensed into each well. Immediately thereafter, the reaction liquid was measured using a SECTOR PR 400 (Meso Scale Discovery) reader. Soluble human IL-6 receptor concentration was calculated from the standard curve response using SOFTmax PRO analysis software (Molecular Devices). Changes in plasma concentration of soluble human IL-6 receptor in normal mice following intravenous administration, determined as described above, are shown in FIG. 45.

As a result, compared to administration of soluble human IL-6 receptor alone, simultaneous administration of soluble human IL-6 receptor with H54/L28-N434W antibody, which has FcRn binding activity at pH 7.4 and does not have Ca-dependent binding activity to soluble human IL-6 receptor had a significantly delayed disappearance of the soluble human IL-6 receptor. In contrast, the disappearance of the soluble human IL-6 receptor was accelerated when the soluble human IL-6 receptor was co-administered with the 6RL#9-N434W or FH4-N434W antibody, which has 100 times or more Ca-dependent binding to the soluble IL receptor. -6 human and FcRn binding activity at pH 7.4 compared to administration of soluble human IL-6 receptor alone. Plasma concentrations of soluble human IL-6 receptor one day after administration of soluble human IL-6 receptor simultaneously with antibody 6RL#9-N434W or FH4-N434W were reduced by 3-fold and 8-fold, respectively, compared with administration of soluble IL-6 receptor -6 persons separately. As a result, it was confirmed that the disappearance of the antigen from plasma can be further accelerated by imparting FcRn binding activity at pH 7.4 to an antibody that binds to the antigen in a calcium-dependent manner.

A 6RL#9-IgG1 or FH4-IgG1 antibody having a 100-fold or more increased Ca-dependent binding activity to the soluble human IL-6 receptor was confirmed to increase the disappearance of the soluble human IL-6 receptor compared to the H54/L28 antibody. -IgG1 having no Ca-dependent binding activity to the soluble human IL-6 receptor. It was confirmed that the antibody 6RL#9-N434W or FH4-N434W, which has a 100-fold or more increased Ca-dependent binding activity to human soluble IL-6 receptor and FcRn binding activity at pH 7.4, accelerates the disappearance to a greater extent. soluble human IL-6 receptor, compared to administering soluble human IL-6 receptor alone. These data indicate that an antibody that binds to an antigen in a Ca-dependent manner dissociates from the antigen in the endosome, similar to an antibody that binds to an antigen in a pH-dependent manner.

[Reference Example 15] Investigation of human germline sequences that bind to calcium ions

(15-1) An antibody that binds to an antigen in a calcium dependent manner

Antibodies that bind to an antigen in a Ca-dependent manner (antibodies with Ca-dependent antigen binding) are antibodies whose interactions with an antigen change depending on the calcium concentration. An antibody with Ca-dependent binding is believed to bind to an antigen via a calcium ion. Thus, the amino acids that form an epitope on one side of the antigen are negatively charged amino acids that can chelate calcium ions, or amino acids that can be a hydrogen bond acceptor. These properties of the amino acids that form the epitope allow targeting of an epitope other than binding molecules, which are obtained by introducing histidine residues and which bind to the antigen in a pH-dependent manner. It is believed that the use of antigen-binding molecules having calcium- and pH-dependent binding properties will allow the formation of antigen-binding molecules that can be individually targeted to various epitopes having broad properties. Thus, if a pool of molecules containing a calcium-binding motif (Ca-library) is constructed from which antigen-binding molecules are derived, it is believed that antibodies with Ca-dependent antigen binding will be generated in an efficient manner.

(15-2) Obtaining Human Germline Sequences

An example of a population of molecules containing a calcium-binding motif is one in which said molecules are antibodies. In other words, a library of antibodies containing a calcium-binding motif may be a Ca-library.

Antibodies with calcium ion dependent binding containing human germline sequences have not been described. Thus, germline antibody sequences having human germline sequences were cloned using cDNA derived from embryonic spleen polyRNA (Clontech) as a template to assess whether antibodies having human germline sequences bind to calcium ion. Cloned DNA fragments were inserted into expression vectors of animal cells. The nucleotide sequences of the constructed expression vectors were determined in a manner known to those skilled in the art. SEQ IDs are shown in Table 34. By PCR, polynucleotides encoding SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), SEQ ID NO: 8 (Vk4), and SEQ ID NO: 4 (Vk5) was linked to a polynucleotide encoding the natural kappa chain constant region (SEQ ID NO: 100). Linked DNA fragments were inserted into animal cell expression vectors. Moreover, the polynucleotides encoding SEQ ID NO: 113 (Vk1), SEQ ID NO: 114 (Vk2), SEQ ID NO: 115 (Vk3), SEQ ID NO: 116 (Vk4), and SEQ ID NO: 117 (Vk5 ) was linked by PCR to a polynucleotide encoding a polypeptide (SEQ ID NO: 11) having a two amino acid deletion at the C-terminus of IgG1. The resulting DNA fragments were inserted into expression vectors of animal cells. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art.

(15-3) Expression and purification of antibodies

Constructed expression vectors of animal cells into which DNA fragments having five types of human germline sequences are inserted were introduced into animal cells. The expression of the antibody was carried out in the following way. Cells from the human embryonic kidney cell line Freestyle 293-F (Invitrogen) were suspended in Freestyle 293 Expression Medium (Invitrogen) and seeded at a cell density of 1.33×10 ⁶ cells/mL (3 mL) into each well of a plate with 6 cells. The resulting plasmids were introduced into cells by lipofection. Cells were cultured for four days in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm). From culture supernatants prepared as described above, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. The absorbance of purified antibody solutions at 280 was measured using a spectrophotometer. Antibody concentrations were calculated from the determined values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4: 2411-2423).

(15-4) Evaluation of antibodies having human germline sequences for their calcium ion binding activity

Purified antibodies were evaluated for their calcium ion binding activity. The thermal denaturation intermediate temperature (Tm value) was measured by differential scanning calorimetry (DSC) as an indicator for the study of calcium ion binding to an antibody (MicroCal VP-Capillary DSC, MicroCal). The thermal denaturation intermediate temperature (Tm value) is an indicator of stability. It becomes higher when the protein is stabilized by binding to calcium ions, compared with no binding of calcium ions (J. Biol. Chem. (2008) 283, 37, 25140-25149). The activity of calcium ion binding to the antibody was investigated by examining changes in the Tm value of the antibody, depending on changes in the concentration of calcium ions in the antibody solution. The purified antibody was dialyzed (EasySEP, TOMY) using an external solution of 20 mM Tris-HCl, 150 mM NaCl, and 2 mM CaCl ₂ (pH 7.4), or 20 mM Tris-HCl, 150 mM NaCl, and 3 μM CaCl ₂ (pH 7.4). DSC measurement was performed at a heating rate of 240°C/h from 20 to 115°C using an antibody solution prepared at a concentration of approximately 0.1 mg/ml with dialysate as the test substance. The intermediate thermal denaturation temperatures (Tm values) of the Fab domains of each antibody, calculated from the DSC denaturation curve, are shown in Table 35.

The result showed that the Tm values of Fab domains of antibodies having the sequence Vk1, Vk2, Vk3 or Vk4 did not vary depending on the concentration of calcium ions in solutions containing the Fab domain. Meanwhile, the Tm value for the Fab domain of an antibody having the Vk5 sequence varied depending on the concentration of calcium ions in the solution containing the Fab domain. This demonstrates that the Vk5 sequence binds to calcium ions.

[Reference Example 16] Human Vk5 sequence evaluation (hVk5)

(16-1) Sequence hVk5

The only hVk5 sequence registered in the Kabat database is the hVk5-2 sequence. In the following, hVk5 and hVk5-2 are used as synonyms.

WO 2010/136598 describes that the relative abundance of the hVk5-2 sequence in the germline sequence is 0.4%. There are also other reports that the relative abundance of the hVk5-2 sequence in the germline sequence is 0-0.06% (J. Mol. Biol. (2000) 296, 57-86; Proc. Natl. Acad. Sci. (2009 ) 106, 48, 20216-20221). As described above, since the hVk5-2 sequence is a sequence with a low frequency of occurrence in the germline sequence, it was believed to be ineffective for generating a calcium-dependent binding antibody from an antibody library composed of human germline sequences or B cells. obtained by immunizing a mouse expressing human antibodies. Thus, it was thought possible to construct a Ca library containing the human hVk5-2 sequence. However, the feasibility of this possibility is unknown, since no report on the physical properties of the hVk5-2 sequence has been published.

(16-2) Construction, expression and purification of the non-glycosylated form of the hVk5-2 sequence

The hVk5-2 sequence has an N-glycosylation sequence at amino acid position 20 (Kabat numbering). Sugar chains attached to proteins exhibit heterogeneity. Thus, it is desirable to eliminate glycosylation from the point of view of the homogeneity of the substance. In this context, a variant of hVk5-2_L65 (SEQ ID NO: 118) was constructed in which the Asn (N) residue at position 20 (Kabat numbering) was replaced by Thr (T). Amino acid substitution was performed in a manner known to those skilled in the art using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). DNA encoding the hVk5-2_L65 variant was inserted into an animal expression vector. An animal expression vector into which engineered DNA encoding the hVk5-2_L65 variant is inserted, in combination with an animal expression vector having an insert for expression of CIM_H (SEQ ID NO: 117) as a heavy chain, was inserted into animal cells by the method described in Reference Example 6. An antibody containing hVk5-2_L65 and CIM_H, which were expressed in animal cells into which the vectors were introduced, was purified by the method described in Reference Example 6.

(16-3) Evaluation of an antibody having a non-glycosylated hVk5-2 sequence for physical properties

The isolated antibody having the modified hVk5-2_L65 sequence was analyzed by ion exchange chromatography to examine whether it is less heterogeneous than the antibody having the original hVk5-2 sequence prior to modification. The ion exchange chromatography procedure is shown in Table 36. The result of the analysis showed that hVk5-2_L65 modified at the glycosylation site was less heterogeneous than the original hVk5-2 sequence, as shown in FIG. 46.

Further, evaluation of whether an antibody containing the less heterogeneous hVk5-2_L65 sequence binds to calcium ion was evaluated by the method described in Reference Example 15. The result showed that the Tm value for the Fab domain of an antibody having hVk5-2_L65 with an altered glycosylation site, also varied depending on the concentration of calcium ions in antibody solutions, as shown in Table 37. In particular, it was demonstrated that the Fab domain of an antibody having hVk5-2_L65 with an altered glycosylation site binds to a calcium ion.

[Reference Example 17] Evaluation of the calcium ion binding activity of antibody molecules having a CDR sequence of hVk5-2 sequence

(17-1) Construction, expression and purification of modified antibodies having a CDR sequence from the hVk5-2 sequence

The hVk5-2_L65 sequence is a sequence with altered amino acids in the glycosylation site of the framework region of the human Vk5-2 sequence. As described in Reference Example 16, it was demonstrated that calcium ion binding occurred even after the glycosylation site was changed. Meanwhile, from the viewpoint of immunogenicity, it is generally desirable that the framework sequence is a germline sequence. Thus, the inventors of the present invention evaluated whether the framework sequence of an antibody could be replaced with a framework sequence of a non-glycosylated germline sequence while maintaining the calcium ion binding activity of the antibody.

Polynucleotides encoding chemically synthesized sequences that contain an altered framework sequence from the sequence hVk5-2, hVk1, hVk2, hVk3 or hVk4 (CaVk1 (SEQ ID NO: 119), CaVk2 (SEQ ID NO: 120), CaVk3 (SEQ ID NO : 121) or CaVk4 (SEQ ID NO: 122), respectively) were linked by PCR to a polynucleotide encoding the constant region (SEQ ID NO: 100) of the native kappa chain. Linked DNA fragments were inserted into animal cell expression vectors. The sequences of the engineered variants were confirmed in a manner known to those skilled in the art. Each plasmid constructed as described above was inserted into animal cells in combination with a plasmid into which a polynucleotide encoding a CIM_H heavy chain (SEQ ID NO: 117) was inserted, by the method described in Example 6. Expressed antibody molecules of interest were purified from culture animal cell media into which the plasmids were introduced.

(17-2) Evaluation of altered antibodies having a CDR sequence from the hVk5-2 sequence for their calcium ion binding activity

The assessment of whether calcium ions bind to altered antibodies having a CDR sequence from the hVk5-2 sequence and framework sequences from germline sequences other than hVk5-2 (hVk1, hVk2, hVk3 and hVk4) was assessed using the method described in Example 6. The evaluation result is shown in Table 38. It was found that the value of the Tm Fab domain of each of the altered antibodies varied depending on the concentration of calcium ions in the antibody solutions. This demonstrates that antibodies having a different framework sequence from the framework sequences from the hVk5-2 sequence also bind to calcium ions.

It has been demonstrated that the thermal denaturation temperature (Tm value), as an indicator of the thermal stability of the Fab domain of each antibody modified to have a CDR sequence from the hVk5-2 sequence and a non-hVk5-2 (hVk1 , hVk2, hVk3, or hVk4) is higher than the thermal denaturation temperature of the Fab domain of the parent antibody having the hVk5-2 sequence. This result shows that antibodies having a CDR sequence from the hVk5-2 sequence and an hVk1, hVk2, hVk3 or hVk4 framework sequence not only have calcium ion binding activity, but are also excellent molecules in terms of thermal stability.

[Reference Example 18] Identification of the calcium ion binding site in the human germline hVk5-2 sequence

(18-1) Construction of a mutation site in the CDR sequence from the hVk5-2 sequence

As described in Reference Example 17, it was also shown that antibodies having a light chain obtained by introducing the CDR domain of the hVk5-2 sequence into the framework sequence of a different germline sequence bind to a calcium ion. This result indicates that in hVk5-2, the calcium ion binding site is located in its CDR. Amino acids that bind to the calcium ion, i.e. chelate the calcium ion, include negatively charged amino acids and amino acids that can be a hydrogen bond acceptor. Thus, it was investigated whether antibodies having a mutant hVk5-2 sequence with an Ala (A) substitution of an Asp (D) or Glu (E) residue in the CDR sequence of the hVk5-2 sequence bind to calcium ion.

(18-2) Construction of hVk5-2 variant sequences with Ala substitution, and antibody expression and purification

Antibody molecules were prepared to contain a light chain with an Asp and/or Glu residue replaced by an Ala residue in the CDR sequence from the hVk5-2 sequence. As described in Reference Example 16, the non-glycosylated hVk5-2_L65 variant exhibited calcium ion binding and was assumed to be equivalent to the hVk5-2 sequence in terms of calcium ion binding. In this example, amino acid substitutions were made in hVk5-2_L65 as a template sequence. The engineered variants are shown in Table 39. Amino acid substitutions were performed by methods known to those skilled in the art, such as using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, or the Infusion Advantage PCR Cloning Kit (TAKARA) for constructing expression vectors for altered light chains having an amino acid substitution.

The nucleotide sequences of the constructed expression vectors were verified in a manner known to those skilled in the art. Expression vectors designed for altered light chains were transiently introduced, in combination with the heavy chain expression vector CIM_H (SEQ ID NO: 117), into human embryonic kidney-derived HEK293H (Invitrogen) or FreeStyle293 (Invitrogen) cells for expression. antibodies. From the resulting culture supernatants, antibodies were purified using rProtein A SepharoseTM Fast Flow (GE Healthcare) in a manner known to those skilled in the art. The absorbance of purified antibody solutions at 280 nm was measured using a spectrophotometer. Antibody concentrations were calculated from the determined values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4: 2411-2423).

(18-3) Evaluation of antibodies having an Ala substitution in the hVk5-2 sequence for calcium ion binding activity

A study was made whether the obtained purified antibodies bind to calcium ions by the method described in Reference Example 15. The result is shown in Table 40. It was found that some antibodies having an Asp or Glu residue substitution in the CDR sequence from the hVk5-2 sequence with an Ala residue , which cannot be involved in the binding or chelation of calcium ions, have a Fab domain whose Tm did not vary depending on the concentration of calcium ions in antibody solutions. It was shown that the substitution sites in which the change to Ala did not change Tm (positions 32 and 92 (Kabat numbering)) are significantly important for the binding of calcium ions by the antibody.

[Reference Example 19] Evaluation of antibodies having an hVk1 sequence with a calcium ion binding motif for calcium ion binding

(19-1) Construction of hVk1 sequence with calcium binding motif, and expression and purification of antibodies

The result described in Reference Example 18 for calcium binding activity in the case of Ala substitution demonstrates that Asp or Glu residues in the CDR sequence of the hVk5-2 sequence were important for calcium binding. Thus, the present inventors evaluated whether the antibody could bind to calcium ion when the residues at positions 30, 31, 32, 50 and 92 (Kabat numbering) were separately introduced into a different germline variable region sequence. Specifically, the LfVk1_Ca variant (SEQ ID NO: 131) was constructed by substituting residues at positions 30, 31, 32, 50 and 92 (Kabat numbering) in the hVk5-2 sequence for residues at positions 30, 31, 32, 50 and 92 ( Kabat numbering) hVk1 sequence (human germline sequence). In particular, it was investigated whether antibodies having the hVk1 sequence in which only 5 residues from the hVk5-2 sequence are inserted can bind calcium. Variants were generated in the same manner as described in Reference Example 17. The resulting LfVk1_Ca and LfVk1 light chain variants having the hVk1 light chain sequence (SEQ ID NO: 132) were co-expressed with the CIM_H heavy chain (SEQ ID NO: 117). Antibodies were expressed and purified in the same manner as described in Reference Example 18.

(19-2) Evaluation of antibodies having a human hVk1 sequence with a calcium ion binding motif for calcium ion binding activity

Whether the purified antibody prepared as described above binds calcium ions was evaluated by the method described in Reference Example 15. The results are shown in Table 41. The Tm value for the Fab domain of an antibody having LfVk1 with the hVk1 sequence did not vary, depending on on the calcium concentration in antibody solutions. Meanwhile, the Tm of an antibody having the LfVk1_Ca sequence shifted by 1° C. or more when the calcium concentration in the antibody solutions changed. Thus, an antibody having LfVk1_Ca was shown to bind to calcium. The results described above demonstrate that the complete hVk5-2 CDR sequence is not required, while the residues introduced to construct the LfVk1_Ca sequence alone are sufficient for calcium ion binding.

[Reference Example 20] Construction of an Antibody Molecule Population (Ca Library) with a Calcium Ion Binding Motif Embedded in the Variable Region to Efficiently Obtain Binding Antibodies That Bind to an Antigen in a Ca Concentration-Dependent Manner

Preferred calcium binding motifs include, for example, the hVk5-2 sequence and the CDR sequence, as well as residues at positions 30, 31, 32, 50 and 92 (Kabat numbering). Other calcium-binding motifs include the EF-hand motif, which is possessed by calcium-binding proteins (eg, calmodulin) and C-type lectin (eg, ASGPR).

The Ca-library consists of heavy and light chain variable regions. The human antibody sequence was used as the variable region and the calcium binding motif was introduced into the light chain variable region. The hVk1 sequence was chosen as the light chain variable region template for the insertion of the calcium binding motif. An antibody containing the LfVk1_Ca sequence obtained by inserting the hVk5-2 CDR sequence (one of the calcium-binding motifs) into the hVk1 sequence was shown to bind to calcium ions as shown in Reference Example 19. A plurality of amino acids were allowed to be in the template sequence, to diversify the antigen-binding molecules that make up the library. Positions exposed on the surface of the variable region that are likely to interact with the antigen were chosen as positions where a plurality of amino acids were allowed to be. In particular, positions 30, 31, 32, 34, 50, 53, 91, 92, 93, 94, and 96 (Kabat numbering) were chosen as nonstrict residues.

The type and frequency of occurrence of amino acid residues were determined and subsequently allowed to appear. The frequency of occurrence of amino acid residues in the non-strict residues of the hVk1 and hVk3 sequences registered in the Kabat database (KABAT, E.A. ET AL.: "Sequence of proteins of immunological interest", vol. 91, 1991, NIH PUBLICATION) was analyzed. Based on the results of the analysis, the type of amino acids that were allowed to appear in the Ca-library was selected from the amino acids with a higher frequency of occurrence at each position. At this time, amino acids for which it was determined that the frequency of their occurrence is low, based on the results of the analysis, were also selected to avoid deviation of the properties of the amino acids. The frequency of occurrence of the selected amino acids was determined by referring to the results of the analysis in the Kabat database.

A Ca library containing a calcium binding motif emphasizing sequence diversity such that it contains multiple amino acids at each non-motif residue was designed as a Ca library based on amino acids and frequency of occurrence as described above. Detailed designs of the Ca-library are presented in tables 1 and 2 (where the positions in each table correspond to the EU numbering). In addition, if position 92 corresponding to Kabat numbering is Asn (N) for amino acid frequencies as described in Tables 1 and 2, position 94 may be Leu (L) instead of Ser (S).

[Reference Example 21] Obtaining a Ca Library

The antibody heavy chain variable region gene library was amplified by PCR using poly-A RNA derived from human PBMC and commercial human poly-A RNA, etc. as a matrix. As described in Reference Example 20, for antibody light chain variable regions, light chain variable regions were designed that increase the incidence of antibodies that retain a calcium binding motif and can bind to an antigen in a calcium concentration dependent manner. In addition, for amino acid residues among non-strict residues other than residues with an introduced calcium-binding motif, a library of antibody light chain variable regions with uniformly distributed amino acids with a high frequency of occurrence in natural human antibodies was constructed, referring to the information on the frequency of occurrence of amino acids in natural antibodies human (KABAT, E.A. ET AL.: "Seqeuences of proteins of immunological interest", vol. 91, 1991, NIH PUBLICATION). The combination of antibody heavy chain and light chain variable region gene libraries prepared as described above was inserted into a phagemid vector to construct a human antibody phage display library that presents Fab domains composed of human antibody sequences (Methods Mol Biol. (2002) 178, 87-100). The library was constructed using the linker portion connecting the phagemid Fab to the phage pIII protein and the sequences of the phage display library with a trypsin cleavage sequence inserted between the N2 and CT domains of the helper phage pIII protein gene. The sequences of portions of antibody genes isolated from E. coli into which the antibody gene libraries were introduced were identified to obtain sequence information for 290 clones. The intended distribution of amino acids and the distribution of amino acids in the identified sequences is shown in FIG. 52. A library was constructed containing various sequences corresponding to the intended distribution of amino acids.

[Reference Example 22] Investigation of the calcium ion binding activity of the molecules contained in the Ca library

(22-1) Calcium ion binding activity of molecules contained in the Ca-library

As described in Reference Example 14, the hVk5-2 sequence that was shown to bind calcium ions is a sequence with a low frequency of occurrence in the germline sequence. Thus, it was considered insufficient to obtain a calcium binding antibody from an antibody library consisting of human germline sequences or from B cells obtained by immunizing a mouse expressing human antibodies. As a result, a Ca library was constructed according to Reference Example 21. The presence or absence of a clone showing calcium binding in the constructed Ca library was examined.

(22-2) Expression and purification of antibodies

Clones included in the Ca-library were inserted into expression plasmids of animal cells. Antibodies were expressed using the following method. Cells derived from Freestyle 293-F human embryonic kidney cells (Invitrogen) were suspended in Freestyle 293 Expression Medium (Invitrogen) and seeded at a cell density of 1.33×10 ⁶ cells/mL (3 mL) into each well of a plate with 6 cells. The resulting plasmids were introduced into cells by lipofection. Cells were cultured for four days in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm). From the culture supernatants obtained as described above, antibodies were purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) in a manner known to those skilled in the art. The absorbance of purified antibody solutions at 280 nm was measured using a spectrophotometer. Antibody concentrations were calculated from the determined values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4: 2411-2423).

(22-3) Evaluation of the obtained antibodies in relation to the binding of calcium ions

Whether the purified antibody prepared as described above binds to calcium ions was determined by the method described in Reference Example 6. The results are shown in Table 42. The Tm value for the Fab domains of the many antibodies contained in the Ca library varied depending on concentration of calcium ions, demonstrating the presence of molecules that bind calcium ions.

[Reference Example 23] Construction of an antibody library with pH-dependent binding

(23-1) Method for producing antibodies with pH-dependent binding

WO 2009/125825 describes an antibody with pH-dependent antigen binding, the properties of which change in the range of neutral and acidic pH values, due to the introduction of histidine into the antigen-binding molecule. The disclosed pH-dependent binding antibody is prepared by modifying to replace a portion of the amino acid sequence of the antigen-binding molecule of interest with histidine. To more efficiently produce an antibody with pH-dependent binding without first obtaining the antigen-binding molecule of interest to be modified, one way may be to obtain an antigen-binding molecule that binds to the desired antigen from a pool of antigen-binding molecules (referred to as a His-library) with histidine added into the variable region (more preferably, the region potentially involved in antigen binding). It may be possible to efficiently obtain an antigen-binding molecule having the desired properties from a His library, since histidine occurs more frequently in antigen-binding molecules from a His library than from conventional antibody libraries.

(23-2) Construction of a population of antibody molecules (His libraries) with a histidine residue introduced into their variable region to efficiently obtain binding antibodies that bind to the antigen in a pH-dependent manner

First, the positions for embedding histidine in the His library were chosen. WO 2009/125825 describes the production of antibodies with pH-dependent antigen binding by replacing amino acid residues in the sequences of antibodies against IL-6 receptor, IL-6 receptor and IL-31 receptor with histidine. In addition, anti-egg white lysozyme (FEBS Letter 11483, 309, 1, 85-88) and anti-hepcidin (WO 2009/139822) antibodies with pH-dependent antigen binding capability were obtained by replacing the antigen-binding molecule in the amino acid sequence with histidine. The positions where histidine residues were added to the anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-IL-31 receptor antibody, anti-egg white lysozyme antibody, and anti-hepcidin antibody are shown in Table 43. The positions shown in Table 43 can be be cited as candidate positions that control antigen-antibody binding. In addition, in addition to the positions presented in table 43, positions that are likely to come into contact with the antigen were also considered suitable for introducing histidine residues.

In a His library consisting of heavy chain and light chain variable regions, a human antibody sequence was used as the heavy chain variable region, and histidine residues were introduced into the light chain variable region. The positions above and the positions that may be involved in antigen binding, ie. positions 30, 32, 50, 53, 91, 92 and 93 (numbered by Kabat, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH) in the light chain were selected as positions for adding histidine residues to the His library. In addition, the Vk1 sequence was selected as a light chain variable region template sequence for the introduction of histidine residues. A variety of amino acids were allowed to appear in the template sequence to diversify the antigen-binding molecules that make up the library. Positions exposed on the surface of the variable region that are likely to interact with the antigen were selected as positions where multiple amino acids were allowed to appear. In particular, positions 30, 31, 32, 34, 50, 53, 91, 92, 93, 94 and 96 of the light chain were chosen as non-strict residues (Kabat numbering, Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest .NIH).

The type and frequency of occurrence of amino acid residues were determined and subsequently allowed to appear. The frequencies of amino acids in non-strict residues in the hVk1 and hVk3 sequences registered in the Kabat database (KABAT, E.A. ET AL.: "Sequences of proteins of immunological interest", vol. 91, 1991, NIH PUBLICATION) were analyzed. Based on the results of the analysis, the type of amino acids allowed to appear in the His library was selected from the amino acids with a higher frequency of occurrence at each position. At the same time, amino acids for which the frequency of their occurrence was determined to be low based on the results of the analysis were also selected to avoid bias in the properties of the amino acids. The frequency of occurrence of the selected amino acids was determined by referring to the results of the analysis in the Kabat database.

As His libraries, His library 1 was constructed, which is fixed to necessarily include one histidine residue in each CDR, and His library 2, which is more focused on sequence diversity than His library 1, considering amino acids and frequency of occurrence, as described above. Detailed constructions of His libraries 1 and 2 are presented in Tables 3 and 4 (where the positions in each table corresponded to the Kabat numbering). Ser (S) at position 94 can be omitted if Asn (N) is at position 92, corresponding to Kabat numbering, for the amino acid frequency as described in Tables 3 and 4.

[Reference Example 24] Preparation of a phage display library for human antibodies (His library 1) to obtain an antibody that binds to an antigen in a pH-dependent manner.

The antibody heavy chain variable region gene library was amplified by PCR using poly-A RNA derived from human PBMC and commercial human poly-A RNA as a template. An antibody light chain variable gene library constructed as His Library 1 as described in Example 1 was amplified using PCR. The combination of the antibody heavy chain and light chain variable region gene libraries prepared as described above was inserted into a phagemid vector to construct a human antibody phage display library that presents Fab domains composed of human antibody sequences. For the construction method, (Methods Mol Biol. (2002) 178, 87-100) were used as references. The library was constructed using the linker portion connecting the phagemid Fab to the phage pIII protein and the sequences of the phage display library with a trypsin cleavage sequence inserted between the N2 and CT domains of the helper phage pIII protein gene. The sequences of portions of antibody genes isolated from E. coli into which the antibody gene libraries were introduced were identified and sequence information was obtained for 132 clones. The intended distribution of amino acids and the distribution of amino acids in the identified sequences is shown in FIG. 53. A library was constructed containing various sequences corresponding to the intended distribution of amino acids.

[Reference Example 25] Preparation of a Human Antibody Phage Display Library (His Library 2) for Obtaining Antibodies That Bind to Antigen in a pH-Dependent Manner

The antibody heavy chain variable region gene library was amplified by PCR using poly-A RNA derived from human PBMC and commercial human poly-A RNA as a template. As described in Reference Example 23, light chain portions from the variable regions of an antibody were constructed into light chain portions with an increased frequency of occurrence of histidine residues having a high potential to be an antigen contact region to increase the frequency of occurrence of antibodies having pH-dependent capability. antigen binding. In addition, for amino acid residues other than those in which histidine residues are introduced among the non-strict residues, a library of antibody light chain variable regions is constructed with uniformly distributed amino acids with a high frequency of occurrence, identified using information about the frequency of occurrence of amino acids in natural human antibodies. A library of antibody light chain variable region genes constructed as described above was synthesized. The library can be commercially synthesized on consignment terms. The combination of antibody heavy chain and light chain variable region gene libraries prepared as described above was inserted into a phagemid vector to construct a human antibody phage display library that presents Fab domains composed of human antibody sequences in a known manner (Methods Mol Biol. (2002 ) 178, 87-100). The antibody gene portion isolated from E. coli with the introduced antibody gene library was sequenced as described in Reference Example 24.

[Reference Example 26] Effects of Combining FcγRIIb Selective Binding Modification with Other Fc Region Amino Acid Substitutions

An attempt was made to further enhance the selectivity for FcγRIIb by modifying the variant by replacing Pro at position 238 (EU numbering) with Asp, which increases the selectivity for FcγRIIb as found in Example 14.

First, with regard to IL6R-G1d-v1 (SEQ ID NO: 80), which was obtained by making the modification of replacing Pro at position 238 (EU numbering) of IL6R-G1d with Asp, a variant of IL6R-G1d-v4 (SEQ ID NO: 172) in which Leu at position 328 (EU numbering) was changed to Glu to increase selectivity for FcγRIIb as described in Example 14. IL6R-G1d-v4 expressed in combination with IL6R-L (SEQ ID NO: 83), which was used as the L chain, was obtained as described in Reference Example 2. An antibody having an amino acid sequence derived from IL6R-G1d-v4 as the H chain of the antibody obtained in this example is described as IgG1-v4 . The binding activity of IgG1, IgG1-v1, IgG1-v2 and IgG1-v4 to FcγRIIb, assayed as described in Example 14, is shown in Table 44. The modifications in the table correspond to the modifications made to IL6R-G1d.

From the results presented in Table 44, it was expected that since L328E increased FcγRIIb binding activity by 2.3 times compared to IgG1, then combining this substitution with P238D, which similarly increased FcγRIIb binding activity by 4.8 times compared to IgG1 , further increase the degree of increase in FcγRIIb binding activity; however, in reality, the FcγRIIb binding activity of the variant containing the combination of these changes was reduced by 0.47-fold compared to IgG1. This result is an effect that could not be predicted from the respective changes.

Similarly, in IL6R-G1d-v1 (SEQ ID NO: 80), obtained by amending IL6R-G1d by replacing Pro at position 238 (listed according to EU numbering) with Asp, Ser was introduced at position 267 (shown according to EU numbering). ) on Glu and Leu at position 328 (listed according to EU numbering) on Phe as described in Example 2, which increase FcγRIIb binding activity, and IL6R-G1d-v5 variant (SEQ ID NO: 173) was prepared according to the method of Reference Example 2. The resulting antibody having an amino acid sequence derived from IL6R-G1d-v5 as the H chain of the antibody was named IgG1-v5. FcγRIIb binding activity on IgG1, IgG1-v1, IgG1-v3, and IgG1-v5, as assessed according to the method of Example 14, is shown in Table 45.

S267E/L328F, which is the FcγRIIb enhancing modification in Example 14, was added to the P238D variant. Changes in FcγRIIb binding activity before and after making this change are shown in Table 45.

From the results presented in Table 45, it was expected that since S267E/L328F increased FcγRIIb binding activity by 408 times over IgG1, then combining these substitutions with P238D, which similarly increased FcγRIIb binding activity by 4.8 times over IgG1, further increase the degree of increase in FcγRIIb binding activity; however, in reality, similar to the previous example, the FcγRIIb binding activity of the variant containing the combination of these changes was increased only 12-fold or so compared to IgG1. This result also represents an effect that could not be predicted from the effects of the corresponding changes.

These results indicated that although the change from Pro at position 238 (indicated by EU numbering) to Asp alone increased FcγRIIb binding activity, there was no effect when it was combined with other changes that increased FcγRIIb binding activity. The reason for this may be that the structure at the interaction surface between Fc and FcγR is changed when Pro is changed at position 238 (indicated by EU numbering) to Asp, and the effects of the changes observed in the naturally occurring antibody are no longer reflected in the results. . Thus, it was considered extremely difficult to generate an Fc with excellent selectivity for FcγRIIb using an Fc containing substitutions of Pro at position 238 (listed according to EU numbering) with Asp as a template, since information regarding the effects of changes obtained in the case of occurring in the nature of antibodies, information about the effects of changes obtained in the case of naturally occurring antibodies could not be used.

[Reference Example 27] Detailed analysis of FcγRIIb binding for variants with a hinge change in addition to the P238D change

As shown in Reference Example 26, in Fc obtained by replacing Pro at position 238 (indicated by EU numbering) with Asp in naturally occurring human IgG1, the expected combinatorial effect could not be achieved even by combining it with another change predicted for that it further enhances FcγRIIb binding as measured by naturally occurring antibodies. Thus, in order to find variants that further enhance FcγRIIb binding, extensive modifications were made to the altered Fc obtained by replacing Pro at position 238 (indicated by EU numbering) with Asp. IL6R-F11 (SEQ ID NO: 174) was made by changing the Met at position 252 (as indicated by EU numbering) to Tyr and by changing Asn at position 434 (as indicated by EU numbering) to Tyr in IL6R-G1d ( SEQ ID NO: 20), which was used as the H chain of the antibody. Moreover, IL6R-F652 (SEQ ID NO: 175) was made by changing the Pro at position 238 (listed according to EU numbering) to Asp in IL6R-F11. Expression plasmids containing the antibody H chain sequence were generated for each of the antibody H chain sequences obtained by replacing the region near the residue at position 238 (listed according to EU numbering) (positions 234 to 237, and 239 (shown according to EU numbering) ) in IL6R-F652, in each case by 18 amino acids, excluding the original amino acids and Cys. For all antibodies, IL6R-L (SEQ ID NO: 83) was used as the total antibody L chain. These variants were expressed and purified by the method of Reference Example 2. These Fc variants are referred to as PD variants. The interactions of each PD variant with FcγRIIa type R and FcγRIIb 5 were evaluated in detail by the method of Example 14.

A graph showing the results of the analysis of interaction with the corresponding FcγR was built in accordance with the following method. The value obtained by dividing the binding amount of each PD variant to each FcγR by the FcγR binding amount of the pre-modified antibody that was used as control (IL6R-F652/IL6R-L, which has the Pro substitution change at position 238 (indicated by numbering EU) by Asp and then multiplying the result by 100 is shown as the relative binding activity of each PD variant to each FcγR The horizontal axis shows the relative values of the FcγRIIb binding activity for each PD variant and the vertical axis shows the relative values of the FcγRIIa binding activity type R for each variant PD (Fig. 55).

As a result, it was found that the binding of FcγRIIb in the case of eleven types of changes was increased compared to the antibody before the changes were made, and they have the effects of maintaining or enhancing the binding of FcγRIIa type R. The activity of these eleven variants in relation to binding of FcγRIIb and FcγRIIa type R is summarized in table 46. In the table, SEQ ID NO refers to SEQ ID NO of the H chain of the variant being evaluated, and the change refers to the change made to IL6R-F11 (SEQ ID NO: 174).

In FIG. 56 shows the relative values for the FcγRIIb binding activity obtained by further making the above eleven changes to the variant having the P238D change, and the relative values for the FcγRIIb binding activity of the variant obtained by making these changes to the Fc that does not contain P238D. These eleven changes enhanced FcγRIIb binding compared to before they were added to the P238D variant. On the contrary, the effect of reducing FcγRIIb binding was observed for eight of these changes, with the exception of G237F, G237W and S239D, when they were introduced into the variant that does not contain P238D (GpH7-B3/GpL16-k0) used in example 14. Reference example 26 and these results showed that, based on the effects of making changes to naturally occurring IgG1, it is difficult to predict the effects of combining and making the same changes to a variant containing the P238D change. In other words, it is not possible to identify the eight changes identified this time without this study, in which these changes are combined and made into a variant containing the P238D change.

The results of measuring the KD values of the variants listed in Table 46 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIaV by the method of Example 14 are summarized in Table 47. In the table, SEQ ID NO refers to SEQ ID NO of the H chain of the evaluated variant and change refers to change listed in IL6R-F11 (SEQ ID NO: 174). The template used to obtain IL6R-F11, IL6R-G1d/IL6R-L, is indicated by an asterisk (*). Moreover, KD(IIaR)/KD(IIb) and KD(IIaH)/KD(IIb) in the table, respectively, show the value obtained by dividing the KD value of each FcγRIIaR option by the KD value of each FcγRIIb option, and the value obtained by dividing the KD value of each FcγRIIaH variant by the KD value of each FcγRIIb variant. The KD(IIb) of the original polypeptide/KD(IIb) of the modified polypeptide refers to the value obtained by dividing the KD value of the original polypeptide in relation to FcγRIIb by the KD value of each FcγRIIb variant. In addition, Table 47 shows KD values for stronger FcγRIIaR and FcγRIIaH binding activity of each variant/KD values for stronger FcγRIIaR and FcγRIIaH binding activity of the parent polypeptide. In this case, the parent polypeptide refers to the variant that has IL6R-F11 (SEQ ID NO: 27) as the H chain. It was determined that, due to the weak binding of FcγR to IgG, it could not be accurately analyzed using kinetic analysis, and thus the grayed cells in Table 47 show values calculated using Equation 5 of Example 14.

[Equation 5]

KD=CxRmax/(Req-RI)-C

Table 47 shows that for all variants the binding affinity for FcγRIIb was increased compared to IL6R-F11 and the improvement ranged from 1.9-fold to 5.0-fold. The ratio of FcγRIIaR KD value of each variant/FcγRIIb KD value of each variant, and the FcγRIIaH KD value of each variant/FcγRIIb KD value of each variant correspond to FcγRIIb binding activity relative to FcγRIIaR binding activity and FcγRIIaH binding activity, respectively. Therefore, these values indicate the degree of binding selectivity of each variant for FcγRIIb, and a higher value indicates a higher binding selectivity for FcγRIIb. For the parent IL6R-F11/IL6R-L polypeptide, the ratio of FcγRIIaR KD value/FcγRIIb KD value and the FcγRIIaH KD value/FcγRIIb KD value ratio is 0.7 in both cases, and, accordingly, all variants presented in the table 47 demonstrated an increase in binding selectivity for FcγRIIb over the parent polypeptide. When the ratio of KD value for the stronger of the FcγRIIaR and FcγRIIaH variant binding activities/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide is 1 or more, it means that the stronger of the FcγRIIaR and FcγRIIaH binding activities of the variant has an equivalent or reduced binding versus binding at the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Since this value ranged from 0.7 to 5.0 for the variants obtained this time, it can be said that the binding at the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variants obtained this time was substantially the same or reduced compared to original polypeptide. These results showed that, compared to the parent polypeptide, the variants obtained in this case have retained or reduced binding activity for FcγRIIa type R and type H, and increased selectivity for FcγRIIb. Moreover, compared to IL6R-F11, all variants had lower affinity for FcγRIa and FcγRIIIaV.

[Reference Example 28] X-ray diffraction analysis of the complex formed between Fc containing P238D and the extracellular region of FcγRIIb

As shown earlier in Reference Example 27, even though a change is made that is predicted from analysis of naturally occurring IgG1 antibodies to increase FcγRIIb binding activity or FcγRIIb selectivity, in Fc containing P238D, it was found that the binding activity of FcγRIIb is reduced, and the reason for this may be that the structure on the interaction surface between Fc and FcγRIIb is changed due to the introduction of P238D. Thus, in order to establish the cause of this phenomenon, the three-dimensional structure of the complex formed between Fc IgG1 containing the P238D mutation (hereinafter, Fc(P238D)) and the extracellular region of FcγRIIb by X-ray crystallography was determined and compared with the three-dimensional structure of the complex formed between Fc naturally occurring IgG1 (hereinafter Fc(WT)) and the extracellular region of FcγRIIb, and compared the methods of binding. Many reports have been made about the three-dimensional structure of the complex formed between Fc and the extracellular region of FcγR; and analyzed the three-dimensional structures of the Fc(WT) complex/FcγRIIIb extracellular region (Nature, 2000, 400: 267-273; J. Biol. Chem. 2011, 276: 16469-16477), the Fc(WT) complex/FcγRIIIa extracellular region ( Proc. Natl. Acad. Sci. USA, 2011, 108: 12669-126674), and Fc(WT)/FcγRIIa extracellular region complex (J. Imunol. 2011, 187: 3208-3217). Although the three-dimensional structure of the Fc(WT)/FcγRIIb extracellular region has not been analyzed, the three-dimensional structure of the complex formed with Fc(WT) is known for FcγRIIa, and the extracellular regions of FcγRIIa and FcγRIIb share 93% amino acid sequence and have very high homology. Thus, the three-dimensional structure of Fc(WT)/FcγRIIb extracellular region was predicted by modeling using the crystal structure of the Fc(WT)/FcγRIIa extracellular region.

Структура, полученная в результате этого анализа, представлена на фиг. 57. Внеклеточная область FcγRIIb связывается между двумя СН2-доменами Fc, и это было аналогично трехмерным структурам комплексов, образованных между Fc(WT) и соответствующей внеклеточной областью FcγRIIIa, FcγRIIIb или FcγRIIa, проанализированных до настоящего времени.The three-dimensional structure of the Fc(P238D)/FcγRIIb extracellular region was determined by X-ray crystallography at a resolution of 2.6

The structure resulting from this analysis is shown in FIG. 57. The extracellular region of FcγRIIb binds between two Fc CH2 domains and this was similar to the three-dimensional structures of complexes formed between Fc(WT) and the corresponding extracellular region of FcγRIIIa, FcγRIIIb or FcγRIIa analyzed to date.

или менее между отобранной внеклеточной областью FcγRIIb и СН2-доменом В Fc и сравнивали для сравнения межатомного взаимодействия между FcγRIIb и СН2-доменом В в Fc(WT) с межатомным взаимодейсттвием между FcγRIIb и Fc(P238D). Как показано в таблице 48, межатомные взаимодействия между СН2-доменом В Fc и FcγRIIb в Fc(P238D) и Fc(WT) не совпадали.Further, for detailed comparison, the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the structure of the Fc(WT) complex/FcγRIIb extracellular region were superimposed on each other using a least squares approximation based on the distances between pairs of Cα atoms in relation to the extracellular region of FcγRIIb and CH2 -domain A Fc (Fig. 58). In this case, the degree of overlap between the CH2 domains of B Fc was not satisfactory, and conformational differences were found in this part. Moreover, using the crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region and the model structure of the Fc(WT) complex/FcγRIIb extracellular region, pairs of atoms were selected that have a distance of 3.7

or less between a selected extracellular region of FcγRIIb and the CH2 domain B of Fc and compared to compare the interatomic interaction between FcγRIIb and the CH2 domain B in Fc(WT) with the interatomic interaction between FcγRIIb and Fc(P238D). As shown in Table 48, the interatomic interactions between the CH2 domain of Fc and FcγRIIb in Fc(P238D) and Fc(WT) did not match.

Moreover, detailed structures in the P238D region were compared by overlaying the X-ray crystal structure of the Fc(P238D) complex/FcγRIIb extracellular domain with the model structure of the Fc(WT) complex/FcγRIIb extracellular domain using the least squares method based on the Cα atomic distance between the CH2 domains of A and B Fc separately. Since the position of the amino acid residue at position 238 (EU numbering), i.e. the mutagenesis position of Fc(P238D) is changed relative to Fc(WT), it was found that the structure of the loop in the amino acid residue region at position 238 after the hinge region differs between Fc(P238D) and Fc(WT) (Fig. 59). Pro at position 238 (EU numbering) is initially located within Fc(WT), form a hydrophobic core with residues at position 238. However, if Pro at position 238 (EU numbering) is changed to a highly hydrophilic and charged Asp, the presence of an altered Asp residue in the hydrophobic core is energetically advantageous in terms of desolvation. Thus, in Fc(P238D), to eliminate this energetically disadvantageous situation, the amino acid residue at position 238 (listed according to EU numbering) changes its orientation towards the solvent, and this can cause this change in the structure of the loop near the amino acid residue at position 238. More Moreover, since this loop is located close to the S-S-linked hinge region, its structural change will not be limited to local change, and will affect the relative positioning of the A CH2 domain and the Fc B domain. As a result, the interatomic interactions between FcγRIIb and the CH2 domain of Fc are changed. Thus, the predicted effects could not be observed when changes that increase the selectivity and binding activity for FcγRIIb in naturally occurring IgG were combined with an Fc containing the P238D change.

Moreover, as a result of structural changes due to the introduction of P238D into the CH2 domain of Fc A, a hydrogen bond was detected between the Gly backbone at position 237 (listed according to EU numbering), which is adjacent to the mutant P238D and Tyr at position 160 in FcγRIIb (Fig. .60). The residue in FcγRIIa that corresponds to this Tyr160 is Phe; and when binding to FcγRIIa occurs, this hydrogen bond is not formed. Considering that the amino acid at position 160 is one of a small number of differences between FcγRIIa and FcγRIIb at the interface with Fc, and the presence of this hydrogen bond, which is specific to FcγRIIb, presumably resulted in an increase in FcγRIIb binding activity and a decrease in FcγRIIa binding activity in Fc (P238D) and increase its selectivity.

Moreover, in the CH2 domain of B Fc, an electrostatic interaction was observed between Asp at position 270 (listed according to EU numbering) and Arg at position 131 in FcγRIIb (FIG. 61). In FcγRIIa type H, which is one of the allotypes of FcγRIIa, the residue corresponding to Arg at position 131 of FcγRIIb is His, and thus cannot form this electrostatic interaction. This may explain why Fc(P238D) binding activity is reduced in type H FcγRIIa compared to type R FcγRIIa. domain arrangement causes the formation of new interactions that do not occur with the binding of naturally occurring IgG and FcγR, and this could lead to a selective binding profile of P238D variants in relation to FcγRIIb.

[Expression and purification of Fc(P238D)]

Fc containing the change P238D was obtained as follows. First, the Cys at position 220 (listed according to EU numbering) of hIL6R-IgG1-v1 (SEQ ID NO: 80) was changed to Ser. Then, the Fc(P238D) genetic sequence from Glu at position 236 (indicated according to EU numbering) to its C-terminus was cloned by PCR. Using this cloned genetic sequence, production of expression vectors and expression and purification of Fc(P238D) was carried out according to the method of Reference Examples 1 and 2. The Cys at position 220 (listed according to EU numbering) forms a disulfide bond with the L chain Cys in total IgG1. The L chain is not co-expressed when Fc is prepared alone, and thus the Cys residue was changed to Ser to avoid the formation of unnecessary disulfide bonds.

[Expression and purification of the extracellular region of FcγRIIb]

The extracellular region of FcγRIIb was obtained in accordance with the method of example 14.

[Sedum complex Fc(P238D)/extracellular region FcγRIIb]

To 2 mg of the FcγRIIb extracellular region sample prepared for use in crystallization was added 0.29 mg of Endo F1 (Protein Science 1996, 5: 2617-2622) expressed and purified from Escherichia coli as a glutathione-S-transferase fusion protein. The mixture was allowed to stand at room temperature for three days in 0.1 M Bis-Tris buffer at 6.5 and the N-linked oligosaccharide was cleaved off except for the N-acetylglucosamine directly bound to the Asn of the extracellular region of FcγRIIb. Next, a sample of the extracellular domain of FcγRIIb subjected to carbohydrate cleavage treatment, which was concentrated by ultrafiltration with 5000 MWCO, was purified by gel filtration (Superdex200 10/300) using a column equilibrated in 20 mM HEPS at pH 7.5 containing 0.05 M NaCl. Moreover, Fc(P238D) was added to the fraction of the FcγRIIb extracellular region obtained after carbohydrate cleavage, so that the FcγRIIb extracellular region was present in a molar ratio in a slight excess. The mixture, concentrated by ultrafiltration with 10,000 MWCO, was subjected to purification by gel filtration (Superdex200 10/300) using a column equilibrated in 20 mm HEPS at pH 7.5 containing 0.05 M NaCl. Thus, a sample of the Fc(P238D)/FcγRIIb extracellular region complex was obtained.

[Crystallization of the Fc(P238D) complex/extracellular region of FcγRIIb]

Using a sample of the Fc(P238D)/FcγRIIb extracellular region complex, which was concentrated to approximately 10 mg/ml by ultrafiltration with 10,000 MWCO, the complex was crystallized by the sessile drop vapor diffusion method. Hydra II Plus One (MATRIX) was used for crystallization; and as a reservoir solution containing 100 mM Bis-Tris pH 6.5, 17% PEG3350, 0.2 M ammonium acetate, and 2.7% (w/v) D-galactose, a crystallization drop was obtained by mixing the ratio reservoir solution : crystallization sample = 0.2 µl:0.2 µl. The vessel was closed and the crystals were allowed to stand at 20° C., and thus flat crystals were obtained.

[Measurement of X-ray diffraction data in the crystal of the Fc(P238D) complex/extracellular region of FcγRIIb]

. Кристалл относится к пространственной группе P2₁, и имеет следующие параметры ячейки; а=48,85

, b=76,01

, с=115,09

, α=90°, β=100,70°, γ=90°.One of the obtained single crystals of the Fc(P238D)/FcγRIIb extracellular region was immersed in a solution of 100 mM Bis-Tris pH 6.5, 20% PEG3350, ammonium acetate, 2.7% (w/v) D-galactose, 22, 5% (v/v) ethylene glycol. The single crystal was fished out of solution using a pin with a thin nylon loop attached and frozen in liquid nitrogen. Crystal X-ray diffraction data were measured on a Photon Factory BL-1A bremsstrahlung equipment at the High Energy Accelerator Research Organization. During the measurement, the crystal was constantly placed in a flow of nitrogen at -178°C to maintain it frozen, and a total of 225 X-rays were obtained using a Quantum 270 CCD (ADSC) detector connected to the beam line, with a crystal rotation of 0.8° per one once. Determination of cell parameters, indexing of diffraction spots, and processing of diffraction data from the obtained X-ray patterns were performed using the Xia2 program (ССР4 Software Suite), XDS Package (Walfgang Kabsch) and Scala (CCP4 Software Suite); and finally obtained data on the intensity of the diffraction of the crystal at resolutions up to 2.46

. The crystal belongs to the space group P2 ₁ and has the following cell parameters; a=48.85

, b=76.01

, s=115.09

, α=90°, β=100.70°, γ=90°.

[X-ray crystallographic analysis of the Fc(P238D) complex/extracellular region of FcγRIIb]

до 3,0

в этот момент времени. Более того, уточнение структуры с использованием программы Refmac5 (ССР4 Software Suite), и пересмотр модели для выявления карт электронной плотности, коэффициент которых имеет 2Fo-Fc или Fo-Fc, которые вычисляют, исходя из экспериментально определенного структурного фактора Fo, вычисленного структурного фактора Fc и вычисленной фазы с использованием модели, проводили с помощью программы Coot (Paul Emsley). Уточнение модели проводили путем повторения этих стадий. Наконец, в результате включения молекул воды в модель на основе карт электронной плотности, в которых используются 2Fo-Fc или Fo-Fc в качестве коэффициента, и последующего уточнения, кристаллографический фактор достоверности, R-величина, и свободная R-величина модели, содержащей 4846 не являющихся водородом атомами, становились равными 23,7% и 27,6% для 24291 данных интенсивности дифракции при разрешении от 25

до 2,6

, соответственно.The crystal structure of the Fc(P238D) complex/FcγRIIb extracellular region was determined by the molecular displacement method using the Phaser program (CCP4 Software Suite). From the resulting crystal lattice size and the molecular weight of the Fc(P238D)/FcγRIIb extracellular region, it was predicted that the number of complexes in the asymmetric element was one. From the structural coordinates of the PDB code: 3SGJ, which is the crystal structure of the Fc(WT) complex/FcγRIIIa extracellular region, the amino acid residue regions of the A chain at positions 239-340 and the B chain at positions 239-340 were taken as separate coordinates, and they were adopted, respectively, as models for the search for Fc CH2 domains. The amino acid residue regions of the A chain at positions 341-444 and the B chain at positions 341-443 were taken as a single set of coordinates from the same structural coordinates of the PDB code: 3SGJ; and they were adopted as a model for searching for Fc CH3 domains. Finally, from the structural coordinates of the PDB code: 2FCB, which corresponds to the crystal structure of the extracellular region of FcγRIIb, the regions of the A chain amino acid residues at positions 6-178 were chosen and adopted as a model for searching for the extracellular region of FcγRIIb. The orientation and position of each search pattern in the crystal lattice was determined in the order of Fc CH3-domain, FcγRIIb extracellular region, and Fc CH2-domain based on rotation function and translation function to obtain an initial crystal structure model of the Fc(P238D)/FcγRIIb extracellular region. When the obtained initial model was refined into a rigid body that moves two Fc CH2 domains, two Fc CH3 domains, and an FcγRIIb extracellular region, the crystallographic confidence factor, R-value, became 40.4%, and the free R-value became equal to 41.9% for diffraction intensity data from 25

up to 3.0

at this point in time. Moreover, structure refinement using the Refmac5 program (CCP4 Software Suite), and revision of the model to identify electron density maps whose coefficient has 2Fo-Fc or Fo-Fc, which are calculated from the experimentally determined structure factor Fo, the calculated structure factor Fc and calculated phase using the model were performed using the Coot program (Paul Emsley). The model was refined by repeating these steps. Finally, as a result of including water molecules in the electron density map model using 2Fo-Fc or Fo-Fc as a coefficient and then refining, the crystallographic confidence factor, R-value, and free R-value of the model containing 4846 non-hydrogen atoms became 23.7% and 27.6% for 24291 diffraction intensity data at a resolution of 25

up to 2.6

, respectively.

[Obtaining a model structure of the Fc(WT) complex/extracellular region of FcγRIIb]

Based on the structural coordinates of the PDB code: 3RY6, which corresponds to the crystal structure of the Fc(WT)/FcγRIIa extracellular region complex, the Build Mutants function of Discovery Studio 3.1 (Accelrys) was used to introduce mutations to match the FcγRIIb amino acid sequence in FcγRIIa in these structural coordinates. In this case, the optimization level was set to high, the cutoff radius was set to 4.5, five models were obtained, and the model with the best energy estimate among them was set as the model structure for the Fc(WT)/FcγRIIb extracellular region complex.

[Reference Example 29] FcγR binding analysis of Fc variants in which sites of change were determined based on crystal structures.

Based on the results of X-ray diffraction analysis of the complex formed between Fc(P238D) and the extracellular region of FcγRIIb obtained in Reference Example 28, variants were constructed by making complex changes to the regions of the altered Fc having the replacement of Pro at position 238 (indicated by EU numbering) with Asp, which were predicted to affect interaction with FcγRIIb (residues of positions 233, 240, 241, 263, 265, 266, 267, 268, 271, 273, 295, 296, 298, 300, 323, 325, 326, 327, 328, 330, 332 and 334 (listed according to EU numbering)), and investigated whether combinations of changes could be obtained that further enhance FcγRIIb binding, in addition to the P238D change.

IL6R-B3 (SEQ ID NO: 187) was obtained by introducing into IL6R-G1d (SEQ ID NO: 79), obtained according to example 14, a change obtained by replacing Lys at position 439 (indicated according to EU numbering) with Glu. Next, IL6R-BF648 was obtained by introducing into IL6R-B3 a substitution obtained by replacing Pro at position 238 (indicated by EU numbering) with Asp. IL6R-L (SEQ ID NO: 83) was used as the total L chain of the antibody. These expressed antibody variants were purified according to the method of Reference Example 2. The binding of each of these antibody variants to each of the FcγRs (FcγRIa, FcγRIIa type H, FcγRIIa type R, FcγRIIb and FcyRIIIa type V) was assessed in detail by the method of Example 14.

In accordance with the following method, a graph was built to show the results of the analysis of interactions with the corresponding FcγR. The binding value of each variant to each FcγR was divided by the binding value of the pre-modified control antibody (IL6R-BF648/IL6R-L, modified by replacing Pro at position 238 (indicated by EU numbering) with Asp) to each FcγR, and then the resulting value was multiplied by 100 and is shown as a measure of the relative binding activity of each variant to each FcγR. The horizontal axis shows the relative binding activity of each variant to FcγRIIb, and the vertical axis shows the relative binding activity of each variant to type R FcγRIIa (FIG. 62).

As shown in FIG. 62, the results show that of all changes found, 24 types of changes retain or enhance FcγRIIb binding compared to the pre-modified antibody. The association of these variants with each of the FcγRs is shown in Table 49. In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 187). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*).

The results of measuring the KD values of the variants shown in Table 49 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb, and FcγRIIIa type V using the method of Example 14 are summarized in Table 50. In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 187). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*). Moreover, KD(IIaR)/KD(IIb) and KD(IIaH)/KD(IIb) in the table, respectively, correspond to the value obtained by dividing the KD value of each FcγRIIaR option by the KD value of each FcγRIIb option, and the value obtained by dividing the KD value of each FcγRIIaH variant by the KD value of each FcγRIIb variant. The KD(IIb) of a parent polypeptide/KD(IIb) of an altered polypeptide refers to the value obtained by dividing the KD value of the parent polypeptide for FcγRIIb by the KD value of each FcγRIIb variant. In addition, the KD value for the stronger of the FcγRIIaR and FcγRIIaFI binding activities of each variant/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide are shown in Table 50. Here, the parent polypeptide refers to the variant that has IL6R-B3 (SEQ ID NO: 187) as the H chain. It was determined that, due to the weak binding of FcγR to IgG, it could not be accurately analyzed by kinetic analysis, and thus the grayed boxes in Table 50 show values calculated using Equation 5 of Example 14.

[Equation 5]

KD=CxRmax/(Req-RI)-C

Table 50 shows that, compared to IL6R-B3, all variants showed an improvement in affinity for FcγRIIb, and the improvement ranged from 2.1-fold to 9.7-fold. The ratio of KD value of each FcγRIIaR variant/KD value of each FcγRIIb variant, and the ratio of KD value of each FcγRIIaH variant/KD value of each FcγRIIb variant corresponds to FcγRIIb binding activity relative to FcγRIIaR binding activity and FcγRIIaH binding activity, respectively. Therefore, these values indicate the degree of binding selectivity of each variant to FcγRIIb, and a higher value indicates higher binding selectivity to FcγRIIb. Because the ratio of FcγRIIaR KD value/FcγRIIb KD value and the FcγRIIaH KD value/FcγRIIb KD value ratio in the parent IL6R-B3/IL6R-L polypeptide were 0.3 and 0.2, respectively, all variants in Table 50 showed improved binding selectivity for FcγRIIb over the parent polypeptide. When the ratio of the KD value for the stronger of the FcγRIIaR and FcγRIIaH variant binding activities/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide is 1 or more, this means that the stronger of the FcγRIIaR and FcγRIIaH binding activities of the variant has an equivalent or reduced binding versus binding at the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Since this value ranged from 4.6 to 34.0 for the variants obtained this time, it can be said that compared to the original polypeptide, the variants obtained this time had reduced binding with the stronger of the binding activities of FcγRIIaR and FcγRIIaH. These results indicated that, compared to the parent polypeptide, the variants generated this time have retained or reduced type R and type H FcγRIIa binding activity, enhanced FcγRIIb binding activity, and increased FcγRIIb selectivity. Moreover, compared to IL6R-B3, all variants had lower affinity for FcγRIa and FcγRIIIaV.

As for the promising options among the resulting combined options, the factors that ensure their effects are

investigated using the crystal structure. In FIG. 63 shows the crystal structure of the Fc(P238D)/FcγRIIb extracellular region complex. In this figure, the H chain on the left is the Fc A chain and the H chain on the right is the Fc B chain. Here it can be seen that the site at position 233 (indicated according to EU numbering) in the A chain of Fc, is located near Lys at position 113 of FcγRIIb. However, in this crystal structure, the E233 side chain is in a highly mobile state, and its electron density is not clearly visible. Thus, the change obtained by replacing Glu at position 233 (indicated according to EU numbering) with Asp results in a decrease in the degree of freedom of the side chain, since the side chain becomes one carbon atom shorter. As a result, the loss of entropy when forming an interaction with Lys at position 113 of FcγRIIb can be reduced, and therefore, presumably, this leads to an increase in the binding free energy.

Similarly, in FIG. 64 shows the surroundings near the region at position 330 (indicated according to EU numbering) in the structure of the Fc(P238D)/FcγRIIb extracellular region complex. This figure shows that the environment of the region at position 330 (indicated according to EU numbering) of the Fc A chain in Fc (P238D) is a hydrophilic environment consisting of Ser at position 85, Glu at position 86, Lys at position 163, etc. P. FcγRIIb. Thus, the change obtained by replacing Ala at position 330 (indicated according to EU numbering) with either Lys or Arg would presumably result in a stronger interaction with Ser at position 85 or Glu at position 86 in FcγRIIb.

In FIG. 65 shows the structures of Pro at position 271 (indicated according to EU numbering) of the Fc B chain after superposition of the crystal structure of the Fc complex (P238D)/FcγRIIb extracellular region and the Fc complex (WT)/FcγRIIIa extracellular region by least-squares fit based on distances between pairs of Cα atoms with respect to the Fc B-chain. The two structures match well, but have different 3D Pro structures at position 271 (listed according to EU numbering). When the weak electron density in this region in the crystal structure of the Fc(P238D)/FcγRIIb extracellular region is also taken into account, it is assumed that there is a possibility that Pro at position 271 (listed according to EU numbering) in Fc(P238D)/FcγRIIb causes a high voltage in the structure, thus breaking the balance of the loop structure to achieve the optimal structure. Thus, the change obtained by replacing Pro at position 271 (indicated by EU numbering) with Gly mobilizes this loop structure and presumably leads to increased binding by lowering the energy barrier when the optimal structure is allowed to form during interaction with FcγRIIb.

[Example 30] Examining the combinatorial effect of changes that enhance FcγRIIb binding when combined with P238D.

Of the changes obtained according to Reference Examples 27 and 29, changes that enhanced FcγRIIb binding or retained FcγRIIb binding and exhibited effects of suppressing binding to other FcγRs were combined with each other, and their effect was examined.

Particularly advantageous changes selected from Tables 46 and 49 were introduced into the H chain of the IL6R-BF648 antibody similarly to the method of Reference Example 29. IL6R-L was used as the overall L chain, the expressed antibodies were purified according to the method of Example 12. Binding to each from FcγR (FcγRIa, H-type FcγRIIa, R-type FcγRIIa, FcγRIIb, and V-type FcγRIIIa) were evaluated in detail by the method of Example 14.

In accordance with the following method, the relative binding activity was calculated from the results of the analysis of interactions with the corresponding FcγR. The binding value of each variant to each FcγR was divided by the binding value of the pre-modified control antibody (IL6R-BF648/IL6R-L with Pro substitutions at position 238 (listed according to EU numbering) by Asp to each FcγR, and multiplied by 100; and then this value was presented as the relative binding activity of each variant to each FcγR (table 51).

In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 187). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*).

Results of measuring the KD values of the variants presented in Table 51 for FcγRIa, FcγRIIaR, FcγRIIaH, FcγRIIb and FCγRIIIa

type V in the manner of Example 14 are summarized in Tables 51-2 and 52-2. In the table, the change refers to the change made to IL6R-B3 (SEQ ID NO: 187). The template used to obtain IL6R-B3, IL6R-G1d/IL6R-L, is indicated by an asterisk (*). Moreover, KD(IIaR)/KD(IIb) and KD(IIaH)/KD(IIb) in the table, respectively, correspond to the value obtained by dividing the KD value of each FcγRIIaR option by the KD value of each FcγRIIb option, and the value obtained by dividing the KD value of each FcγRIIaH variant by the KD value of each FcγRIIb variant. The KD(IIb) of the original polypeptide/KD(IIb) of the modified polypeptide refers to the value obtained by dividing the KD value of the original polypeptide in relation to FcγRIIb by the KD value of each FcγRIIb variant. In addition, Tables 51-2 and 52-2 show the ratio of KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of each variant/KD value for the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Here, the original polypeptide refers to a variant that has IL6R-B3 (SEQ ID NO: 187) as the H chain. It was determined that, due to the weak binding of FcγR to IgG, it could not be accurately analyzed by kinetic analysis, and thus the shaded boxes in Tables 51-2 and 52-2 show values calculated using Equation 5 of Example 14.

[Equation 5]

KD=CxRmax/(Req-RI)-C

Tables 51-2 and 52-2 show that, compared to IL6R-B3, all variants showed an increase in affinity for FcγRIIb and the improvement ranged from 3.0-fold to 99.0-fold. The ratio of FcγRIIaR KD value of each variant/FcγRIIb KD value of each variant, and the FcγRIIaH KD value of each variant/FcγRIIb KD value of each variant correspond to FcγRIIb binding activity relative to FcγRIIaR binding activity and FcγRIIaH binding activity, respectively. Therefore, these values indicate the degree of binding selectivity of each variant to FcγRIIb, and a higher value indicates higher binding selectivity to FcγRIIb. Since the ratio of FcγRIIaR KD value/FcγRIIb KD value and the FcγRIIaH KD value/FcγRIIb KD value ratio of the parent IL6R-B3/IL6R-L polypeptide were 0.3 and 0.2, respectively, all variants in tables 51-2 and 52-2 showed an increase in the selectivity of binding to FcγRIIb compared to the original polypeptide. When the ratio of KD value for the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variant/KD value for the stronger of the binding activities of the parent polypeptide is 1 or more, it means that the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variant has equivalent or reduced binding compared to with binding at the stronger of the FcγRIIaR and FcγRIIaH binding activities of the parent polypeptide. Since this value ranged from 0.7 to 29.9 for the variants obtained this time, it can be said that the binding at the stronger of the binding activities of the FcγRIIaR and FcγRIIaH variants obtained this time is practically equivalent or reduced compared to the original polypeptide . These results showed that, compared to the parent polypeptide, the variants generated this time have retained or reduced type R and type H FcγRIIa binding activity, increased FcγRIIb binding activity, and increased FcγRIIb selectivity. Moreover, compared to IL6R-B3, all variants had lower affinity for FcγRIa and FcγRIIIaV.

Table 52-2 is a continuation of Table 52-1.

Industrial Applicability

The present invention relates to methods for improving the pharmacokinetics of antigen-binding molecules and to methods for reducing the immunogenicity of antigen-binding molecules. The present invention relates to antibody therapy that does not cause adverse effects in vivo compared to conventional antibodies.

Claims (2203)

1. A method for improving the pharmacokinetics of an antigen binding molecule, wherein the method comprises modifying the Fc region of the antigen binding molecule comprising an antigen binding domain whose a) antigen binding activity varies with pH and b) the Fc region has FcRn binding activity at pH 7.4, Fc - region, the binding activity of which in relation to the activating Fcγ receptor is lower than the binding activity of the Fc region of native human IgG in relation to the activating Fcγ receptor, where said antigen-binding molecule is a human IgG antibody, where the antigen-binding activity is lower at pH 5.8 than at pH 7.4, where the ratio of the KD value for antigen-binding activity at pH 5.8 to the KD value for antigen-binding activity at pH 7.4 (KD (pH 5.8) / KD (pH 7.4)) is 2 or greater, wherein the antigen binding domain contains a histidine residue at each one or more positions selected from the group consisting of H heavy chain variable region positions such as 27, 31, 32, 33, 35, 50, 58, 59, 61, 62, 99, 100b and 102, and positions of the L light chain variable region such as 24, 27, 28, 31, 32, 34, 50, 51, 52, 53, 54, 55, 56, 89, 90, 91, 92, 93, 94 and 95a (Cabot numbering), where the modification of the Fc region includes the replacement of the amino acid of the specified Fc region, according to EU numbering, at any one or more of the positions of: amino acid at position 234 to any of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr and Trp; amino acid at position 235 to any of Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val and Arg; amino acid at position 236 to any of Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro and Tyr; amino acid at position 237 to any of Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr and Arg; amino acid at position 238 to any of Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp and Arg; amino acid at position 239 to any of Gln, His, Lys, Phe, Pro, Trp, Tyr and Arg; amino acid at position 265 to any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val; amino acid at position 266 to any of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp and Tyr; amino acid at position 267 to any of Arg, His, Lys, Phe, Pro, Trp and Tyr; amino acid at position 269 to any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; amino acid at position 270 to any of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; amino acid at position 271 to any of Arg, His, Phe, Ser, Thr, Trp and Tyr; amino acid at position 295 to any of Arg, Asn, Asp, Gly, His, Phe, Ser, Trp and Tyr; amino acid at position 296 to any of Arg, Gly, Lys and Pro; amino acid at position 297 on Ala; amino acid at position 298 to any of Arg, Gly, Lys, Pro, Trp and Tyr; amino acid at position 300 to any of Arg, Lys and Pro; amino acid at position 324 on Lys or Pro; the amino acid at position 325 to any of Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr and Val; the amino acid at position 327 to any of Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; amino acid at position 328 to any of Arg, Asn, Gly, His, Lys, and Pro; amino acid at position 329 to any of Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val and Arg; amino acid at position 330 on Pro or Ser; amino acid at position 331 to any of Arg, Gly and Lys; or amino acid at position 332 to any of Arg, Lys and Pro; where the Fc region according to (b), which has a greater FcRn-binding activity at pH 7.4, contains at least one of the following amino acid substitutions in positions in the Fc domain, according to EU numbering: N434W; N434Y; M252Y/S254T/T256E/N434Y; M252Y/S254T/T256E/N434W; M252Y/N434Y; M252Y/N434W; M252W/N434Y; M252W/N434W; P257L/N434Y; V308F/N434Y; M252Y/V308P/N434Y; M428L/N434S; M252Y/S254T/T256E/V308P/N434W; I332V; I332V/N434Y; G237M/V308F; S254T/N434W; S254T/N434Y; T256E/N434W; T256E/N434Y; S254T/T256E/N434W; S254T/T256E/N434Y; M252Y/S254T/N434W; M252Y/S254T/N434Y; M252Y/T256E/N434W; M252Y/T256E/N434Y; M252Y/S254T/T256E/N434A; M252Y/N434A; M252W/N434A; M252Y/T256Q/N434W; M252Y/T256Q/N434Y; M252F/T256D/N434W; M252F/T256D/N434Y; H433K/N434F/Y436H; I332V/N434W; V308P/N434W; I332V/M428L/N434Y; G385D/Q386P/N389S/N434W; G385D/Q386P/N389S/N434Y; G385H/N434W; G385H/N434Y; N434F; M252Y/S254T/T256E/N434F; M252Y/S254T/T256E/N434H; M252Y/N434F; M252Y/N434H; M428L/N434W; M428L/N434Y; M252Y/S254T/T256E/M428L/N434W; M252Y/S254T/T256E/M428L/N434Y; M252Y/M428L/N434W; M252Y/M428L/N434Y; M252Y/M428L/N434A; M252Y/S254T/T256E/M428L/N434A; T256E/M428L/N434Y; S254T/M428L/N434W; M252Y/T256Q/N434A; M252Y/T256E/N434A; T256Q/N434W; T256Q/N434Y; M252W/T256Q/N434W; M252W/T256Q/N434Y; S254T/T256Q/N434W; M252Y/S254T/T256Q/N434W; M252Y/S254T/T256Q/N434Y; M252Y/T256E/V308P/N434W; M252Y/V308P/M428L/N434Y; M252Y/S254T/T256E/V308P/M428L/N434W; M252W/M428L/N434W; P257L/M428L/N434Y; M252Y/S254T/T256E/M428L/N434F; M252Y/T256E/N434H; M252W/T256Q/P257L/N434Y; P238A/M252Y/V308P/N434Y; M252Y/D265A/V308P/N434Y; M252Y/T307A/V308P/N434Y; M252Y/V303A/V308P/N434Y; M252Y/V308P/D376A/N434Y; M252Y/V305A/V308P/N434Y; M252Y/V308P/Q311A/N434Y; M252Y/V308P/K317A/N434Y; M252Y/V308P/E380A/N434Y; M252Y/V308P/E382A/N434Y; M252Y/V308P/S424A/N434Y; P257L/V308P/M428L/N434Y; M252Y/T256E/V308P/M428L/N434W; M252Y/T256E/M428L/N434W; M252Y/T256E/V308P/N434Y; M252Y/T256E/M428L/N434Y; M252Y/S254T/T256E/V308P/M428L/N434Y; P257L/M428L/N434W; P257A/M428L/N434Y; P257G/M428L/N434Y; P257I/M428L/N434Y; P257M/M428L/N434Y; P257N/M428L/N434Y; P257S/M428L/N434Y; P257T/M428L/N434Y; P257V/M428L/N434Y; M252W/V308P/N434W; S239K/M252Y/V308P/N434Y; M252Y/S298G/V308P/N434Y; M252Y/D270F/V308P/N434Y; M252Y/V308A/N434Y; M252Y/V308F/N434Y; M252Y/V308I/N434Y; M252Y/V308L/N434Y; M252Y/V308M/N434Y; M252Y/V308Q/N434Y; M252Y/V308T/N434Y; P257A/V308P/M428L/N434Y; P257T/V308P/M428L/N434Y; P257V/V308P/M428L/N434Y; M252W/M428I/N434Y; M252W/M428Y/N434Y; M252W/M428F/N434Y; P238A/M252W/N434Y; M252W/D265A/N434Y; M252W/T307Q/N434Y; M252W/V303A/N434Y; M252W/D376A/N434Y; M252W/V305A/N434Y; M252W/Q311A/N434Y; M252W/D312A/N434Y; M252W/K317A/N434Y; M252W/E380A/N434Y; M252W/E382A/N434Y; M252W/S424A/N434Y; S239K/M252W/N434Y; M252W/S298G/N434Y; M252W/D270F/N434Y; M252W/N325G/N434Y; P257A/M428L/N434W; P257T/M428L/N434W; P257V/M428L/N434W; M252W/I332V/N434Y; P257I/Q311I; M252Y/T307A/N434Y; M252Y/T307Q/N434Y; P257L/T307A/M428L/N434Y; P257A/T307A/M428L/N434Y; P257T/T307A/M428L/N434Y; P257V/T307A/M428L/N434Y; P257L/T307Q/M428L/N434Y; P257A/T307Q/M428L/N434Y; P257T/T307Q/M428L/N434Y; P257V/T307Q/M428L/N434Y; M252Y/T307D/N434Y; M252Y/T307F/N434Y; M252Y/T307G/N434Y; M252Y/T307H/N434Y; M252Y/T307I/N434Y; M252Y/T307K/N434Y; M252Y/T307L/N434Y; M252Y/T307M/N434Y; M252Y/T307N/N434Y; M252Y/T307P/N434Y; M252Y/T307R/N434Y; M252Y/T307S/N434Y; M252Y/T307V/N434Y; M252Y/T307W/N434Y; M252Y/T307Y/N434Y; M252Y/K334L/N434Y; M252Y/G385H/N434Y; M252Y/T289H/N434Y; M252Y/Q311H/N434Y; M252Y/D312H/N434Y; M252Y/N315H/N434Y; M252Y/K360H/N434Y; M252Y/L314R/N434Y; M252Y/L314K/N434Y; M252Y/N286E/N434Y; M252Y/L309E/N434Y; M252Y/R255E/N434Y; M252Y/P387E/N434Y; K248I/M428L/N434Y; M252Y/M428A/N434Y; M252Y/M428D/N434Y; M252Y/M428F/N434Y; M252Y/M428G/N434Y; M252Y/M428H/N434Y; M252Y/M428I/N434Y; M252Y/M428K/N434Y; M252Y/M428N/N434Y; M252Y/M428P/N434Y; M252Y/M428Q/N434Y; M252Y/M428S/N434Y; M252Y/M428T/N434Y; M252Y/M428V/N434Y; M252Y/M428W/N434Y; M252Y/M428Y/N434Y; M252W/T307Q/M428Y/N434Y; M252W/Q311A/M428Y/N434Y; M252W/T307Q/Q311A/M428Y/N434Y; M252Y/T307A/M428Y/N434Y; M252Y/T307Q/M428Y/N434Y; M252Y/D270F/N434Y; M252Y/T307A/Q311A/N434Y; M252Y/T307Q/Q311A/N434Y; M252Y/T307A/Q311H/N434Y; M252Y/T307Q/Q311H/N434Y; M252Y/E382A/N434Y; M252Y/E382A/M428Y/N434Y; M252Y/T307A/E382A/M428Y/N434Y; M252Y/T307Q/E382A/M428Y/N434Y; P238A/M252Y/M428F/N434Y; M252Y/V305A/M428F/N434Y; M252Y/N325G/M428F/N434Y; M252Y/D376A/M428F/N434Y; M252Y/E380A/M428F/N434Y; M252Y/E382A/M428F/N434Y; M252Y/E380A/E382A/M428F/N434Y; S239K/M252Y/V308P/E382A/N434Y; M252Y/D270F/V308P/E382A/N434Y; S239K/M252Y/V308P/M428Y/N434Y; M252Y/T307Q/V308P/E382A/N434Y; M252Y/V308P/Q311H/E382A/N434Y; S239K/M252Y/D270F/V308P/N434Y; M252Y/V308P/E382A/M428F/N434Y; M252Y/V308P/E382A/M428L/N434Y; M252Y/V308P/E382A/M428Y/N434Y; M252Y/V308P/M428Y/N434Y; M252Y/V308P/M428F/N434Y; S239K/M252Y/V308P/E382A/M428Y/N434Y; M252Y/V308P/E380A/E382A/M428F/N434Y; M252Y/T256E/E382A/N434Y; M252Y/T256E/M428Y/N434Y; M252Y/T256E/E382A/M428Y/N434Y; S239K/M252Y/V308P/E382A/M428I/N434Y; M252Y/V308P/E380A/E382A/M428I/N434Y; S239K/M252Y/V308P/M428F/N434Y; S239K/M252Y/E380A/E382A/M428F/N434Y; M252Y/Q311A/M428Y/N434Y; M252Y/D312A/M428Y/N434Y; M252Y/Q311A/D312A/M428Y/N434Y; S239K/M252Y/V308P/Q311A/M428Y/N434Y; S239K/M252Y/V308P/D312A/M428Y/N434Y; S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y; M252Y/T256E/T307P/N434Y; M252Y/T307P/M428Y/N434Y; M252W/V308P/M428Y/N434Y; M252Y/T256E/V308P/E382A/N434Y; M252W/V308P/E382A/N434Y; S239K/M252W/V308P/E382A/N434Y; S239K/M252W/V308P/E382A/M428Y/N434Y; S239K/M252W/V308P/E382A/M428I/N434Y; S239K/M252W/V308P/M428F/N434Y; S239K/M252W/E380A/E382A/M428F/N434Y; S239K/M252Y/T307P/M428Y/N434Y; M252Y/T256E/Q311A/D312A/M428Y/N434Y; S239K/M252Y/M428Y/N434Y; S239K/M252Y/D270F/M428Y/N434Y; S239K/M252Y/D270F/V308P/M428Y/N434Y; S239K/M252Y/V308P/M428I/N434Y; S239K/M252Y/V308P/N325G/E382A/M428I/N434Y; S239K/M252Y/D270F/V308P/N325G/N434Y; S239K/M252Y/D270F/T307P/V308P/N434Y; S239K/M252Y/T256E/D270F/V308P/N434Y; S239K/M252Y/D270F/T307Q/V308P/N434Y; S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y; S239K/M252Y/D270F/T307Q/Q311A/N434Y; S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y; S239K/M252Y/D270F/V308P/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/N434Y; S239K/M252Y/D270F/V308P/L309E/N434Y; S239K/M252Y/D270F/V308P/P387E/N434Y; S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y; S239K/M252Y/D270F/V308P/N325G/M428I/N434Y; S239K/M252Y/T256E/D270F/V308P/M428I/N434Y; S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/N434Y; S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/M428I/N434Y; S239K/M252Y/D270F/V308P/L309E/M428I/N434Y; S239K/M252Y/D270F/V308P/M428L/N434Y; S239K/M252Y/V308P/M428L/N434Y; S239K/M252Y/V308P/L309P/M428L/N434Y; S239K/M252Y/V308P/L309R/M428L/N434Y; S239K/M252Y/L309P/M428L/N434Y; S239K/M252Y/L309R/M428L/N434Y; S239K/M252Y/T307Q/V308P/M428L/N434Y; S239K/M252Y/D270F/V308P/L309E/P387E/N434Y; S239K/M252Y/T256E/D270F/V308P/N325G/M428I/N434Y; S239K/M252Y/T307Q/N434Y; P257V/T307Q/M428I/N434Y; P257V/T307Q/M428V/N434Y; P257V/T307Q/N325G/M428L/N434Y; P257V/T307Q/Q311A/M428L/N434Y; P257V/T307Q/Q311A/N325G/M428L/N434Y; P257V/V305A/T307Q/M428L/N434Y; S239K/M252Y/E258H/D270F/T307Q/V308P/Q311A/N434Y; S239K/M252Y/D270F/V308P/N325G/E382A/M428I/N434Y; M252Y/P257V/T307Q/M428I/N434Y; S239K/M252W/V308P/M428Y/N434Y; S239K/M252W/V308P/Q311A/M428Y/N434Y; S239K/M252W/T256E/V308P/M428Y/N434Y; S239K/M252W/N286E/V308P/M428Y/N434Y; S239K/M252Y/T256E/D270F/N286E/V308P/N434Y; S239K/M252W/T307P/M428Y/N434Y; S239K/M252W/T256E/V308P/Q311A/M428Y/N434Y; S239K/M252W/T256E/N286E/V308P/M428Y/N434Y; P257V/T307A/Q311A/M428L/N434Y; P257V/V305A/T307A/M428L/N434Y; M252Y/V308P/L309E/N434Y; M252Y/V308P/L309D/N434Y; M252Y/V308P/L309A/N434Y; M252W/V308P/L309E/M428Y/N434Y; M252W/V308P/L309D/M428Y/N434Y; M252W/V308P/L309A/M428Y/N434Y; S239K/M252Y/N286E/V308P/M428I/N434Y; S239K/M252Y/N286E/V308P/N434Y; S239K/M252Y/T307Q/Q311A/M428I/N434Y; S239K/M252Y/T256E/V308P/N434Y; S239K/M252Y/D270F/N286E/T307Q/Q311A/M428I/N434Y; S239K/M252Y/D270F/T307Q/Q311A/P387E/M428I/N434Y; S239K/M252Y/D270F/N286E/T307Q/V308P/Q311A/M428I/N434Y; S239K/M252Y/D270F/N286E/T307Q/V308P/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/Q311A/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/P387E/M428I/N434Y; P257V/T307Q/M428L/N434W; P257V/T307A/M428L/N434W; P257A/T307Q/L309P/M428L/N434Y; P257V/T307Q/L309P/M428L/N434Y; P257A/T307Q/L309R/M428L/N434Y; P257V/T307Q/L309R/M428L/N434Y; P257V/N286E/M428L/N434Y; P257V/P387E/M428L/N434Y; P257V/T307H/M428L/N434Y; P257V/T307N/M428L/N434Y; P257V/T307G/M428L/N434Y; P257V/T307P/M428L/N434Y; P257V/T307S/M428L/N434Y; P257V/N286E/T307A/M428L/N434Y; P257V/T307A/P387E/M428L/N434Y; S239K/M252Y/T307P/N325G/M428Y/N434Y; M252Y/T307A/Q311H/K360H/N434Y; M252Y/T307A/Q311H/P387E/N434Y; M252Y/T307A/Q311H/M428A/N434Y; M252Y/T307A/Q311H/E382A/N434Y; M252Y/T307W/Q311H/N434Y; S239K/P257A/V308P/M428L/N434Y; P257A/V308P/L309E/M428L/N434Y; P257V/T307Q/N434Y; M252Y/P257V/T307Q/M428V/N434Y; M252Y/P257V/T307Q/M428L/N434Y; M252Y/P257V/T307Q/N434Y; M252Y/P257V/T307Q/M428Y/N434Y; M252Y/P257V/T307Q/M428F/N434Y; P257V/T307Q/Q311A/N325G/M428V/N434Y; P257V/T307Q/Q311A/N325G/N434Y; P257V/T307Q/Q311A/N325G/M428Y/N434Y; P257V/T307Q/Q311A/N325G/M428F/N434Y; P257A/T307Q/M428V/N434Y; P257A/T307Q/N434Y; P257A/T307Q/M428Y/N434Y; P257A/T307Q/M428F/N434Y; P257V/N286E/T307Q/Q311A/N325G/M428L/N434Y; P257A/Q311A/M428L/N434Y; P257A/Q311H/M428L/N434Y; P257A/T307Q/Q311A/M428L/N434Y; P257A/T307A/Q311A/M428L/N434Y; P257A/T307Q/Q311H/M428L/N434Y; P257A/T307A/Q311H/M428L/N434Y; P257A/N286E/T307Q/M428L/N434Y; P257A/N286E/T307A/M428L/N434Y; S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y; S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y; P257S/T307A/M428L/N434Y; P257M/T307A/M428L/N434Y; P257N/T307A/M428L/N434Y; P257I/T307A/M428L/N434Y; P257V/T307Q/M428Y/N434Y; P257V/T307Q/M428F/N434Y; S239K/P257V/V308P/M428L/N434Y; P257V/T307Q/V308P/N325G/M428L/N434Y; P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y; P257V/T307A/V308P/N325G/M428L/N434Y; P257A/V308P/N325G/M428L/N434Y; P257A/T307A/V308P/M428L/N434Y; P257A/T307Q/V308P/M428L/N434Y; P257V/N286E/T307Q/N325G/M428L/N434Y; T256E/P257V/N434Y; T256E/P257T/N434Y; S239K/P257T/V308P/M428L/N434Y; P257T/V308P/N325G/M428L/N434Y; T256E/P257T/V308P/N325G/M428L/N434Y; P257T/V308P/N325G/E382A/M428L/N434Y; P257T/V308P/N325G/P387E/M428L/N434Y; P257T/V308P/L309P/N325G/M428L/N434Y; P257T/V308P/L309R/N325G/M428L/N434Y; T256E/P257V/T307Q/M428L/N434Y; P257V/T307Q/E382A/M428L/N434Y; P257V/T307Q/P387E/M428L/N434Y; P257L/V308P/N434Y; P257T/T307Q/N434Y; P257V/T307Q/N325G/N434Y; P257V/T307Q/Q311A/N434Y; P257V/V305A/T307Q/N434Y; P257V/N286E/T307A/N434Y; P257V/T307Q/L309R/Q311H/M428L/N434Y; P257V/V308P/N325G/M428L/N434Y; S239K/P257V/V308P/Q311H/M428L/N434Y; P257V/V305A/T307A/N325G/M428L/N434Y; S239K/D270F/T307P/N325G/M428Y/N434Y; P257T/T307A/M428V/N434Y; P257A/M428V/N434Y; P257A/T307A/M428V/N434Y; P257S/T307Q/M428L/N434Y; P257V/N297A/T307Q/M428L/N434Y; P257V/N286A/T307Q/M428L/N434Y; P257V/T307Q/N315A/M428L/N434Y; P257V/T307Q/N384A/M428L/N434Y; P257V/T307Q/N389A/M428L/N434Y; P257V/N434Y; P257T/N434Y; P257V/N286E/N434Y; P257T/N286E/N434Y; P257V/N286E/T307Q/N434Y; P257V/N286E/T307Q/M428Y/N434Y; P257V/V305A/T307Q/M428Y/N434Y; P257T/T307Q/M428Y/N434Y; P257V/T307Q/V308P/N325G/M428Y/N434Y; P257T/T307Q/M428F/N434Y; P257A/N286E/T307Q/M428F/N434Y; P257A/N286E/T307Q/M428Y/N434Y; T250A/P257V/T307Q/M428L/N434Y; T250F/P257V/T307Q/M428L/N434Y; T250I/P257V/T307Q/M428L/N434Y; T250M/P257V/T307Q/M428L/N434Y; T250S/P257V/T307Q/M428L/N434Y; T250V/P257V/T307Q/M428L/N434Y; T250W/P257V/T307Q/M428L/N434Y; T250Y/P257V/T307Q/M428L/N434Y; M252Y/Q311A/N434Y; S239K/M252Y/S254T/V308P/N434Y; M252Y/T307Q/Q311A M252Y/S254T/N286E/N434Y; M252Y/S254T/V308P/N434Y; M252Y/S254T/T307A/N434Y; M252Y/S254T/T307Q/N434Y; M252Y/S254T/Q311A/N434Y; M252Y/S254T/Q311H/N434Y; M252Y/S254T/T307A/Q311A/N434Y; M252Y/S254T/T307Q/Q311A/N434Y; M252Y/S254T/M428I/N434Y; M252Y/T256E/T307A/Q311H/N434Y; M252Y/T256Q/T307A/Q311H/N434Y; M252Y/S254T/T307A/Q311H/N434Y; M252Y/N286E/T307A/Q311H/N434Y; M252Y/T307A/Q311H/M428I/N434Y; M252Y/T256E/T307Q/Q311H; M252Y/N286E/T307A/Q311A; M252Y/N286E/T307Q/Q311A; M252Y/N286E/T307Q/Q311H; M252Y/T256E/N286E/N434Y; S239K/M252Y/V308P; S239K/M252Y/V308P/E382A; S239K/M252Y/T256E/V308P; S239K/M252Y/N286E/V308P; S239K/M252Y/N286E/V308P/M428I; M252Y/N286E/M428L/N434Y; M252Y/S254T/E382A/N434Y; S239K/M252Y/S254T/V308P/M428I/N434Y; S239K/M252Y/M428I/N434Y; M252Y/D312A/E382A/M428Y/N434Y; M252Y/E382A/P387E/N434Y; M252Y/D312A/P387E/N434Y; M252Y/P387E/M428Y/N434Y; M252Y/T256Q/E382A/N434Y; M252Y/N286E/V308P/N434Y; M252Y/V305T/T307P/V308I/L309A/N434Y; M252Y/T307P/V308I/L309A/N434Y; S239K/N434W; S239K/V308F/N434Y; S239K/M252Y/N434Y; S239K/M252Y/S254T/N434Y; S239K/M252Y/T307A/Q311H/N434Y; S239K/M252Y/T256Q/N434Y; S239K/M252W/N434W; S239K/P257A/N286E/T307Q/M428L/N434Y; S239K/P257A/T307Q/M428L/N434Y; V308P; M252Y/V279L/V308P/N434Y; S239K/M252Y/V279L/V308P/N434Y; M252Y/V282D/V308P/N434Y; S239K/M252Y/V282D/V308P/N434Y; M252Y/V284K/V308P/N434Y; S239K/M252Y/V284K/V308P/N434Y; M252Y/K288S/V308P/N434Y; S239K/M252Y/K288S/V308P/N434Y; M252Y/V308P/G385R/N434Y; S239K/M252Y/V308P/G385R/N434Y; M252Y/V308P/Q386K/N434Y; S239K/M252Y/V308P/Q386K/N434Y; L235G/G236R/S239K/M252Y/V308P/N434Y; G236R/S239K/M252Y/V308P/N434Y; S239K/M252Y/V308P/L328R/N434Y; S239K/M252Y/N297A/V308P/N434Y; P238D/M252Y/V308P/N434Y; S239K/M252Y/N286E/N434Y; S239K/M252Y/T256E/N434Y; S239K/M252Y/Q311A/N434Y; P238D/M252Y/N434Y; L235K/S239K/M252Y/V308P/N434Y; L235R/S239K/M252Y/V308P/N434Y; G237K/S239K/M252Y/V308P/N434Y; G237R/S239K/M252Y/V308P/N434Y; P238K/S239K/M252Y/V308P/N434Y; P238R/S239K/M252Y/V308P/N434Y; S239K/M252Y/V308P/P329K/N434Y; S239K/M252Y/V308P/P329R/N434Y; S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y; M252Y/N286A/V308P/N434Y; M252Y/N286D/V308P/N434Y; M252Y/N286F/V308P/N434Y; M252Y/N286G/V308P/N434Y; M252Y/N286H/V308P/N434Y; M252Y/N286I/V308P/N434Y; M252Y/N286K/V308P/N434Y; M252Y/N286L/V308P/N434Y; M252Y/N286M/V308P/N434Y; M252Y/N286P/V308P/N434Y; M252Y/N286Q/V308P/N434Y; M252Y/N286R/V308P/N434Y; M252Y/N286S/V308P/N434Y; M252Y/N286T/V308P/N434Y; M252Y/N286V/V308P/N434Y; M252Y/N286W/V308P/N434Y; M252Y/N286Y/V308P/N434Y; M252Y/K288A/V308P/N434Y; M252Y/K288D/V308P/N434Y; M252Y/K288E/V308P/N434Y; M252Y/K288F/V308P/N434Y; M252Y/K288G/V308P/N434Y; M252Y/K288H/V308P/N434Y; M252Y/K288I/V308P/N434Y; M252Y/K288L/V308P/N434Y; M252Y/K288M/V308P/N434Y; M252Y/K288N/V308P/N434Y; M252Y/K288P/V308P/N434Y; M252Y/K288Q/V308P/N434Y; M252Y/K288R/V308P/N434Y; M252Y/K288V/V308P/N434Y; M252Y/K288W/V308P/N434Y; M252Y/K288Y/V308P/N434Y; S239K/M252Y/V308P/N325G/N434Y; M252Y/N286E/T307Q/Q311A/N434Y; S239K/M252Y/N286E/T307Q/Q311A/N434Y; M252Y/V308P/L328E/N434Y; M252Y/N286E/V308P/M428I/N434Y; M252Y/N286E/T307Q/Q311A/M428I/N434Y; M252Y/N286E/T307Q/V308P/Q311A/N434Y; M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y; S239K/M252Y/N286E/T307Q/Q311A/M428I/N434Y; P238D/M252Y/N286E/T307Q/Q311A/M428I/N434Y; S239K/M252Y/T307Q/Q311A/N434Y; P238D/M252Y/T307Q/Q311A/N434Y; P238D/M252Y/N286E/T307Q/Q311A/N434Y; P238D/M252Y/N325S/N434Y; P238D/M252Y/N325M/N434Y; P238D/M252Y/N325L/N434Y; P238D/M252Y/N325I/N434Y; P238D/M252Y/Q295M/N434Y; P238D/M252Y/N325G/N434Y; M252Y/T307Q/V308P/Q311A/N434Y; M252Y/T307Q/V308P/Q311A/M4281/N434Y; P238D/M252Y/A327G/N434Y; P238D/M252Y/L328D/N434Y; P238D/M252Y/L328E/N434Y; L235K/G237R/S239K/M252Y/V308P/N434Y; L235K/P238K/S239K/M252Y/V308P/N434Y; T307A/Q311A/N434Y; T307Q/Q311A/N434Y; T307A/Q311H/N434Y; T307Q/Q311H/N434Y; M252Y/L328E/N434Y; G236D/M252Y/L328E/N434Y; M252Y/S267M/L328E/N434Y; M252Y/S267L/L328E/N434Y; P238D/M252Y/T307P/N434Y; M252Y/T307P/Q311A/N434Y; M252Y/T307P/Q311H/N434Y; P238D/T250A/M252Y/N434Y; P238D/T250F/M252Y/N434Y; P238D/T250G/M252Y/N434Y; P238D/T250H/M252Y/N434Y; P238D/T250I/M252Y/N434Y; P238D/T25UL/M252Y/N434Y; P238D/T250M/M252Y/N434Y; F751 5.30E-07 P238D/T250N/M252Y/N434Y; P238D/T250Q/M252Y/N434Y; P238D/T250S/M252Y/N434Y; P238D/T250V/M252Y/N434Y; P238D/T250W/M252Y/N434Y; P238D/T250Y/M252Y/N434Y; P238D/N434Y; L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y; L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y; P238D/T307Q/Q311A/N434Y; P238D/M252Y/T307Q/L309E/Q311A/N434Y; T307A/L309E/Q311A/N434Y; T307Q/L309E/Q311A/N434Y; T307A/L309E/Q311H/N434Y; T307Q/L309E/Q311H/N434Y; M252Y/T256A/K434Y; M252Y/E272A/N434Y; M252Y/K274A/N434Y; M252Y/V282A/N434Y; M252Y/N286A/N434Y; M252Y/K338A/N434Y; M252Y/K340A/N434Y; M252Y/E345A/N434Y; M252Y/N361A/N434Y; M252Y/Q362A/N434Y; M252Y/S375A/N434Y; M252Y/Y391A/N434Y; M252Y/D413A/N434Y; M252Y/L309A/N434Y; M252Y/L309H/N434Y; M252Y/S254T/N286E/T307Q/Q311A/N434Y; M252Y/S254T/T307Q/L309E/Q311A/N434Y; M252Y/N315A/N434Y; M252Y/N315D/N434Y; M252Y/N315E/N434Y; M252Y/N315F/N434Y; M252Y/N315G/N434Y; M252Y/N315I/N434Y; M252Y/N315K/N434Y; M252Y/N315L/N434Y; M252Y/N315M/N434Y; M252Y/N315Q/N434Y; M252Y/N315R/N434Y; M252Y/N315S/N434Y; M252Y/N315T/N434Y; M252Y/N315V/N434Y; M252Y/N315W/N434Y; M252Y/N315Y/N434Y; M252Y/N325A/N434Y; M252Y/N384A/N434Y; M252Y/N389A/N434Y; M252Y/N389A/N390A/N434Y; M252Y/S254T/T256S/N434Y; M252Y/A378V/N434Y; M252Y/E380S/N434Y; M252Y/E382V/N434Y; M252Y/S424E/N434Y; M252Y/N434Y/Y436I; M252Y/N434Y/T437R; P238D/T250V/M252Y/T307P/N434Y; P238D/T250V/M252Y/T307Q/Q311A/N434Y; P238D/M252Y/N286E/N434Y; L235K/S239K/M252Y/N434Y; L235R/S239K/M252Y/N434Y; P238D/T250Y/M252Y/W313Y/N434Y; P238D/T250Y/M252Y/W313F/N434Y; P238D/T250V/M252Y/1253V/N434Y; P238D/T250V/M252Y/R255A/N434Y; P238D/T250V/M252Y/R255D/N434Y; P238D/T250V/M252Y/R255E/N434Y; P238D/T250V/M252Y/R255F/N434Y; P238D/T250V/M252Y/R255H/N434Y; P238D/T250V/M252Y/K2551/M434Y; P238D/T250V/M252Y/R255K/N434Y; P238D/T250V/M252Y/R255L/N434Y; P238D/T250V/M252Y/R255M/N434Y; P238D/T250V/M252Y/R255N/N434Y; P238D/T250V/M252Y/R255Q/N434Y; P238D/T250V/ M252Y/ R255S/ N434Y; P238D/T250V/M252Y/R255W/N434Y; P238D/T250V/M252Y/R255Y/N434Y; M252Y/D280A/N434Y; M252Y/D280E/N434Y; M252Y/D280G/N434Y; M252Y/D280H/N434Y; M252Y/D280K/N434Y; M252Y/D280N/N434Y; M252Y/D280Q/N434Y; M252Y/D280R/N434Y; M252Y/D280S/N434Y; M252Y/D280T/N434Y; M252Y/N384A/N389A/N434Y; G236A/S239D/M252Y/N286E/T307Q/Q311A/N434Y; L235K/S239K/M252Y/T307Q/Q311A/N434Y; L235R/S239K/M252Y/T307Q/Q311A/N434Y; L235K/S239K/M252Y/N286E/N434Y; L235R/S239K/M252Y/N286E/N434Y; M252Y/N434Y/Y436A; M252Y/N434Y/Y436E; M252Y/N434Y/Y436F; M252Y/N434Y/Y436G; M252Y/N434Y/Y436H; M252Y/N434Y/Y436K; M252Y/N434Y/Y436L; M252Y/N434Y/Y436M; M252Y/N434Y/Y436N; M252Y/N434Y/Y436S; M252Y/N434Y/Y436T; M252Y/N434Y/Y436V; M252Y/N434Y/Y436W; M252Y/S254T/N434Y/Y436I; L235K/S239K/M252Y/N434Y/Y436I; L235R/S239K/M252Y/N434Y/Y436I; L235K/S239K/M252Y/N286E/T307Q/Q311A/N315E/N434Y; L235R/S239K/M252Y/N286E/T307Q/Q311A/N315E/N434Y; M252Y/N315D/N384A/N389A/N434Y; M252Y/N315E/N384A/N389A/N434Y; M252Y/N315D/G316A/N434Y; M252Y/N315D/G316D/N434Y; M252Y/N315D/G316E/N434Y; M252Y/N315D/G316F/N434Y; M252Y/N315D/G316H/N434Y; M252Y/N315D/G316I/N434Y; M252Y/N315D/G316K/N434Y; M252Y/N315D/G316L/N434Y; M252Y/N315D/G316M/N434Y; M252Y/N315D/G316N/N434Y; M252Y/N315D/G316P/N434Y; M252Y/N315D/G316Q/N434Y; M252Y/N315D/G316R/N434Y; M252Y/N315D/G316S/N434Y; M252Y/N315D/G316T/N434Y; M252Y/N315D/G316V/N434Y; M252Y/N315D/G316W/N434Y; M252Y/N286S/N434Y; M252Y/D280E/N384A/N389A/N434Y; M252Y/D280G/N384A/N389A/N434Y; M252Y/N286S/N384A/N389A/N434Y; M252Y/N286E/N384A/N389A/N434Y; L235K/S239K/M252Y/N286E/N434Y/Y436I; L235R/S239K/M252Y/N286E/N434Y/Y436I; L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436I; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436I; L235K/S239K/M252Y/S254T/N434Y/Y436I; L235R/S239K/M252Y/S254T/N434Y/Y436I; M252Y/T307Q/Q311A/N434Y/Y436I; M252Y/S254T/T307Q/Q311A/N434Y/Y436I; P238D/T250V/M252Y/N286E/N434Y; P238D/T250V/M252Y/N434Y/Y436I; T250V/M252Y/N434Y; L234R/P238D/T250V/M252Y/N434Y; G236K/P238D/T250V/M252Y/N434Y; G237K/P238D/T250V/M252Y/N434Y; G237R/P238D/T250V/M252Y/N434Y; P238D/S239K/T250V/M252Y/N434Y; L235K/S239K/M252Y/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V; P238D/T250V/M252Y/N434Y/Y436V; M252Y/N286E/T307Q/Q311A/N434Y/Y436V; L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436V; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/N434Y/Y436V; T250V/M252Y/V308P/N434Y/Y436V; T250V/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; M252Y/N434Y/H435K/Y436V; M252Y/N434Y/H435N/Y436V; M252Y/N434Y/H435R/Y436V; M252Y/S254A/N434Y; M252Y/S254G/N434Y; M252Y/S254H/N434Y; M252Y/S2541/N434Y; M252Y/S254Q/N434Y; M252Y/S254V/N434Y; P238D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; P238D/T250V/M252Y/N286E/N434Y/Y436V; L235R/S239K/M252Y/V308P/N434Y/Y436V; L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V; L235R/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V; M252Y/T307Q/Q311A/N434Y/Y436V; M252Y/N325G/N434Y; L235R/P238D/S239K/M252Y/N434Y; L235R/P238D/S239K/M252Y/N434Y/Y436V; K248I/T250V/M252Y/N434Y/Y436V; K248Y/T250V/M252Y/N434Y/Y436V; T250V/M252Y/E258H/N434Y/Y436V; L235R/S239K/T250V/M252Y/N434Y/Y436V; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/M252Y/T307A/Q311H/N434Y; T250V/M252Y/V308P/N434Y; L235R/S239K/T250V/M252Y/V308P/N434Y; T250V/M252Y/T307Q/V308P/Q311A/N434Y; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y; L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y; T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; P238D/T250V/M252Y/N325G/N434Y; P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y; P238D/T250V/M252Y/N325A/N434Y; S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V; S239D/T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V; M252Y/T307A/Q311H/N434Y/Y436V; L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V; G236A/S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V; G236A/S239D/T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V; P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y/Y436V; P238D/N434W; L235K/S239K/M252Y/V308P/N434Y/Y436V; L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/N286E/T307Q/Q311A/K434Y/Y436V; L235K/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V; L235K/P238D/S239K/M252Y/N434Y; L235K/P238D/S239K/M252Y/N434Y/Y436V; L235K/S239K/T250V/M252Y/N434Y/Y436V; L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/T307A/Q311H/N434Y; L235K/S239K/T250V/M252Y/V308P/N434Y; L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y; L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y; L235K/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/T307A/Q311H/N434Y/Y436V; T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V; L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434; Y436V; L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V; L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; M252Y/Q386E/N434Y/Y436V; M252Y/Q386R/N434Y/Y436V; M252Y/Q386S/N434Y/Y436V; M252Y/P387E/N434Y/Y436V; M252Y/P387R/N434Y/Y436V; M252Y/P387S/N434Y/Y436V; M252Y/V422E/N434Y/Y436V; M252Y/V422R/N434Y/Y436V; M252Y/V422S/N434Y/Y436V; M252Y/S424E/N434Y/Y436V; M252Y/S424R/N434Y/Y436V; M252Y/N434Y/Y436V/Q438E; M252Y/N434Y/Y436V/Q438R; M252Y/N434Y/Y436V/Q438S; M252Y/N434Y/Y436V/S440E; M252Y/N434Y/Y436V/S440R; S239D/M252Y/N434Y/Y436V; M252Y/K326D/L328Y/N434Y/Y436V; S239D/M252Y/K326D/L328Y/N434Y/Y436V; K248N/M252Y/N434Y; M252Y/E380N/E382S/N434Y; M252Y/E382N/N384S/N434Y; M252Y/S424N/N434Y; M252Y/N434Y/Y436N/Q438T; M252Y/N434Y/Q438N; M252Y/N434Y/S440N; M252Y/N434Y/S442N; M252Y/S383N/G385S/N434Y; M252Y/Q386T/N434Y; M252Y/G385N/P387S/N434Y; S239D/M252Y/N434Y; M252Y/K326D/L328Y/N434Y; S239D/M252Y/K326D/L328Y/N434Y; S239D/M252Y/T307Q/Q311A/N434Y; M252Y/T307Q/Q311A/K326D/L328Y/N434Y; S239D/M252Y/T307Q/Q311A/K326D/L328Y/N434Y; M252Y/V422E/S424R/N434Y/Y436V; M252Y/V422S/S424R/N434Y/Y436V; M252Y/N434Y/Y436V/Q438R/S440E; M252Y/V422D/N434Y/Y436V; M252Y/V422K/N434Y/Y436V; M252Y/V422T/N434Y/Y436V; M252Y/V422Q/N434Y/Y436V; M252Y/S424K/N434Y/Y436V; M252Y/N434Y/Y436V/Q438K; M252Y/N434Y/Y436V/S440D; M252Y/N434Y/Y436V/S440Q; M252Y/S424N/N434Y/Y436V; M252Y/T307Q/Q311A/S424N/N434Y; T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/V422E/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/S424R/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/V422E/S424R/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/S440E; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S440E; M252Y/T307Q/N434Y/Y436V; M252Y/T307A/N434Y/Y436V; M252Y/Q311A/N434Y/Y436V; M252Y/Q311H/N434Y/Y436V; M252Y/T307Q/Q311H/N434Y/Y436V; M252Y/T307A/Q311A/N434Y/Y436V; L235R/S239K/M252Y/V422E/N434Y/Y436V; L235R/S239K/M252Y/V422S/N434Y/Y436V; L235R/S239K/M252Y/S424R/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V/Q438R; L235R/S239K/M252Y/N434Y/Y436V/S440E; L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V; L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S424N/N434Y/Y436V; M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E; M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S 440E; T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E; M252Y/T307Q/Q311A/N434Y/Q438R/S440E; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Q438R/S440E; L235R/S239K/M252Y/S424N/N434Y; L235R/S239K/M252Y/T307Q/Q311A/S424N/N434Y; L235R/S239K/T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V; M252Y/N434Y/Q438R/S440E; L235R/S239K/M252Y/N434Y/Q438R/S440E; S424N/N434W; V308F/S424N/N434Y; I332V/S424N/N434Y; P238D/T250Y/M252Y/N434Y/Y436V; P238D/T250Y/M252Y/T307Q/Q311A/N434Y; P238D/T250Y/M252Y/T307Q/Q311A/M434Y/Y436V; P238D/T250Y/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; P2571/N434H; V308F; V259I/V308F/M428L; E380A/M428L/N434S; T307A/M428L/N434S; T307A/E380A/M428L/N434S; T307A/E380A/N434H; M252Y/H4330/N434Y/Y436V/Q438R/S440E; M252Y/H433E/N434Y/Y436V/Q438R/S440E; M252Y/N434Y/Y436V/T437A/Q438R/S440E; M252Y/N434Y/Y436V/T437G/Q438R/S440E; M252Y/N434Y/Y436V/Q438R/K439D/S440E; M252Y/N434Y/Y436V/Q438R/S440E/L441A; M252Y/N434Y/Y436V/Q438R/S440E/L441E; M252Y/S254T/N434Y/Y436V/Q438R/S440E; M252Y/T256E/N434Y/Y436V/Q438R/S440E; M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E; T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E; T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; M252Y/T256Q/N434Y/Y436V/Q438R/S440E; M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E; T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; I332E/M428L/N434S; L251A/M252Y/N434Y/Y436V; L251H/M252Y/N434Y/Y436V; L251N/M252Y/N434Y/Y436V; L251S/M252Y/N434Y/Y436V; L251T/M252Y/N434Y/Y436V; L251V/M252Y/N434Y/Y436V; M252Y/I253V/N434Y/Y436V; M252Y/N434Y/Y436V/Q438R/S440D; M252Y/N434Y/Y436V/Q438K/S440E; M252Y/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/Q311H/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307A/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307Q/Q311H/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/H433D/N434Y/Y436V; L235R/S239K/H433D/N434W/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440D; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/V422A/S424A/N434Y/Y436V; L235R/S239K/M252Y/V422L/S424L/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V/Q438A/S440A; L235R/S239K/M252Y/N434Y/Y436V/Q438L/S440L; L235R/S239K/M252Y/V422A/S424A/H433D/N434Y/Y436V; L235R/S239K/M252Y/V422L/S424L/H433D/N434Y/Y436V; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438A/S440A; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438L/S440L; G237K/S239K/M252Y/N434Y/Y436V; G237R/S239K/M252Y/N434Y/Y436V; S239K/M252Y/P329K/N434Y/Y436V; S239K/M252Y/P329R/N434Y/Y436V; M252Y/L328Y/N434Y; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440E; L234A/L235A/M252Y/N434Y/Y436V; L234A/L235A/M252Y/N297A/N434Y/Y436V; L234A/L235A/T25OV/M252Y/T307Q/V3O8P/Q311A/N434Y/Y436V; L234A/L235A/T25OV/M252Y/N297A/T307Q/V308P/Q311A/N434Y/Y436V; L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/N434Y/Y436V; L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440D; L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D; L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436T/Q438K/S440E; L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440E; L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E; L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E; L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438R/S440E; G236R/M252Y/L328R/N434Y/Y436V; L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436V; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V; L235R/S239K/M252Y/T256E/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436T; or L235R/S239K/M252Y/N434Y/Y436F. 2. The method of claim 1, wherein the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F). 3. The method of claim 1, which comprises replacing the amino acid of said Fc region at one or more of amino acid positions 235, 237, 238, 239, 270, 298, 325 and 329 as indicated by EU numbering. 4. A method for improving the pharmacokinetics of an antigen binding molecule, wherein the method comprises modifying the Fc region of the antigen binding molecule comprising a) an antigen binding domain whose antigen binding activity varies with pH and b) an Fc region that has FcRn binding activity at pH 7.4 in an Fc region in which one of the two polypeptides constituting the Fc region has FcRn binding activity at pH 7.4 and the other has no FcRn binding activity at pH 7.4, where said antigen-binding molecule is a human IgG antibody, where the antigen-binding activity is lower at pH 5.8 than at pH 7.4, where the ratio of the KD value for antigen-binding activity at pH 5.8 to the KD value for antigen-binding activity at pH 7.4 (KD (pH 5.8) / KD (pH 7.4)) is 2 or greater, wherein the antigen binding domain contains a histidine residue at each one or more positions selected from the group consisting of H heavy chain variable region positions such as 27, 31, 32, 33, 35, 50, 58, 59, 61, 62, 99, 100b and 102, and positions of the L light chain variable region such as 24, 27, 28, 31, 32, 34, 50, 51, 52, 53, 54, 55, 56, 89, 90, 91, 92, 93, 94 and 95a (Cabot numbering); where the Fc region according to (b), which has a greater FcRn-binding activity at pH 7.4, contains at least one of the following amino acid substitutions in positions in the Fc domain, according to EU numbering: N434W; N434Y; M252Y/S254T/T256E/N434Y; M252Y/S254T/T256E/N434W; M252Y/N434Y; M252Y/N434W; M252W/N434Y; M252W/N434W; P257L/N434Y; V308F/N434Y; M252Y/V308P/N434Y; M428L/N434S; M252Y/S254T/T256E/V308P/N434W; I332V; I332V/N434Y; G237M/V308F; S254T/N434W; S254T/N434Y; T256E/N434W; T256E/N434Y; S254T/T256E/N434W; S254T/T256E/N434Y; M252Y/S254T/N434W; M252Y/S254T/N434Y; M252Y/T256E/N434W; M252Y/T256E/N434Y; M252Y/S254T/T256E/N434A; M252Y/N434A; M252W/N434A; M252Y/T256Q/N434W; M252Y/T256Q/N434Y; M252F/T256D/N434W; M252F/T256D/N434Y; H433K/N434F/Y436H; I332V/N434W; V308P/N434W; I332V/M428L/N434Y; G385D/Q386P/N389S/N434W; G385D/Q386P/N389S/N434Y; G385H/N434W; G385H/N434Y; N434F; M252Y/S254T/T256E/N434F; M252Y/S254T/T256E/N434H; M252Y/N434F; M252Y/N434H; M428L/N434W; M428L/N434Y; M252Y/S254T/T256E/M428L/N434W; M252Y/S254T/T256E/M428L/N434Y; M252Y/M428L/N434W; M252Y/M428L/N434Y; M252Y/M428L/N434A; M252Y/S254T/T256E/M428L/N434A; T256E/M428L/N434Y; S254T/M428L/N434W; M252Y/T256Q/N434A; M252Y/T256E/N434A; T256Q/N434W; T256Q/N434Y; M252W/T256Q/N434W; M252W/T256Q/N434Y; S254T/T256Q/N434W; M252Y/S254T/T256Q/N434W; M252Y/S254T/T256Q/N434Y; M252Y/T256E/V308P/N434W; M252Y/V308P/M428L/N434Y; M252Y/S254T/T256E/V308P/M428L/N434W; M252W/M428L/N434W; P257L/M428L/N434Y; M252Y/S254T/T256E/M428L/N434F; M252Y/T256E/N434H; M252W/T256Q/P257L/N434Y; P238A/M252Y/V308P/N434Y; M252Y/D265A/V308P/N434Y; M252Y/T307A/V308P/N434Y; M252Y/V303A/V308P/N434Y; M252Y/V308P/D376A/N434Y; M252Y/V305A/V308P/N434Y; M252Y/V308P/Q311A/N434Y; M252Y/V308P/K317A/N434Y; M252Y/V308P/E380A/N434Y; M252Y/V308P/E382A/N434Y; M252Y/V308P/S424A/N434Y; P257L/V308P/M428L/N434Y; M252Y/T256E/V308P/M428L/N434W; M252Y/T256E/M428L/N434W; M252Y/T256E/V308P/N434Y; M252Y/T256E/M428L/N434Y; M252Y/S254T/T256E/V308P/M428L/N434Y; P257L/M428L/N434W; P257A/M428L/N434Y; P257G/M428L/N434Y; P257I/M428L/N434Y; P257M/M428L/N434Y; P257N/M428L/N434Y; P257S/M428L/N434Y; P257T/M428L/N434Y; P257V/M428L/N434Y; M252W/V308P/N434W; S239K/M252Y/V308P/N434Y; M252Y/S298G/V308P/N434Y; M252Y/D270F/V308P/N434Y; M252Y/V308A/N434Y; M252Y/V308F/N434Y; M252Y/V308I/N434Y; M252Y/V308L/N434Y; M252Y/V308M/N434Y; M252Y/V308Q/N434Y; M252Y/V308T/N434Y; P257A/V308P/M428L/N434Y; P257T/V308P/M428L/N434Y; P257V/V308P/M428L/N434Y; M252W/M428I/N434Y; M252W/M428Y/N434Y; M252W/M428F/N434Y; P238A/M252W/N434Y; M252W/D265A/N434Y; M252W/T307Q/N434Y; M252W/V303A/N434Y; M252W/D376A/N434Y; M252W/V305A/N434Y; M252W/Q311A/N434Y; M252W/D312A/N434Y; M252W/K317A/N434Y; M252W/E380A/N434Y; M252W/E382A/N434Y; M252W/S424A/N434Y; S239K/M252W/N434Y; M252W/S298G/N434Y; M252W/D270F/N434Y; M252W/N325G/N434Y; P257A/M428L/N434W; P257T/M428L/N434W; P257V/M428L/N434W; M252W/I332V/N434Y; P257I/Q311I; M252Y/T307A/N434Y; M252Y/T307Q/N434Y; P257L/T307A/M428L/N434Y; P257A/T307A/M428L/N434Y; P257T/T307A/M428L/N434Y; P257V/T307A/M428L/N434Y; P257L/T307Q/M428L/N434Y; P257A/T307Q/M428L/N434Y; P257T/T307Q/M428L/N434Y; P257V/T307Q/M428L/N434Y; M252Y/T307D/N434Y; M252Y/T307F/N434Y; M252Y/T307G/N434Y; M252Y/T307H/N434Y; M252Y/T307I/N434Y; M252Y/T307K/N434Y; M252Y/T307L/N434Y; M252Y/T307M/N434Y; M252Y/T307N/N434Y; M252Y/T307P/N434Y; M252Y/T307R/N434Y; M252Y/T307S/N434Y; M252Y/T307V/N434Y; M252Y/T307W/N434Y; M252Y/T307Y/N434Y; M252Y/K334L/N434Y; M252Y/G385H/N434Y; M252Y/T289H/N434Y; M252Y/Q311H/N434Y; M252Y/D312H/N434Y; M252Y/N315H/N434Y; M252Y/K360H/N434Y; M252Y/L314R/N434Y; M252Y/L314K/N434Y; M252Y/N286E/N434Y; M252Y/L309E/N434Y; M252Y/R255E/N434Y; M252Y/P387E/N434Y; K248I/M428L/N434Y; M252Y/M428A/N434Y; M252Y/M428D/N434Y; M252Y/M428F/N434Y; M252Y/M428G/N434Y; M252Y/M428H/N434Y; M252Y/M428I/N434Y; M252Y/M428K/N434Y; M252Y/M428N/N434Y; M252Y/M428P/N434Y; M252Y/M428Q/N434Y; M252Y/M428S/N434Y; M252Y/M428T/N434Y; M252Y/M428V/N434Y; M252Y/M428W/N434Y; M252Y/M428Y/N434Y; M252W/T307Q/M428Y/N434Y; M252W/Q311A/M428Y/N434Y; M252W/T307Q/Q311A/M428Y/N434Y; M252Y/T307A/M428Y/N434Y; M252Y/T307Q/M428Y/N434Y; M252Y/D270F/N434Y; M252Y/T307A/Q311A/N434Y; M252Y/T307Q/Q311A/N434Y; M252Y/T307A/Q311H/N434Y; M252Y/T307Q/Q311H/N434Y; M252Y/E382A/N434Y; M252Y/E382A/M428Y/N434Y; M252Y/T307A/E382A/M428Y/N434Y; M252Y/T307Q/E382A/M428Y/N434Y; P238A/M252Y/M428F/N434Y; M252Y/V305A/M428F/N434Y; M252Y/N325G/M428F/N434Y; M252Y/D376A/M428F/N434Y; M252Y/E380A/M428F/N434Y; M252Y/E382A/M428F/N434Y; M252Y/E380A/E382A/M428F/N434Y; S239K/M252Y/V308P/E382A/N434Y; M252Y/D270F/V308P/E382A/N434Y; S239K/M252Y/V308P/M428Y/N434Y; M252Y/T307Q/V308P/E382A/N434Y; M252Y/V308P/Q311H/E382A/N434Y; S239K/M252Y/D270F/V308P/N434Y; M252Y/V308P/E382A/M428F/N434Y; M252Y/V308P/E382A/M428L/N434Y; M252Y/V308P/E382A/M428Y/N434Y; M252Y/V308P/M428Y/N434Y; M252Y/V308P/M428F/N434Y; S239K/M252Y/V308P/E382A/M428Y/N434Y; M252Y/V308P/E380A/E382A/M428F/N434Y; M252Y/T256E/E382A/N434Y; M252Y/T256E/M428Y/N434Y; M252Y/T256E/E382A/M428Y/N434Y; S239K/M252Y/V308P/E382A/M428I/N434Y; M252Y/V308P/E380A/E382A/M428I/N434Y; S239K/M252Y/V308P/M428F/N434Y; S239K/M252Y/E380A/E382A/M428F/N434Y; M252Y/Q311A/M428Y/N434Y; M252Y/D312A/M428Y/N434Y; M252Y/Q311A/D312A/M428Y/N434Y; S239K/M252Y/V308P/Q311A/M428Y/N434Y; S239K/M252Y/V308P/D312A/M428Y/N434Y; S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y; M252Y/T256E/T307P/N434Y; M252Y/T307P/M428Y/N434Y; M252W/V308P/M428Y/N434Y; M252Y/T256E/V308P/E382A/N434Y; M252W/V308P/E382A/N434Y; S239K/M252W/V308P/E382A/N434Y; S239K/M252W/V308P/E382A/M428Y/N434Y; S239K/M252W/V308P/E382A/M428I/N434Y; S239K/M252W/V308P/M428F/N434Y; S239K/M252W/E380A/E382A/M428F/N434Y; S239K/M252Y/T307P/M428Y/N434Y; M252Y/T256E/Q311A/D312A/M428Y/N434Y; S239K/M252Y/M428Y/N434Y; S239K/M252Y/D270F/M428Y/N434Y; S239K/M252Y/D270F/V308P/M428Y/N434Y; S239K/M252Y/V308P/M428I/N434Y; S239K/M252Y/V308P/N325G/E382A/M428I/N434Y; S239K/M252Y/D270F/V308P/N325G/N434Y; S239K/M252Y/D270F/T307P/V308P/N434Y; S239K/M252Y/T256E/D270F/V308P/N434Y; S239K/M252Y/D270F/T307Q/V308P/N434Y; S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y; S239K/M252Y/D270F/T307Q/Q311A/N434Y; S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y; S239K/M252Y/D270F/V308P/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/N434Y; S239K/M252Y/D270F/V308P/L309E/N434Y; S239K/M252Y/D270F/V308P/P387E/N434Y; S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y; S239K/M252Y/D270F/V308P/N325G/M428I/N434Y; S239K/M252Y/T256E/D270F/V308P/M428I/N434Y; S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/N434Y; S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/M428I/N434Y; S239K/M252Y/D270F/V308P/L309E/M428I/N434Y; S239K/M252Y/D270F/V308P/M428L/N434Y; S239K/M252Y/V308P/M428L/N434Y; S239K/M252Y/V308P/L309P/M428L/N434Y; S239K/M252Y/V308P/L309R/M428L/N434Y; S239K/M252Y/L309P/M428L/N434Y; S239K/M252Y/L309R/M428L/N434Y; S239K/M252Y/T307Q/V308P/M428L/N434Y; S239K/M252Y/D270F/V308P/L309E/P387E/N434Y; S239K/M252Y/T256E/D270F/V308P/N325G/M428I/N434Y; S239K/M252Y/T307Q/N434Y; P257V/T307Q/M428I/N434Y; P257V/T307Q/M428V/N434Y; P257V/T307Q/N325G/M428L/N434Y; P257V/T307Q/Q311A/M428L/N434Y; P257V/T307Q/Q311A/N325G/M428L/N434Y; P257V/V305A/T307Q/M428L/N434Y; S239K/M252Y/E258H/D270F/T307Q/V308P/Q311A/N434Y; S239K/M252Y/D270F/V308P/N325G/E382A/M428I/N434Y; M252Y/P257V/T307Q/M428I/N434Y; S239K/M252W/V308P/M428Y/N434Y; S239K/M252W/V308P/Q311A/M428Y/N434Y; S239K/M252W/T256E/V308P/M428Y/N434Y; S239K/M252W/N286E/V308P/M428Y/N434Y; S239K/M252Y/T256E/D270F/N286E/V308P/N434Y; S239K/M252W/T307P/M428Y/N434Y; S239K/M252W/T256E/V308P/Q311A/M428Y/N434Y; S239K/M252W/T256E/N286E/V308P/M428Y/N434Y; P257V/T307A/Q311A/M428L/N434Y; P257V/V305A/T307A/M428L/N434Y; M252Y/V308P/L309E/N434Y; M252Y/V308P/L309D/N434Y; M252Y/V308P/L309A/N434Y; M252W/V308P/L309E/M428Y/N434Y; M252W/V308P/L309D/M428Y/N434Y; M252W/V308P/L309A/M428Y/N434Y; S239K/M252Y/N286E/V308P/M428I/N434Y; S239K/M252Y/N286E/V308P/N434Y; S239K/M252Y/T307Q/Q311A/M428I/N434Y; S239K/M252Y/T256E/V308P/N434Y; S239K/M252Y/D270F/N286E/T307Q/Q311A/M428I/N434Y; S239K/M252Y/D270F/T307Q/Q311A/P387E/M428I/N434Y; S239K/M252Y/D270F/N286E/T307Q/V308P/Q311A/M428I/N434Y; S239K/M252Y/D270F/N286E/T307Q/V308P/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/Q311A/M428I/N434Y; S239K/M252Y/D270F/N286E/V308P/P387E/M428I/N434Y; P257V/T307Q/M428L/N434W; P257V/T307A/M428L/N434W; P257A/T307Q/L309P/M428L/N434Y; P257V/T307Q/L309P/M428L/N434Y; P257A/T307Q/L309R/M428L/N434Y; P257V/T307Q/L309R/M428L/N434Y; P257V/N286E/M428L/N434Y; P257V/P387E/M428L/N434Y; P257V/T307H/M428L/N434Y; P257V/T307N/M428L/N434Y; P257V/T307G/M428L/N434Y; P257V/T307P/M428L/N434Y; P257V/T307S/M428L/N434Y; P257V/N286E/T307A/M428L/N434Y; P257V/T307A/P387E/M428L/N434Y; S239K/M252Y/T307P/N325G/M428Y/N434Y; M252Y/T307A/Q311H/K360H/N434Y; M252Y/T307A/Q311H/P387E/N434Y; M252Y/T307A/Q311H/M428A/N434Y; M252Y/T307A/Q311H/E382A/N434Y; M252Y/T307W/Q311H/N434Y; S239K/P257A/V308P/M428L/N434Y; P257A/V308P/L309E/M428L/N434Y; P257V/T307Q/N434Y; M252Y/P257V/T307Q/M428V/N434Y; M252Y/P257V/T307Q/M428L/N434Y; M252Y/P257V/T307Q/N434Y; M252Y/P257V/T307Q/M428Y/N434Y; M252Y/P257V/T307Q/M428F/N434Y; P257V/T307Q/Q311A/N325G/M428V/N434Y; P257V/T307Q/Q311A/N325G/N434Y; P257V/T307Q/Q311A/N325G/M428Y/N434Y; P257V/T307Q/Q311A/N325G/M428F/N434Y; P257A/T307Q/M428V/N434Y; P257A/T307Q/N434Y; P257A/T307Q/M428Y/N434Y; P257A/T307Q/M428F/N434Y; P257V/N286E/T307Q/Q311A/N325G/M428L/N434Y; P257A/Q311A/M428L/N434Y; P257A/Q311H/M428L/N434Y; P257A/T307Q/Q311A/M428L/N434Y; P257A/T307A/Q311A/M428L/N434Y; P257A/T307Q/Q311H/M428L/N434Y; P257A/T307A/Q311H/M428L/N434Y; P257A/N286E/T307Q/M428L/N434Y; P257A/N286E/T307A/M428L/N434Y; S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y; S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y; P257S/T307A/M428L/N434Y; P257M/T307A/M428L/N434Y; P257N/T307A/M428L/N434Y; P257I/T307A/M428L/N434Y; P257V/T307Q/M428Y/N434Y; P257V/T307Q/M428F/N434Y; S239K/P257V/V308P/M428L/N434Y; P257V/T307Q/V308P/N325G/M428L/N434Y; P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y; P257V/T307A/V308P/N325G/M428L/N434Y; P257A/V308P/N325G/M428L/N434Y; P257A/T307A/V308P/M428L/N434Y; P257A/T307Q/V308P/M428L/N434Y; P257V/N286E/T307Q/N325G/M428L/N434Y; T256E/P257V/N434Y; T256E/P257T/N434Y; S239K/P257T/V308P/M428L/N434Y; P257T/V308P/N325G/M428L/N434Y; T256E/P257T/V308P/N325G/M428L/N434Y; P257T/V308P/N325G/E382A/M428L/N434Y; P257T/V308P/N325G/P387E/M428L/N434Y; P257T/V308P/L309P/N325G/M428L/N434Y; P257T/V308P/L309R/N325G/M428L/N434Y; T256E/P257V/T307Q/M428L/N434Y; P257V/T307Q/E382A/M428L/N434Y; P257V/T307Q/P387E/M428L/N434Y; P257L/V308P/N434Y; P257T/T307Q/N434Y; P257V/T307Q/N325G/N434Y; P257V/T307Q/Q311A/N434Y; P257V/V305A/T307Q/N434Y; P257V/N286E/T307A/N434Y; P257V/T307Q/L309R/Q311H/M428L/N434Y; P257V/V308P/N325G/M428L/N434Y; S239K/P257V/V308P/Q311H/M428L/N434Y; P257V/V305A/T307A/N325G/M428L/N434Y; S239K/D270F/T307P/N325G/M428Y/N434Y; P257T/T307A/M428V/N434Y; P257A/M428V/N434Y; P257A/T307A/M428V/N434Y; P257S/T307Q/M428L/N434Y; P257V/N297A/T307Q/M428L/N434Y; P257V/N286A/T307Q/M428L/N434Y; P257V/T307Q/N315A/M428L/N434Y; P257V/T307Q/N384A/M428L/N434Y; P257V/T307Q/N389A/M428L/N434Y; P257V/N434Y; P257T/N434Y; P257V/N286E/N434Y; P257T/N286E/N434Y; P257V/N286E/T307Q/N434Y; P257V/N286E/T307Q/M428Y/N434Y; P257V/V305A/T307Q/M428Y/N434Y; P257T/T307Q/M428Y/N434Y; P257V/T307Q/V308P/N325G/M428Y/N434Y; P257T/T307Q/M428F/N434Y; P257A/N286E/T307Q/M428F/N434Y; P257A/N286E/T307Q/M428Y/N434Y; T250A/P257V/T307Q/M428L/N434Y; T250F/P257V/T307Q/M428L/N434Y; T250I/P257V/T307Q/M428L/N434Y; T250M/P257V/T307Q/M428L/N434Y; T250S/P257V/T307Q/M428L/N434Y; T250V/P257V/T307Q/M428L/N434Y; T250W/P257V/T307Q/M428L/N434Y; T250Y/P257V/T307Q/M428L/N434Y; M252Y/Q311A/N434Y; S239K/M252Y/S254T/V308P/N434Y; M252Y/T307Q/Q311A M252Y/S254T/N286E/N434Y; M252Y/S254T/V308P/N434Y; M252Y/S254T/T307A/N434Y; M252Y/S254T/T307Q/N434Y; M252Y/S254T/Q311A/N434Y; M252Y/S254T/Q311H/N434Y; M252Y/S254T/T307A/Q311A/N434Y; M252Y/S254T/T307Q/Q311A/N434Y; M252Y/S254T/M428I/N434Y; M252Y/T256E/T307A/Q311H/N434Y; M252Y/T256Q/T307A/Q311H/N434Y; M252Y/S254T/T307A/Q311H/N434Y; M252Y/N286E/T307A/Q311H/N434Y; M252Y/T307A/Q311H/M428I/N434Y; M252Y/T256E/T307Q/Q311H; M252Y/N286E/T307A/Q311A; M252Y/N286E/T307Q/Q311A; M252Y/N286E/T307Q/Q311H; M252Y/T256E/N286E/N434Y; S239K/M252Y/V308P; S239K/M252Y/V308P/E382A; S239K/M252Y/T256E/V308P; S239K/M252Y/N286E/V308P; S239K/M252Y/N286E/V308P/M428I; M252Y/N286E/M428L/N434Y; M252Y/S254T/E382A/N434Y; S239K/M252Y/S254T/V308P/M428I/N434Y; S239K/M252Y/M428I/N434Y; M252Y/D312A/E382A/M428Y/N434Y; M252Y/E382A/P387E/N434Y; M252Y/D312A/P387E/N434Y; M252Y/P387E/M428Y/N434Y; M252Y/T256Q/E382A/N434Y; M252Y/N286E/V308P/N434Y; M252Y/V305T/T307P/V308I/L309A/N434Y; M252Y/T307P/V308I/L309A/N434Y; S239K/N434W; S239K/V308F/N434Y; S239K/M252Y/N434Y; S239K/M252Y/S254T/N434Y; S239K/M252Y/T307A/Q311H/N434Y; S239K/M252Y/T256Q/N434Y; S239K/M252W/N434W; S239K/P257A/N286E/T307Q/M428L/N434Y; S239K/P257A/T307Q/M428L/N434Y; V308P; M252Y/V279L/V308P/N434Y; S239K/M252Y/V279L/V308P/N434Y; M252Y/V282D/V308P/N434Y; S239K/M252Y/V282D/V308P/N434Y; M252Y/V284K/V308P/N434Y; S239K/M252Y/V284K/V308P/N434Y; M252Y/K288S/V308P/N434Y; S239K/M252Y/K288S/V308P/N434Y; M252Y/V308P/G385R/N434Y; S239K/M252Y/V308P/G385R/N434Y; M252Y/V308P/Q386K/N434Y; S239K/M252Y/V308P/Q386K/N434Y; L235G/G236R/S239K/M252Y/V308P/N434Y; G236R/S239K/M252Y/V308P/N434Y; S239K/M252Y/V308P/L328R/N434Y; S239K/M252Y/N297A/V308P/N434Y; P238D/M252Y/V308P/N434Y; S239K/M252Y/N286E/N434Y; S239K/M252Y/T256E/N434Y; S239K/M252Y/Q311A/N434Y; P238D/M252Y/N434Y; L235K/S239K/M252Y/V308P/N434Y; L235R/S239K/M252Y/V308P/N434Y; G237K/S239K/M252Y/V308P/N434Y; G237R/S239K/M252Y/V308P/N434Y; P238K/S239K/M252Y/V308P/N434Y; P238R/S239K/M252Y/V308P/N434Y; S239K/M252Y/V308P/P329K/N434Y; S239K/M252Y/V308P/P329R/N434Y; S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y; M252Y/N286A/V308P/N434Y; M252Y/N286D/V308P/N434Y; M252Y/N286F/V308P/N434Y; M252Y/N286G/V308P/N434Y; M252Y/N286H/V308P/N434Y; M252Y/N286I/V308P/N434Y; M252Y/N286K/V308P/N434Y; M252Y/N286L/V308P/N434Y; M252Y/N286M/V308P/N434Y; M252Y/N286P/V308P/N434Y; M252Y/N286Q/V308P/N434Y; M252Y/N286R/V308P/N434Y; M252Y/N286S/V308P/N434Y; M252Y/N286T/V308P/N434Y; M252Y/N286V/V308P/N434Y; M252Y/N286W/V308P/N434Y; M252Y/N286Y/V308P/N434Y; M252Y/K288A/V308P/N434Y; M252Y/K288D/V308P/N434Y; M252Y/K288E/V308P/N434Y; M252Y/K288F/V308P/N434Y; M252Y/K288G/V308P/N434Y; M252Y/K288H/V308P/N434Y; M252Y/K288I/V308P/N434Y; M252Y/K288L/V308P/N434Y; M252Y/K288M/V308P/N434Y; M252Y/K288N/V308P/N434Y; M252Y/K288P/V308P/N434Y; M252Y/K288Q/V308P/N434Y; M252Y/K288R/V308P/N434Y; M252Y/K288V/V308P/N434Y; M252Y/K288W/V308P/N434Y; M252Y/K288Y/V308P/N434Y; S239K/M252Y/V308P/N325G/N434Y; M252Y/N286E/T307Q/Q311A/N434Y; S239K/M252Y/N286E/T307Q/Q311A/N434Y; M252Y/V308P/L328E/N434Y; M252Y/N286E/V308P/M428I/N434Y; M252Y/N286E/T307Q/Q311A/M428I/N434Y; M252Y/N286E/T307Q/V308P/Q311A/N434Y; M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y; S239K/M252Y/N286E/T307Q/Q311A/M428I/N434Y; P238D/M252Y/N286E/T307Q/Q311A/M428I/N434Y; S239K/M252Y/T307Q/Q311A/N434Y; P238D/M252Y/T307Q/Q311A/N434Y; P238D/M252Y/N286E/T307Q/Q311A/N434Y; P238D/M252Y/N325S/N434Y; P238D/M252Y/N325M/N434Y; P238D/M252Y/N325L/N434Y; P238D/M252Y/N325I/N434Y; P238D/M252Y/Q295M/N434Y; P238D/M252Y/N325G/N434Y; M252Y/T307Q/V308P/Q311A/N434Y; M252Y/T307Q/V308P/Q311A/M4281/N434Y; P238D/M252Y/A327G/N434Y; P238D/M252Y/L328D/N434Y; P238D/M252Y/L328E/N434Y; L235K/G237R/S239K/M252Y/V308P/N434Y; L235K/P238K/S239K/M252Y/V308P/N434Y; T307A/Q311A/N434Y; T307Q/Q311A/N434Y; T307A/Q311H/N434Y; T307Q/Q311H/N434Y; M252Y/L328E/N434Y; G236D/M252Y/L328E/N434Y; M252Y/S267M/L328E/N434Y; M252Y/S267L/L328E/N434Y; P238D/M252Y/T307P/N434Y; M252Y/T307P/Q311A/N434Y; M252Y/T307P/Q311H/N434Y; P238D/T250A/M252Y/N434Y; P238D/T250F/M252Y/N434Y; P238D/T250G/M252Y/N434Y; P238D/T250H/M252Y/N434Y; P238D/T250I/M252Y/N434Y; P238D/T25UL/M252Y/N434Y; P238D/T250M/M252Y/N434Y; F751 5.30E-07 P238D/T250N/M252Y/N434Y; P238D/T250Q/M252Y/N434Y; P238D/T250S/M252Y/N434Y; P238D/T250V/M252Y/N434Y; P238D/T250W/M252Y/N434Y; P238D/T250Y/M252Y/N434Y; P238D/N434Y; L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y; L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y; P238D/T307Q/Q311A/N434Y; P238D/M252Y/T307Q/L309E/Q311A/N434Y; T307A/L309E/Q311A/N434Y; T307Q/L309E/Q311A/N434Y; T307A/L309E/Q311H/N434Y; T307Q/L309E/Q311H/N434Y; M252Y/T256A/K434Y; M252Y/E272A/N434Y; M252Y/K274A/N434Y; M252Y/V282A/N434Y; M252Y/N286A/N434Y; M252Y/K338A/N434Y; M252Y/K340A/N434Y; M252Y/E345A/N434Y; M252Y/N361A/N434Y; M252Y/Q362A/N434Y; M252Y/S375A/N434Y; M252Y/Y391A/N434Y; M252Y/D413A/N434Y; M252Y/L309A/N434Y; M252Y/L309H/N434Y; M252Y/S254T/N286E/T307Q/Q311A/N434Y; M252Y/S254T/T307Q/L309E/Q311A/N434Y; M252Y/N315A/N434Y; M252Y/N315D/N434Y; M252Y/N315E/N434Y; M252Y/N315F/N434Y; M252Y/N315G/N434Y; M252Y/N315I/N434Y; M252Y/N315K/N434Y; M252Y/N315L/N434Y; M252Y/N315M/N434Y; M252Y/N315Q/N434Y; M252Y/N315R/N434Y; M252Y/N315S/N434Y; M252Y/N315T/N434Y; M252Y/N315V/N434Y; M252Y/N315W/N434Y; M252Y/N315Y/N434Y; M252Y/N325A/N434Y; M252Y/N384A/N434Y; M252Y/N389A/N434Y; M252Y/N389A/N390A/N434Y; M252Y/S254T/T256S/N434Y; M252Y/A378V/N434Y; M252Y/E380S/N434Y; M252Y/E382V/N434Y; M252Y/S424E/N434Y; M252Y/N434Y/Y436I; M252Y/N434Y/T437R; P238D/T250V/M252Y/T307P/N434Y; P238D/T250V/M252Y/T307Q/Q311A/N434Y; P238D/M252Y/N286E/N434Y; L235K/S239K/M252Y/N434Y; L235R/S239K/M252Y/N434Y; P238D/T250Y/M252Y/W313Y/N434Y; P238D/T250Y/M252Y/W313F/N434Y; P238D/T250V/M252Y/1253V/N434Y; P238D/T250V/M252Y/R255A/N434Y; P238D/T250V/M252Y/R255D/N434Y; P238D/T250V/M252Y/R255E/N434Y; P238D/T250V/M252Y/R255F/N434Y; P238D/T250V/M252Y/R255H/N434Y; P238D/T250V/M252Y/K2551/M434Y; P238D/T250V/M252Y/R255K/N434Y; P238D/T250V/M252Y/R255L/N434Y; P238D/T250V/M252Y/R255M/N434Y; P238D/T250V/M252Y/R255N/N434Y; P238D/T250V/M252Y/R255Q/N434Y; P238D/T250V/ M252Y/ R255S/ N434Y; P238D/T250V/M252Y/R255W/N434Y; P238D/T250V/M252Y/R255Y/N434Y; M252Y/D280A/N434Y; M252Y/D280E/N434Y; M252Y/D280G/N434Y; M252Y/D280H/N434Y; M252Y/D280K/N434Y; M252Y/D280N/N434Y; M252Y/D280Q/N434Y; M252Y/D280R/N434Y; M252Y/D280S/N434Y; M252Y/D280T/N434Y; M252Y/N384A/N389A/N434Y; G236A/S239D/M252Y/N286E/T307Q/Q311A/N434Y; L235K/S239K/M252Y/T307Q/Q311A/N434Y; L235R/S239K/M252Y/T307Q/Q311A/N434Y; L235K/S239K/M252Y/N286E/N434Y; L235R/S239K/M252Y/N286E/N434Y; M252Y/N434Y/Y436A; M252Y/N434Y/Y436E; M252Y/N434Y/Y436F; M252Y/N434Y/Y436G; M252Y/N434Y/Y436H; M252Y/N434Y/Y436K; M252Y/N434Y/Y436L; M252Y/N434Y/Y436M; M252Y/N434Y/Y436N; M252Y/N434Y/Y436S; M252Y/N434Y/Y436T; M252Y/N434Y/Y436V; M252Y/N434Y/Y436W; M252Y/S254T/N434Y/Y436I; L235K/S239K/M252Y/N434Y/Y436I; L235R/S239K/M252Y/N434Y/Y436I; L235K/S239K/M252Y/N286E/T307Q/Q311A/N315E/N434Y; L235R/S239K/M252Y/N286E/T307Q/Q311A/N315E/N434Y; M252Y/N315D/N384A/N389A/N434Y; M252Y/N315E/N384A/N389A/N434Y; M252Y/N315D/G316A/N434Y; M252Y/N315D/G316D/N434Y; M252Y/N315D/G316E/N434Y; M252Y/N315D/G316F/N434Y; M252Y/N315D/G316H/N434Y; M252Y/N315D/G316I/N434Y; M252Y/N315D/G316K/N434Y; M252Y/N315D/G316L/N434Y; M252Y/N315D/G316M/N434Y; M252Y/N315D/G316N/N434Y; M252Y/N315D/G316P/N434Y; M252Y/N315D/G316Q/N434Y; M252Y/N315D/G316R/N434Y; M252Y/N315D/G316S/N434Y; M252Y/N315D/G316T/N434Y; M252Y/N315D/G316V/N434Y; M252Y/N315D/G316W/N434Y; M252Y/N286S/N434Y; M252Y/D280E/N384A/N389A/N434Y; M252Y/D280G/N384A/N389A/N434Y; M252Y/N286S/N384A/N389A/N434Y; M252Y/N286E/N384A/N389A/N434Y; L235K/S239K/M252Y/N286E/N434Y/Y436I; L235R/S239K/M252Y/N286E/N434Y/Y436I; L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436I; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436I; L235K/S239K/M252Y/S254T/N434Y/Y436I; L235R/S239K/M252Y/S254T/N434Y/Y436I; M252Y/T307Q/Q311A/N434Y/Y436I; M252Y/S254T/T307Q/Q311A/N434Y/Y436I; P238D/T250V/M252Y/N286E/N434Y; P238D/T250V/M252Y/N434Y/Y436I; T250V/M252Y/N434Y; L234R/P238D/T250V/M252Y/N434Y; G236K/P238D/T250V/M252Y/N434Y; G237K/P238D/T250V/M252Y/N434Y; G237R/P238D/T250V/M252Y/N434Y; P238D/S239K/T250V/M252Y/N434Y; L235K/S239K/M252Y/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V; P238D/T250V/M252Y/N434Y/Y436V; M252Y/N286E/T307Q/Q311A/N434Y/Y436V; L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436V; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/N434Y/Y436V; T250V/M252Y/V308P/N434Y/Y436V; T250V/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; M252Y/N434Y/H435K/Y436V; M252Y/N434Y/H435N/Y436V; M252Y/N434Y/H435R/Y436V; M252Y/S254A/N434Y; M252Y/S254G/N434Y; M252Y/S254H/N434Y; M252Y/S2541/N434Y; M252Y/S254Q/N434Y; M252Y/S254V/N434Y; P238D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; P238D/T250V/M252Y/N286E/N434Y/Y436V; L235R/S239K/M252Y/V308P/N434Y/Y436V; L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V; L235R/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V; M252Y/T307Q/Q311A/N434Y/Y436V; M252Y/N325G/N434Y; L235R/P238D/S239K/M252Y/N434Y; L235R/P238D/S239K/M252Y/N434Y/Y436V; K248I/T250V/M252Y/N434Y/Y436V; K248Y/T250V/M252Y/N434Y/Y436V; T250V/M252Y/E258H/N434Y/Y436V; L235R/S239K/T250V/M252Y/N434Y/Y436V; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/M252Y/T307A/Q311H/N434Y; T250V/M252Y/V308P/N434Y; L235R/S239K/T250V/M252Y/V308P/N434Y; T250V/M252Y/T307Q/V308P/Q311A/N434Y; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y; L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y; T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; P238D/T250V/M252Y/N325G/N434Y; P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y; P238D/T250V/M252Y/N325A/N434Y; S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V; S239D/T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V; M252Y/T307A/Q311H/N434Y/Y436V; L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V; G236A/S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V; G236A/S239D/T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V; P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y/Y436V; P238D/N434W; L235K/S239K/M252Y/V308P/N434Y/Y436V; L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/N286E/T307Q/Q311A/K434Y/Y436V; L235K/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V; L235K/P238D/S239K/M252Y/N434Y; L235K/P238D/S239K/M252Y/N434Y/Y436V; L235K/S239K/T250V/M252Y/N434Y/Y436V; L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/T307A/Q311H/N434Y; L235K/S239K/T250V/M252Y/V308P/N434Y; L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y; L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y; L235K/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V; L235K/S239K/M252Y/T307A/Q311H/N434Y/Y436V; T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V; L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434; Y436V; L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V; L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V; M252Y/Q386E/N434Y/Y436V; M252Y/Q386R/N434Y/Y436V; M252Y/Q386S/N434Y/Y436V; M252Y/P387E/N434Y/Y436V; M252Y/P387R/N434Y/Y436V; M252Y/P387S/N434Y/Y436V; M252Y/V422E/N434Y/Y436V; M252Y/V422R/N434Y/Y436V; M252Y/V422S/N434Y/Y436V; M252Y/S424E/N434Y/Y436V; M252Y/S424R/N434Y/Y436V; M252Y/N434Y/Y436V/Q438E; M252Y/N434Y/Y436V/Q438R; M252Y/N434Y/Y436V/Q438S; M252Y/N434Y/Y436V/S440E; M252Y/N434Y/Y436V/S440R; S239D/M252Y/N434Y/Y436V; M252Y/K326D/L328Y/N434Y/Y436V; S239D/M252Y/K326D/L328Y/N434Y/Y436V; K248N/M252Y/N434Y; M252Y/E380N/E382S/N434Y; M252Y/E382N/N384S/N434Y; M252Y/S424N/N434Y; M252Y/N434Y/Y436N/Q438T; M252Y/N434Y/Q438N; M252Y/N434Y/S440N; M252Y/N434Y/S442N; M252Y/S383N/G385S/N434Y; M252Y/Q386T/N434Y; M252Y/G385N/P387S/N434Y; S239D/M252Y/N434Y; M252Y/K326D/L328Y/N434Y; S239D/M252Y/K326D/L328Y/N434Y; S239D/M252Y/T307Q/Q311A/N434Y; M252Y/T307Q/Q311A/K326D/L328Y/N434Y; S239D/M252Y/T307Q/Q311A/K326D/L328Y/N434Y; M252Y/V422E/S424R/N434Y/Y436V; M252Y/V422S/S424R/N434Y/Y436V; M252Y/N434Y/Y436V/Q438R/S440E; M252Y/V422D/N434Y/Y436V; M252Y/V422K/N434Y/Y436V; M252Y/V422T/N434Y/Y436V; M252Y/V422Q/N434Y/Y436V; M252Y/S424K/N434Y/Y436V; M252Y/N434Y/Y436V/Q438K; M252Y/N434Y/Y436V/S440D; M252Y/N434Y/Y436V/S440Q; M252Y/S424N/N434Y/Y436V; M252Y/T307Q/Q311A/S424N/N434Y; T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/V422E/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/S424R/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/V422E/S424R/N434Y/Y436V; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/S440E; T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S440E; M252Y/T307Q/N434Y/Y436V; M252Y/T307A/N434Y/Y436V; M252Y/Q311A/N434Y/Y436V; M252Y/Q311H/N434Y/Y436V; M252Y/T307Q/Q311H/N434Y/Y436V; M252Y/T307A/Q311A/N434Y/Y436V; L235R/S239K/M252Y/V422E/N434Y/Y436V; L235R/S239K/M252Y/V422S/N434Y/Y436V; L235R/S239K/M252Y/S424R/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V/Q438R; L235R/S239K/M252Y/N434Y/Y436V/S440E; L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V; L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S424N/N434Y/Y436V; M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E; M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S 440E; T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E; M252Y/T307Q/Q311A/N434Y/Q438R/S440E; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Q438R/S440E; L235R/S239K/M252Y/S424N/N434Y; L235R/S239K/M252Y/T307Q/Q311A/S424N/N434Y; L235R/S239K/T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V; L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V; M252Y/N434Y/Q438R/S440E; L235R/S239K/M252Y/N434Y/Q438R/S440E; S424N/N434W; V308F/S424N/N434Y; I332V/S424N/N434Y; P238D/T250Y/M252Y/N434Y/Y436V; P238D/T250Y/M252Y/T307Q/Q311A/N434Y; P238D/T250Y/M252Y/T307Q/Q311A/M434Y/Y436V; P238D/T250Y/M252Y/T307Q/V308P/Q311A/N434Y/Y436V; P2571/N434H; V308F; V259I/V308F/M428L; E380A/M428L/N434S; T307A/M428L/N434S; T307A/E380A/M428L/N434S; T307A/E380A/N434H; M252Y/H4330/N434Y/Y436V/Q438R/S440E; M252Y/H433E/N434Y/Y436V/Q438R/S440E; M252Y/N434Y/Y436V/T437A/Q438R/S440E; M252Y/N434Y/Y436V/T437G/Q438R/S440E; M252Y/N434Y/Y436V/Q438R/K439D/S440E; M252Y/N434Y/Y436V/Q438R/S440E/L441A; M252Y/N434Y/Y436V/Q438R/S440E/L441E; M252Y/S254T/N434Y/Y436V/Q438R/S440E; M252Y/T256E/N434Y/Y436V/Q438R/S440E; M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E; T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E; T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; M252Y/T256Q/N434Y/Y436V/Q438R/S440E; M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E; T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; I332E/M428L/N434S; L251A/M252Y/N434Y/Y436V; L251H/M252Y/N434Y/Y436V; L251N/M252Y/N434Y/Y436V; L251S/M252Y/N434Y/Y436V; L251T/M252Y/N434Y/Y436V; L251V/M252Y/N434Y/Y436V; M252Y/I253V/N434Y/Y436V; M252Y/N434Y/Y436V/Q438R/S440D; M252Y/N434Y/Y436V/Q438K/S440E; M252Y/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307Q/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/Q311H/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307A/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T307Q/Q311H/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256N/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/H433D/N434Y/Y436V; L235R/S239K/H433D/N434W/Y436V/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440D; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440D; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/V422A/S424A/N434Y/Y436V; L235R/S239K/M252Y/V422L/S424L/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436V/Q438A/S440A; L235R/S239K/M252Y/N434Y/Y436V/Q438L/S440L; L235R/S239K/M252Y/V422A/S424A/H433D/N434Y/Y436V; L235R/S239K/M252Y/V422L/S424L/H433D/N434Y/Y436V; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438A/S440A; L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438L/S440L; G237K/S239K/M252Y/N434Y/Y436V; G237R/S239K/M252Y/N434Y/Y436V; S239K/M252Y/P329K/N434Y/Y436V; S239K/M252Y/P329R/N434Y/Y436V; M252Y/L328Y/N434Y; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440D; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440E; L234A/L235A/M252Y/N434Y/Y436V; L234A/L235A/M252Y/N297A/N434Y/Y436V; L234A/L235A/T25OV/M252Y/T307Q/V3O8P/Q311A/N434Y/Y436V; L234A/L235A/T25OV/M252Y/N297A/T307Q/V308P/Q311A/N434Y/Y436V; L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E; L235R/G236R/S239K/M252Y/N434Y/Y436V; L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440D; L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D; L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D; L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436T/Q438K/S440E; L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440E; L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E; L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E; L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438R/S440E; G236R/M252Y/L328R/N434Y/Y436V; L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/H433D/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440E; L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438R/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438K/S440E; L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436F/Q438R/S440E; L235R/S239K/M252Y/S254T/N434Y/Y436V; L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V; L235R/S239K/M252Y/T256E/N434Y/Y436V; L235R/S239K/M252Y/N434Y/Y436T; or L235R/S239K/M252Y/N434Y/Y436F. 5. The method according to any one of paragraphs. 1-4, where the antigen-binding domain is the variable region of an antibody.

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